Robotic method for coating a multiwell plate by a polyelectrolyte multilayer film

Abstract

The invention concerns a robotic method for coating the bottom surface of at least one well of a multiwell plate by a polyelectrolyte multilayer film, the multiwell plate obtainable according to the method and the use thereof for cell culture.

Claims

1. A multiwell plate obtainable by a method comprising n successive sequences, n being an integer from 1 to 2000, wherein each sequence comprises the steps of: a) robotic deposit of a volume V.sub.PE.sup.1 of a solution of a first polyelectrolyte PE.sup.1 on the bottom surface of at least one well of a multiwell plate, wherein the first polyelectrolyte PE.sup.1 is either a cationic polymer comprising amino groups, or an anionic polymer, then b) robotic aspiration of an aspirated volume V.sub.aspPE.sup.1 of said solution of PE.sup.1, wherein the aspirated volume V.sub.aspPE.sup.1 is higher than or equal to V.sub.PE.sup.1, then c) robotic deposit of a volume V.sub.PE.sup.2 of a solution of a second polyelectrolyte PE.sup.2 on said bottom surface, wherein the second polyelectrolyte PE.sup.2 is a cationic polymer comprising amino groups when PE.sup.1 is an anionic polymer, or PE.sup.2 is an anionic polymer when PE.sup.1 is a cationic polymer comprising amino groups, then d) robotic aspiration of an aspirated volume V.sub.aspPE.sup.2 of said solution of PE.sup.2, wherein the aspirated volume V.sub.aspPE.sup.2 is higher than or equal to V.sub.PE.sup.2.

2. The multiwell plate according to claim 1, wherein, for each sequence of the method, at steps a), b), c), d), the bottom surface is tilted with an inclination angle α from 5 to 40° relative to the horizontal plane.

3. The multiwell plate according to claim 1, wherein the anionic polymer is selected from the group consisting of poly(acrylic) acid, poly(methacrylic) acid, poly(glutamic) acid, polyuronic acid, glycosaminoglycans, poly(aspartic acid) and Polystyrene sulfonate, any combination of the polyamino-acids (in the D and/or L forms), and mixtures thereof.

4. The multiwell plate according to claim 1, wherein the cationic polymer comprising amino group is selected from the group consisting of poly(lysine), poly(diallydimethylammonium chloride), poly(allylamine), poly(ethylene)imine, chitosan, polyarginine, Poly(ornithine), polyhistidine, poly(mannosamine), polyallylamine hydrochloride, any combination of the polyamino acids (in the D and/or L forms), and mixtures thereof.

5. The multiwell plate according to claim 1, wherein the polyelectrolyte multilayer film is a poly(L-lysine)/hyaluronan sodium film, a polystyrene sulfonate/polyallylamine hydrochloride film, a poly(L-lysine)/poly(L-glutamic acid) film or a chitosan/poly(L-glutamic acid) film.

6. The multiwell plate according to claim 1, wherein the anionic polymer comprises carboxylic groups, and wherein the method comprises, after the n sequences, the following steps: e) reacting said amino and carboxylic groups in the presence of a coupling agent, so as to form amide bonds and to cross-link the polyelectrolyte multilayer film, then f) treating said cross-linked polyelectrolyte multilayer film with a protein containing solution, so as to incorporate said protein on and inside said cross-linked polyelectrolyte multilayer film.

7. The multiwell plate according to claim 1, wherein the method comprises, after the n sequences, a step e′) of robotic deposit of a volume V.sub.PE.sup.3 of a solution of a third polyelectrolyte PE.sup.3 on said bottom surface, wherein: polyelectrolyte PE.sup.3 is linked to at least a peptide, and the third polyelectrolyte PE.sup.3 is a cationic polymer comprising amino groups when PE.sup.2 is an anionic polymer, or PE.sup.3 is an anionic polymer when PE.sup.2 is a cationic polymer comprising amino groups.

8. A multiwell plate comprising wells, wherein the bottom surface of m wells is coated by a polyelectrolyte multilayer film, wherein m is an integer from 1 to the number of wells of the multiwell plate, the polyelectrolyte multilayer film comprising n layer pairs, n being an integer from 1 to 2000 and each layer pair comprising a layer of a first polyelectrolyte PE.sup.1 and a layer of a second polyelectrolyte PE.sup.2 of opposite charge, wherein the first polyelectrolyte PE.sup.1 is either a cationic polymer comprising amino groups, or an anionic polymer, the second polyelectrolyte PE.sup.2 is a cationic polymer comprising amino groups when PE.sup.1 is an anionic polymer, or PE.sup.2 is an anionic polymer when PE.sup.1 is a cationic polymer comprising amino groups, the polyelectrolyte multilayer film presenting a coefficient of variation CV of its mean thickness less than or equal to 20.3%, wherein $\mathrm{CV}=\frac{\mathrm{SD}}{\mathrm{hMEAN}}\times 100$ SD being the standard deviation $\mathrm{SD}=\sqrt{\frac{{.Math.}_1^m{\left(\mathrm{hWELLi}-\mathrm{hMEAN}\right)}^2}{\left(m-1\right)}}$ $\mathrm{hMEAN}=\frac{{.Math.}_1^m\mathrm{hWELL}}{m}$ $h_{\mathrm{WELL}}=\frac{\left(\mathrm{hN}+\mathrm{hW}+\mathrm{hC}+\mathrm{hE}+\mathrm{hS}\right)}{S}$ hN, hW, hC, hE and hS being film thicknesses determined at the positions N, W, C, E, S inside each well as shown in FIG. 3.

9. The multiwell plate according to claim 8, wherein the anionic polymer is selected from the group consisting of poly(acrylic) acid, poly(methacrylic) acid, poly(glutamic) acid, polyuronic acid, glycosaminoglycans, poly(aspartic acid) and Polystyrene sulfonate, any combination of the polyamino-acids (in the D and/or L forms), and mixtures thereof.

10. The multiwell plate according to claim 8, wherein the cationic polymer comprising amino group is selected from the group consisting of poly(lysine), poly(diallydimethylammonium chloride), poly(allylamine), poly(ethylene)imine, chitosan, polyarginine, Poly(ornithine), polyhistidine, poly(mannosamine), polyallylamine hydrochloride, any combination of the polyamino acids (in the D and/or L forms), and mixtures thereof.

11. The multiwell plate according to claim 8, wherein the polyelectrolyte multilayer film is a poly(L-lysine)/hyaluronan sodium film, a polystyrene sulfonate/polyallylamine hydrochloride film, a poly(L-lysine)/poly(L-glutamic acid) film or a chitosan/poly(L-glutamic acid) film.

12. The multiwell plate according to claim 8, further comprising a layer of a third polyelectrolyte PE.sup.3 deposited on the top of the polyelectrolyte multilayer film, wherein the third polyelectrolyte PE.sup.3 is linked to at least a peptide, and the third polyelectrolyte PE.sup.3 is a cationic polymer comprising amino groups when the second polyelectrolyte PE.sup.2 is an anionic polymer, or the third polyelectrolyte PE.sup.3 is an anionic polymer when the second polyelectrolyte PE.sup.2 is a cationic polymer comprising amino groups.

13. The multiwell plate according to claim 8, wherein the anionic polymer comprises carboxylic groups, and the polyelectrolyte multilayer film is cross-linked via amide bonds or derivatives thereof formed from the carboxylic groups and the amino groups of the polyelectrolyte multilayer film.

14. The multiwell plate according to claim 13, wherein a protein is incorporated on and inside the cross-linked polyelectrolyte multilayer film.

15. A multiwell plate comprising wells, wherein the bottom surface of m wells is coated by a polyelectrolyte multilayer film, wherein m is an integer from 1 to the number of wells of the multiwell plate, the polyelectrolyte multilayer film comprising n layer pairs, n being an integer from 1 to 2000 and each layer pair comprising a layer of a first polyelectrolyte PE.sup.1 and a layer of a second polyelectrolyte PE.sup.2 of opposite charge, wherein the first polyelectrolyte PE.sup.1 is either a cationic polymer comprising amino groups, or an anionic polymer, the second polyelectrolyte PE.sup.2 is a cationic polymer comprising amino groups when PE.sup.1 is an anionic polymer, or PE.sup.2 is an anionic polymer when PE.sup.1 is a cationic polymer comprising amino groups, the polyelectrolyte multilayer film presenting a standard deviation SD less than or equal to 3.9%, wherein $\mathrm{SD}=\sqrt{\frac{{.Math.}_1^m{\left(\mathrm{hWELLi}-\mathrm{hMEAN}\right)}^2}{\left(m-1\right)}}$ $\mathrm{hMEAN}=\frac{{.Math.}_1^m\mathrm{hWELL}}{m}$ $h_{\mathrm{WELL}}=\frac{\left(\mathrm{hN}+\mathrm{hW}+\mathrm{hC}+\mathrm{hE}+\mathrm{hS}\right)}{S}$ hN, hW, hC, hE and hS being film thicknesses determined at the positions N, W, C, E, S inside each well as shown in FIG. 3.

16. The multiwell plate according to claim 15, wherein the anionic polymer is selected from the group consisting of poly(acrylic) acid, poly(methacrylic) acid, poly(glutamic) acid, polyuronic acid, glycosaminoglycans, poly(aspartic acid) and Polystyrene sulfonate, any combination of the polyamino-acids (in the D and/or L forms), and mixtures thereof.

17. The multiwell plate according to claim 15, wherein the cationic polymer comprising amino group is selected from the group consisting of poly(lysine), poly(diallydimethylammonium chloride), poly(allylamine), poly(ethylene)imine, chitosan, polyarginine, Poly(ornithine), polyhistidine, poly(mannosamine), polyallylamine hydrochloride, any combination of the polyamino acids (in the D and/or L forms), and mixtures thereof.

18. The multiwell plate according to claim 15, further comprising a layer of a third polyelectrolyte PE.sup.3 deposited on the top of the polyelectrolyte multilayer film, wherein the third polyelectrolyte PE.sup.3 is linked to at least a peptide, and the third polyelectrolyte PE.sup.3 is a cationic polymer comprising amino groups when the second polyelectrolyte PE.sup.2 is an anionic polymer, or the third polyelectrolyte PE.sup.3 is an anionic polymer when the second polyelectrolyte PE.sup.2 is a cationic polymer comprising amino groups.

19. The multiwell plate according to claim 15, wherein the anionic polymer comprises carboxylic groups, and the polyelectrolyte multilayer film is cross-linked by amide bonds or derivatives thereof.

20. The multiwell plate according to claim 15, wherein a protein is incorporated on and inside the cross-linked polyelectrolyte multilayer film.

Description

[0117] The following figures and examples illustrate the invention, but should not be regarded as limiting the scope of the application.

[0118] FIG. 1. Schematic of the layer-by-layer deposit at high throughput in multiple well cell culture plates. Working principle of process when the tilting embodiment is operated. The plate is tilted during all the deposit and aspiration steps in all four major deposit and rinsing steps: polycation and its rinsing, polyanion and its rinsing.

[0119] FIG. 2: Illustration of a multiwell plate and definition of the (X,Y, Z) coordinates, the (X,Y) coordinates of each well center being known for commercially available cell culture plates; Z0, the initial position of the tip during the dispense of solutions, needs to be defined by the user.

[0120] FIG. 3: Definition of the 4 pole positions (N, W, E, S) and center position (C) that are selected to assess the film thickness homogeneity inside each well. The imaging process of each of the 5 positions in each individual well was automatized using a custom-made macro using the confocal microscope software.

[0121] FIG. 4: Fluorescence intensity acquired via high resolution imaging in function of the distance Z from the bottom surface of the well. The film thickness h=Z2−Z1 was measured using a custom-made macro using Image J.

[0122] FIG. 5: Film thickness measured at the different pole positions (N, W, C, E, S) for each well in the case of hand-made films using a multichannel pipette (comparative example). The mean+Standard deviation (mean±SD) of hN, hW, hC, hE, and hS, respectively (for m=48 wells) are plotted for each position.

[0123] FIG. 6: CV(%) measured at the different pole positions (N, W, C, E, S) for each well in the case of hand-made films using a multichannel pipette (comparative example).

[0124] FIG. 7: Box plot of the mean thickness per well (hWELL) over m independent wells in the case of hand-made films using a multichannel pipette (comparative example).

[0125] FIG. 8: CV of hWELL (with m=48 wells) in the case of hand-made films using a multichannel pipette (comparative example).

[0126] For FIGS. 5 to 8, data ware pooled for two independent experiments, each with 24 wells per multiwell plate (m=48 wells in total).

[0127] FIG. 9: Film thickness (μm)±SD measured at the different pole positions (N, W, C, E, S) for each well in the case of robot-made films for the condition NT_0% of example 1.

[0128] FIG. 10: CV(%) measured at the different pole positions (N, W, C, E, S) for each well in the case of robot-made films for the condition NT_0% of example 1.

[0129] FIG. 11: Film thickness (μm)±SD measured at the different pole positions (N, W, C, E, S) for each well in the case of robot-made films for the condition NT_10% of example 1.

[0130] FIG. 12: CV(%) measured at the different pole positions (N, W, C, E, S) for each well in the case of robot-made films for the condition NT_10% of example 1.

[0131] FIG. 13: Film thickness (μm)±SD measured at the different pole positions (N, W, C, E, S) for each well in the case of robot-made films for the condition T_0% of example 1.

[0132] FIG. 14: CV(%) measured at the different pole positions (N, W, C, E, S) for each well in the case of robot-made films for the condition T_0% of example 1.

[0133] FIG. 15: Film thickness (μm)±SD measured at the different pole positions (N, W, C, E, S) for each well in the case of robot-made films for the condition T_10% of example 1.

[0134] FIG. 16: CV(%) measured at the different pole positions (N, W, C, E, S) for each well in the case of robot-made films for the condition T_10% of example 1.

[0135] FIG. 17: Film thickness (μm)±SD measured at the different pole positions (N, W, C, E, S) for each well in the case of robot-made films for the condition surface wet of example 1.

[0136] FIG. 18: CV(%) measured at the different pole positions (N, W, C, E, S) for each well in the case of robot-made films for the condition surface wet of example 1.

[0137] For FIGS. 9 to 18, data are mean±SD and CV for 9 wells in total per condition, from two independent experiments.

[0138] FIG. 19: Mean thickness per well (hWELL) for (PLUHA) 12 film made in 96-well plates using the robotic arm. Mean thickness per well was calculated, for each individual microwell, as the mean of the 5 positions. All mean thickness measurements were plotted as box plot for each of the 5 experimental conditions. Data are: from 33 microwells (from 3 independent experiments) for NT conditions; for 81 wells (from 4 independent experiments) for T conditions, and from 12 wells for Surface Wet conditions (Example 1) Data representation: Data are represented as box plots showing 1st quartile, median, 3rd quartile, the limits being 10 and 90% and the extreme values 5 and 95%, respectively

[0139] FIG. 20: CV for (PLUHA) 12 film made in 96-well plates using the robotic arm corresponding to the Mean thickness per well illustrated in FIG. 19 (example 1).

[0140] FIGS. 21 to 25: For each experimental condition (FIG. 21: NT_0%-FIG. 22: NT_10%-FIG. 23: T_0%-FIG. 24: T_10%-FIG. 25: surface wet), 3 representative curves of the fluorescence intensities (acquired using the tile scan option of the confocal microscope) of all pixels in each well are shown (continuous, dashed and dotted black lines) (example 1). The standard deviation of their height is given in Table 2.

[0141] FIGS. 26 to 29: Thickness measurements at the five pole positions (N, W, C, E, S) and at different distances (Z-step) between the end of the tip and the bottom surface for each of the 4 experimental positions (FIG. 26: NT_0%-FIG. 27: NT_10%-FIG. 28: T_0%-FIG. 29: T_10%). The end of the tip was positioned at 4 different heights above the bottom of the microplate, from Z0=+0.1 to +1 mm by steps of +0.3 mm. (eg+0.1; +0.4; +0.7 and +1 mm above the bottom of the well (n=6 well per condition). Each box plot represents a total of 30 thickness measurements on 6 independent wells at 5 positions inside each well.

[0142] FIG. 30: % of surface area covered (calculated from tile scan images of the microwells) by C2C12 cells for the three experimental conditions as a function of the BMP-2 initial concentration of loading. Data are mean±SD of at least 10 wells (example 3)

[0143] FIG. 31: ALP activity (a.u.) by the cells for the four experimental conditions as a function of the BMP-2 initial concentration of loading (example 3). Bioactivity of matrix-bound BMP-2 on C2C12 cells. (PLL/HA) films built using the robotic arm were crosslinked (with EDC30), post-loaded with BMP-2 at 5, 10, 25 and 50 μg/mL were assessed for their bioactivity. C2C12 cells plated at 5000 cells/well in growth medium were stained for ALP after 3 days of culture. (Example 3)

[0144] FIG. 32: ALP activity (a.u.) of stem cells for the T_10% condition as a function of the BMP-7 initial concentration of loading. Bioactivity of matrix-bound BMP-7 on D1 stem cells. Matrix-bound BMP-7 was loaded on crosslinked (PLUHA) 12 films (crosslinked with EDC10), which were prepared using the robotic arm in the T_10% condition. D1 murine mesenchymal stem cells were plated in each microwell and cultured up to 2 days in GM, before being switch in DM for 7 additional days. ALP activity was quantified by enzymatic assay. 4 increasing BMP-7 loading concentrations from 2.5 to 50 μg/mL were tested in comparison to the film in the absence of BMP-7. ALP expression at day 3 is plotted as a function of the BMP-7 loaded dose in the polyelectrolyte films. Data are mean±SD of three independent wells for each experimental condition.

[0145] FIG. 33: Film thickness of (PSS/PAH) polyelectrolyte films as a function of n (number of layer pairs)

[0146] (PSS/PAH) films containing an increasing number of layer pairs from 10 to 40 were deposited using the robotic arm with the condition T_10% on a silicon substrate using PDMS microwells. After film deposit, the PDMS wells were removed and the samples were probed by AFM. Film thickness are measured after scratching of the films. Data are mean±SD of 25 measurements (5 independent measurement per sample, 5 samples for each experimental condition). The linear fit of the data (Y=6,34X−7,11, R=0,974) confirms the linear growth of these films.

[0147] FIG. 34. Bioactivity of matrix-bound BMPs on the BMP responsive skeletal myoblasts (C2C12 cells) assessed by visual observations using a scanner. ALP activity of C2C12 myoblasts cultured for 3 days on BMP-loaded films (crosslinked to EDC70) was visualized at high throughput for each single well by the intensity of the staining. 5 different BMPs were studied (BMP-2, BMP-4, BMP-7, BMP-9, and BV265) and 4 different BMPs loaded quantities (initial BMP concentration in solution from 2.5 to 20 μg/mL).

[0148] FIG. 35: Bioactivity of matrix-bound BMPs on the BMP responsive skeletal myoblasts (C2C12 cells). ALP activity (a.u.) was measured as a function of the initial concentration of BMP in solution during the loading phase in the biomimetic films. Matrix-bound BMPs were loaded on crosslinked (PLL/HA) 12 films (crosslinked with EDC70), which were prepared using the robotic arm in the T_10% condition. C2C12 myoblasts were plated in each microwell and cultured for 3 days in GM. ALP activity was quantified measuring the absorbance at 570 nm using a Tecan Infinite 1000 microplate reader in a multiple reading mode (mean value of 76 different positions in each single microwell). For each BMP (BMP-2, BMP-4, BMP-7, BMP-9, and BV265, 4 increasing BMPs loading concentrations from 2.5 to 20 μg/mL were tested in comparison to the film in the absence of BMPs. ALP expression at day 3 is plotted as a function of the initial BMPs concentration in solution. Data are mean±SD of two independent wells for each experimental condition.

[0149] FIG. 36. Tile scan imaging of a single microwell (6.4 mm in diameter) coated with a PGA/PLL film made of 5 layer pairs. The film was visualized by using PLL-FITC.

[0150] FIG. 37. Number of C2C12 myoblast cells (per mm2) adhering on the on the PGA-peptide ending biomimetic films after 1 H of culture in a serum-free medium. 4 different conditions of film crosslinking were studied (CLO, CL5, CL10, CL30) and four different PGA/PGA-RGD peptide were studied (no peptide, ratio 2/1 ratio 1/2 and only PGA-peptide). Data are mean+SD of three independent well for each experimental conditions.

[0151] FIG. 38. Quantification of the myoblast cell spreading area (same experimental conditions as for figure XY. Cell spreading area was automatically quantified using a custom-made macro using Image J ton calculate the cell area covered by the cells and deduce the mean cell spreading area.

EXAMPLES

Polyelectrolytes

[0152] Different types of polyelectrolytes were used for the film buildup: Poly(L-lysine) hydrobromide (PLL, Sigma, Aldrich, St Quentin Fallavier, France), Poly(allylamine hydrochloride), chitosan (CHI, FMC Biopolymers) and poly(ethylene imine) (PEI, Sigma Aldrich, France) as polycations; Hyaluronic acid (HA, Lifecore Biomedical, USA), Polystyrene sulfonate (PSS) and poly(L-glutamic acid) (PGA, both from Sigma Aldrich, France) as polyanions. PGA was grafted to a RGD containing peptide as described in Picart et al, Adv. Funct. Mat 2005:15, 83-94)

[0153] Four different polycation/polyanion couples were selected: [0154] the (PLL/HA) films is a model system of exponentially growing films. For films made of 12 layer pairs, the thickness should be around 1.5 to 2 μm, i.e. close to the resolution limit of detection by confocal laser scanning microscopy (CLSM). [0155] (PSS/PAH) films are another model system known to growth linearly with the number of deposited layers. [0156] (CHI/PGA) films were chosen as third polyelectrolyte films to show the potentiality of the robot with other polyelectrolytes. [0157] (PLL/PGA) films were chosen as fourth polyelectrolyte films to show the potentiality of the robot to deposit other polyanions and to do high throughput screening of cell adhesion and spreading on films that were prepared using the robot and a film having as final layer a mixture of PGA and PGA-RGD.

Buffers for Film Buildup

[0158] For (PLL/HA) film buildup, PLL (0.5 mg/mL), HA (1 mg/mL) and PEI (2 mg/mL) were dissolved in a HEPES-NaCl buffer (20 mM Hepes at pH 7.4, 0.15 M NaCl). In order to improve film adsorption to the substrates, a first layer of PEI was deposited, followed by an HA layer. Afterwards, the cyclic deposit method of polycation (PLL) and polyanion (HA) intercalated with rinsing steps started until the desired number of layers was reached. All rinsing steps during film buildup were performed with 0.15 M NaCl at pH 6.5.

[0159] For (PLL/PGA) film buildup, PLL and PGA were dissolved at 1 mg/mL in the Hepes-NaCl buffer.

[0160] For (PSS/PAH) film buildup, PAH and PSS were dissolved at 5 mg/mL in a Tris-NaCl buffer (pH 7.4 containing X 0.15 M of NaCl.sup.−For CHI/PGA film buildup, CHI and PGA were dissolved in a 0.1 M sodium acetate buffer at pH 5 containing 0.15 M NaCl. For imaging, CHI was fluorescently labelled with Alexa Fluor 568 (Invitrogen, Amine Reactive Probe) in accordance with manufacturer's protocol excepting a 2 h reaction at pH 6.0. Product purification and removal of unbound dyes was carried out using a Sephadex G-25 size exclusion column (PD-10, Amersham Bioscience, Sweden). Films made of 12 layer pairs were imaged in air using the Zeiss LSM 700 confocal microscope with a 10× objective.

Comparative Example: Film Deposit by Hand Using a Multiple-Channel Pipette

[0161] LbL films were built by hand in 96-well cell culture plates using a multichannel micropipette (Eppendorf Research® pro 300, Germany), typically a channel with 8 tips. The polyelectrolytes were dispensed in each well and incubated for 8 min. Polycation and polyanion dispense was intercalated with 2 rinsing steps of 2 min. The liquid was dispensed carefully by tiling of the plate. It was thrown away by reversing the plate upside down. (PLL/HA) films made of 12 pairs of layers were manually deposited in 24 wells using a 8-arm multichannel pipette. PLL-FITC was used to stain the film.

[0162] First, this procedure is tedious and requires the experimentalist to be highly focused for several hours. Besides, it is time consuming since the total time needed for film deposit may be very long: it is proportional to the number of deposited layer pairs n. For instance, it takes up to two full days of work to manually prepare a film made of 24 pairs of layers.

[0163] In addition, polyelectrolyte film deposition by hand reveals to be highly heterogeneous inside a single well, as can be observed on the film thickness analysis at the different pole positions (FIGS. 5 to 8). The coefficient of variation for the different pole positions varied between 10 to 23% in each specific location, and the mean thickness per well varied of the order of 20% between independent wells (for two independent experiments pooled together). Without wishing to be bound by a theory, the inventors assumed that these spatial heterogeneities may originate from capillary effects, which are known to be important at such length scale.

Example 1: Automated (PLL/HA) Film Buildup Using a Liquid Handling Machine

[0164] A large set of in situ physico-chemical characterization and biological studies was performed in the same plate: i) LbL deposit (example 1), ii) characterization of the LbL film homogeneity in situ (example 1), iii) loading of bioactive proteins and its characterization (example 3), iv) assessment of the bioactivity of the protein-loaded LbL films on cell cultures in situ in microplates: short term adhesion and ALP activity were quantified at high throughput using optical microscopy and spectroscopy (example 3).

Automated Film Buildup Using a Liquid Handling Machine

[0165] LbL films were directly deposited in 96-wells cell culture plates (Reference 655986, Greiner bio-one, Germany) for subsequent characterization in situ (in liquid or air) by confocal microscopy and using a fluorescence/absorbance microplate reader. A protocol was developed to deposit layer-by-layer films at high-throughput in multiple-well plates using an automated liquid handling machine (TECAN Freedom EVO® 100) (FIG. 1).

[0166] The film buildup with this equipment consisted in sequences of polycation and polyanion dispense intercalated with rinsing steps. The principle consisted in using a liquid handling pipetting arm. This liquid handling arm pipetted the liquids in their respective reservoirs and dispensed them in selected wells of the multiple-well plate (FIG. 2). The trough containing the polyelectrolytes and the rinsing solution were deposited on the worktable. Three through were used: one for the polycation, one for the polyanion and one for the rinsing solutions (two in case the rinsing solutions would be different) and a trash. The multiple-well culture plates was/were deposited on the worktable.

[0167] We created an option for tilting the plate during dispense and aspiration steps (FIG. 1), using a commercially available tilting plate carrier: the multiple-well plate can be tilted at an angle α that is defined by the user and can typically vary between 5 and 20 degrees, and was 20 degrees in the examples hereafter. This condition will be named hereafter “Tilting” (T) versus “Non Tilting” (NT) for the standard position of the plate on the worktable (α=0 degree).

[0168] First, using a custom-made macro with the robot software, we defined the number of wells and specific positions where we wanted the layer-by-layer film to be deposited.

[0169] A sequence, i.e. a pair of layers, was made of the following steps: [0170] the liquid handling arm aspirated the polyelectrolyte from the trough and dispense it in the selected wells (typically 50 μL) using a tip (FIG. 2), this step being called “the dispense”, [0171] incubation time in the polyelectrolyte solution of 6 min, [0172] the liquid in each well was aspirated back and dispensed in the trash. During this step, it is possible to add an additional aspiration volume. [0173] In the examples below, the defined volume is the additional aspiration volume, defined as a % of excess volume with respect to the volume initially deposited in that specific well (0, 5, 10, 15 or 20%, respectively corresponding to 1.00×V.sub.aspPE, 1.05×V.sub.aspPE, 1 0.15×V.sub.aspPE and 1.20×V.sub.aspPE, as defined above (PE being either PE.sup.1 or PE.sup.2)); for example: if the additional aspiration is fixed to 10%, the robot will aspirate back 55 μL for an initial dispensed volume of 50 μL. [0174] 2 rinsing steps were done following the same procedure, except that the liquid was then aspirated from the rinsing trough (rinsing volume of 80 μL). [0175] the liquid arm aspirated the oppositely-charged polyelectrolyte (typically 50 μL) from the trough and dispensed it in the selected wells, [0176] incubation time of the oppositely-charged polyelectrolyte of 6 min. [0177] the polyelectrolyte in each well was aspirated back and dispensed in the trash. [0178] x rinsing steps (typically 2) of the oppositely-charged polyelectrolytes (typically 80 μL) were done following the same procedure, except that the liquid was then aspirated from the rinsing trough (rinsing volume of 80 μL).

[0179] For more precision in the pipetting, the dispenses may be achieved with the most appropriate pipetting tip, such as 200 μL. The aspiration may be done with a 1 mL tip.

[0180] This sequence was repeated n times to build a layer-by-layer film made of n layer pairs.

Experimental Conditions

[0181] We compared five experimental conditions:

[0182] By controlling the tilting of the microplate (Non Tilting or Tilting condition with an inclination angle α of 20° relative to the horizontal plane, respectively “NT” or “T”) and the additional aspiration volume (fixed to 0% or 10%): There are four conditions in total named hereafter:

NT_0%; NT_10%; T_0%; T_10%.

[0183] The 5th and last condition is a Non tilting/0% additional aspiration (i.e. (V.sub.aspPE.sup.1=V.sub.PE.sup.1 and V.sub.aspPE.sup.2=V.sub.PE.sup.2) but with a permanent excess volume during the buildup method (i.e. V.sup.wet). We name it hereafter “Surface Wet” condition.

[0184] For this condition, a volume of PE.sup.1 polyelectrolyte solution was dispensed inside each well at the very beginning of the experiment (for a well of a 96-well plate, this volume was set to 30 μL). This allows a constant volume of solution to be left inside each well, in order to ensure that the surface always remains covered by the liquid. By doing so, we aim to avoid local differences in the height of the liquid film above the polyelectrolyte film.

Pipetting Speed

[0185] The pipetting speed can be controlled by the user in the working range of the robotic arm. Typical pipetting speed were set to 400-800 μL/s for the dispense step and 30-150 μL/s for the aspiration steps.

Characterization of Film Homogeneity Inside a Well (Tile Scans and Transverse Sections)

[0186] Provided that one of the film components is labelled with a fluorescent dye, it is possible to image the global film homogeneity inside a given well using a Tile scan option of a confocal microscope (Zeiss LSM 700, Le Peck, France) and a 10× objective. This option enables to automatically scan the well by acquiring subsets of images.

Film Thickness “h”

[0187] We used PLL-FITC for (PLL/HA) films and (CHI-FITC) for (CHI/PGA) films to visualize the films. In fact, for exponentially growing films, it is known that the last layer is able to diffuse within the whole film. Thus, the film thickness can be easily measured by measuring the thickness of the fluorescence band.

[0188] To assess the film homogeneity inside each well at high spatial resolution, we measured film thicknesses at five different positions inside each well (North, West, Center, East and South, respectively N, W, C, E, S, FIG. 3). The center was approximately the center of the well, as assessed by the user. The other poles were located at +2 mm from the center, respectively, in the X and Y directions. To measure film thickness at these positions (the total number of positions being equal to the total number of wells×5), we automated the acquisition of the transverse sections at 0.36 μm intervals using a 63× oil objective and a custom-made macro with Zen software (Zeiss). Then, each thickness was automatically deduced from the fluorescence intensity profile (FIG. 4), using a custom-made macro on Image J (NIH. Bethesda). A typical fluorescent intensity profile starts off at the noise level (close to 0), increases to a peak value as the focal plane goes deeper into the film, and then returns to the noise level. In brief, the maximum of intensity (F.sub.MAX) was first determined. We applied a threshold coefficient C so that I=C×F.sub.MAX. We then deduced the Z position (Z1, Z2) at which this line intercepts the fluorescence intensity profile. The film thickness h can be easily deduced: h=Z2−Z1 (in μm). Of note, this macro was validated by comparing film thicknesses measured manually, by atomic force microscopy and by the macro on the same image.

[0189] The film thicknesses were determined at the different positions N, W, C, E, S inside each well and the corresponding thicknesses hN, hW, hC, hE and hS were determined.

Mean Thickness Per Well “hWELL”

[0190] The mean thickness per well hWELL was calculated as the sum of all 5 thickness measurements at the pole positions (N, W, E, S) and at the center (C), divided by 5. Thus, it was calculated using the formula:

[00001]$h_{\mathrm{WELL}}=\frac{\left(\mathrm{hN}+\mathrm{hW}+\mathrm{hC}+\mathrm{hE}+\mathrm{hS}\right)}{S}$

Mean Thickness of m Independent Wells “hMEAN”
hMEAN was calculated as:

[00002]$\mathrm{hMEAN}=\frac{{.Math.}_1^m\mathrm{hWELL}}{m}$

Standard Deviation “SD” of the Mean Thickness Per Well (hWELL), for m Independent Wells
SD was calculated as:

[00003]$\mathrm{SD}=\sqrt{\frac{{.Math.}_1^m{\left(\mathrm{hWELLi}-\mathrm{hMEAN}\right)}^2}{\left(m-1\right)}}$

Coefficient of Variation “CV” of the Mean Thickness

[0191] The Coefficient of Variation (CV) of the mean thickness for each of the 5 positions or for each well was calculated by:

[00004]$\mathrm{CV}=\frac{\mathrm{SD}}{\mathrm{hMEAN}}\times 100$

CV enables to compare samples independently of their absolute thickness values.

Film Deposit in PDMS Microwells on Silicon Wafers for Ex Situ Characterization

[0192] Film built on silicon wafers (2″ diameter, Dow Corning, USA) were needed for ex-situ characterization using infrared spectroscopy, profilometry and AFM microscopy.

[0193] We designed a custom-made silicon substrate with polydimethylsiloxane (PDMS, Sylgard 184 kit, Dow Corning) wells of similar size than those of the 96-well plates. PDMS was mixed with curing agent (10:1) during 10 min and placed in a desiccator for 20 min to remove bubbles. Then, it was introduced in a mold, degassed again during 20 min and placed in the oven at 65° C. for at least 4 h, before being carefully removed from the mold and cut in rectangular pieces. Circular holes of the same diameter and position as in 96 well plates were made. Both silicon and PDMS substrates were UV treated (PSD-UV ozone cleaning system, Novascan Technologies) for 10 min to increase the bonding strength between them. Thereafter, the treated faces were set in contact, pressed to adhere and introduced in an oven at 100° C. for 1 h30 to be glued together. The (PSS/PAH) and (PLL/HA) polyelectrolyte films were then deposited using the robotic arm as described above.

[0194] For ex-situ film characterization an additional drying step was mandatory.

[0195] For (PLL/HA) films, the crosslinked films stored in Hepes-Nacl buffer were rinsed with MilliQ water and then dried.

[0196] For (PSS/PAH) films, the films were simply rinsed with MilliQ water and then dried. Film drying was done in an incubator for 2 h at 37° C. At the end of the procedure, the PDMS mold was removed and the films were kept at 4° C. Before each FTIR and profilometric analysis, the films were placed in an incubator at 37° C. for 1 h in order to eliminate any possible effect of humidity variation. Dry films were also characterized by AFM.

Ex Situ Analysis Using Infrared Spectroscopy, Profilometry and Atomic Force Microscopy

FTIR Analysis

[0197] Experiments were made using a Vertex 70 spectrophotometer (Bruker Optics Gmbh, Ettlingen, Germany) in the transmission mode using a sensitive MCT (Mercury-Cadmium-Telluride) detector. Prior to film analysis, a background signal was acquired after introducing a bare silicon substrate in the sample compartment using the transmission accessory. Dried films built on silicon substrate were placed in a sample holder and their spectra was acquired by summing 256 interferograms. Spectra analysis was made using OPUS Software v6.5 (Bruker, Germany), removing H.sub.2O and CO2 contributions and correcting the baseline manually, always choosing the same reference points in each spectrum. For each condition, a final spectrum is an average of 3 different spectra of the same sample (but from different wells).

Profilometry

[0198] Thickness measurements of films built on a silicon substrate were performed using a profilometer (Dektak XT, Bruker Corporation, USA). Five samples of each condition, corresponding to 5 different wells built at the same time, were scratched to create a physical step and three measurements per sample were acquired with the software Vision 64® (v 5.4, Bruker Corporation, USA). Scans of 30 s with a length of 1000 μm were performed with a stylus of 12.5 μm in radius and with a force set to 1 mg. Thus, film thickness for each condition was an average value of 15 measurements.

AFM

[0199] AFM images of the polyelectrolyte films deposited on silicon were obtained in tapping mode by means of a DI 3100 AFM (Veeco) with NanoScope IIIa controller using silicon cantilever (OMCL-AC240TS, Olympus). The film-coated substrates were washed in water and air-dried before observation. Substrate topographies were imaged with 512×512 pixels at a frequency of 1 Hz.

Influence of the Tilting (“T”)/Non Tilting (“NT”), of the Additional Aspiration and of the “Surface Wet” Condition on Film Homogeneity in a Single Well and Between Wells

[0200] Film homogeneity inside each well was assessed at the 5 pole positions (FIGS. 9 to 18). The results are provided at tables 2 and 3 below.

TABLE-US-00002 TABLE 2 Experimental values measured for all the parameter studied for the 5 different experimental conditions. PARAMETER NT_0% NT_10% T_0% T_10% SW Mean ± SD of CVs of the 5 19.3 ± 9.8  20.6 ± 5.4  7.1 ± 39   5.1 ± 0.5   6.6 ± 1.6 positions CV of (hWELL) (%) 18.3  20.3 14   6.8   5.3 Global Film Homogeneity  0.742   1.004  0.172   0.177   0.494 (Tile scans) Cell spreading (mm.sup.2) for 1200 ± 486 1828 ± 697 1620 ± 512 BMP50 Total surface covered (%) for   7.6 ± 0.3  25.6 ± 3.9  13.0 ± 1.6 BMP50 ALP bioactivity 85  83 84  83  84

[0201] In order to facilitate comparison of the five conditions, based on the experimental values, a score was attributed for each criteria, the higher the score, the better the parameter. A total mean score was then calculated.

TABLE-US-00003 TABLE 3 Score for all the parameters studied for the 5 different experimental conditions. PARAMETER NT_0% NT_10% T_0% T_10% SW Mean ± SD of CVs 2 2 4 5 5 of the 5 positions CV of (hWELL) (%) 2 2 3 5 5 Global Film 2 1 5 5 3.5 Homogeneity (Tile scans) Cell spreading (mm.sup.2) 1.5 5 4 for BMP50 Total surface 1.5 5 3.5 covered (%) for BMP50 ALP bioactivity 5 5 5 5 5 TOTAL SCORE 2.8 2.2 4.25 5 4.3

[0202] The ranking of table 3 gives the three first best conditions: T_10%>SW>T_0%. The NT_0% and NT_10% are very close and well below the others.

[0203] Accordingly, it appears both from the absolute thickness measurements (FIGS. 9, 11, 13, 15 and 17) as well as from the CV for each pole (FIGS. 10, 12, 14, 16 and 18) that: [0204] the “surface wet” condition (first embodiment describes above), and [0205] the conditions with tilting (second embodiment describe above), [0206] either with no additional aspiration (V.sub.aspPE.sup.1=V.sub.PE.sup.1 and V.sub.aspPE.sup.2=V.sub.PE.sup.2), [0207] or with 10% additional aspiration (V.sub.aspPE.sup.1=1.10V.sub.PE.sup.1 and V.sub.aspPE.sup.2=1.10V.sub.PE.sup.2), lead to more homogeneous films.

[0208] In addition, the mean thickness per well (FIG. 19) is also much less variable for the experiments with tilting, especially the condition with tilting and 10% addition aspiration, and for the surface wet condition.

[0209] CV values (FIG. 20) are systematically above 15% for the “non tilting” (NT) conditions, between 10 and 15% for the T_0% condition and less than 8% for both T_10% and for surface Wet.

[0210] A global view of the wells using PLL-FITC to visualize the films and the tile scan option of the confocal microscope software provided complimentary information on the global film homogeneity in each well. Three representative histogram of fluorescence intensities are plotted in FIGS. 21 to 25. The obtained images, as well as the histograms, clearly showed that the fluorescence distribution is more homogeneous in the T_0% (FIG. 23) and T_10% (FIG. 24) conditions and in the Surface Wet condition (FIG. 25) compared to the “non tilting” (NT) conditions.

Influence of the Positioning of the Pipetting Tips on the Film Thickness

[0211] Before the beginning of the experiment, the user needs to define a reference position in (X,Y,Z) in order to define the initial coordinates of the dispense and aspiration steps. The definition of (X,Y) coordinates is straightforward, knowing the coordinates of the centers of wells of a 96-well plate.

[0212] The following procedure was followed to control the Z-position of the tip at the vicinity of the plate bottom during solution dispense in the well and aspiration from the well. Prior to the beginning of the experiment, the tip was positioned in close vicinity to the bottom of the plate. The tip was first set in contact with the microplate until there was absolutely no movement possible for the microplate. Then, the tip was elevated in Z by one step (100 μm with the used robot). This position was set as reference Z position, Z0.

[0213] We thus investigated whether and how the Z-positioning of the tip influences the film thickness measurement. To this end, films were built at four different Z positions with stepwise increase of 0.3 mm from the reference position Z0 (ie 0.1 mm above the bottom of the plate) (FIGS. 26 to 29).

[0214] The dispersion in film thickness increased with Z for the NT conditions and the T_0% but the values remained almost independent of Z for the T_10% condition. Therefore, this latter condition appears to be more flexible for the user, who does not need to be highly precise in the optimization of Z0.

[0215] As regards the “Surface Wet” condition, since there is a permanent liquid film inside each well, there is no need to precisely adjust the initial Z-position Z0 of the pipette tip prior to the experiments.

Example 2: Automated (PSS/PAH) or (CHI/PGA) Film Buildup Using a Liquid Handling Machine

[0216] The automated deposit method was also applied to other types of polyelectrolyte films. We selected a polyelectrolyte system, namely (PSS/PAH) films, that is known to grow linearly and is considered as a “model system”. As anticipated, their dry thickness was found to growth linearly with the number of deposited layer pairs (FIG. 33) up to around 250 nm for a film made of 40 pairs of layers.

[0217] We also checked for another polyelectrolyte system made of (CHI/PGA) that the polyelectrolyte film can be efficiently deposited at the bottom of the wells. The thickness of a (CHI/PGA) 12 film was 2.26±0.14 μm as measured from confocal microscopy imaging.

Example 3: Bioactive Proteins Loading in (PLL/HA) Films

[0218] The homogeneity of proteins that were post-loaded in the polyelectrolyte films prepared in examples 1 and 2 was assessed.

Film Crosslinking and Loading of Bioactive Proteins in (PLL/HA) Films

[0219] Bioactive proteins were loaded in (PLL/HA) films as previously described in Crouzier T at al., Small 2009, 5:598-608.

[0220] The films were first chemically crosslinked in a 0.15 M NaCl solution at pH 5.5 using 1-Ethyl-3-(3-Dimethylamino-propyl)Carbodiimide (EDC, final concentration of 10, 30 or 70 mg/mL) and N-Hydrosulfosuccinimide sodium salt (Sulfo-NHS, final concentration of 11 mg/mL) as catalyzer. The films were incubated at 4° C. overnight, then thoroughly washed the HEPES-NaCl buffer.

[0221] The (PLL/HA) polyelectrolyte films were manually loaded with bone morphogenetic proteins (BMP-2, BMP-7, BMP-4, BMP-9 or two BMP chimeras, namely chimera 1 and chimera 2) as bioactive proteins at acidic pH using a multi-channel pipette, following as previously described in Crouzier T at al., Small 2009, 5:598-608.

Cell Culture and Cell Response to the Bioactive Polyelectrolyte Films

[0222] We used BMP-2 responsive cells, C2C12 skeletal myoblasts (<25 passages, obtained from the American Type Culture Collection, ATCC), to assess the bioactivity of the polyelectrolyte films. Cells were cultured as previously described (Crouzier T at al., Small 2009, 5:598-608) in tissue culture Petri dishes, in a 1:1 Dulbecco's Modified Eagle Medium (DMEM):Ham's F12 medium (Gibco, Invitrogen, France) supplemented with 10% fetal bovine serum (FBS, PAA Laboratories, France) and 100 U/mL penicillin G and 100 μg/mL streptomycin (Gibco, Invitrogen, France) in a 37° C., 5% CO.sub.2 incubator. Then, 15 000 cells/cm.sup.2 in their medium were seeded in each well. After 4 h and 24 h of adhesion, phase contrast images were acquired and the samples were also fixed in 4% paraformaldehyde (Sigma Aldrich, St Quentin Fallavier, France). The nuclei were stained using DAPI (Life technologies and the actin cytoskeleton using Rhodamine-phalloidin (Sigma Aldrich).

D1 Murine Mesenchymal Stem Cell LD1) Culture

[0223] D1 cell culture was first done for 2 days in growth medium (89% aMEM (sigma M4526), 10% FBS with 1% antibiotics (penicillin streptomycin mix, 15140122 Invitrogen)) followed by 7 days in differentiation medium (growth medium supplemented with 50 μg/ml L-Ascorbic acid 2-phosphate and sesquimagnesium salt hydrate (Sigma A8960) and 10 mM β-Glycerol phosphate disodium salt pentahydrate (Sigma 50020). 9375 cells were seeded in each well. After the cell culture was stopped, ALP activity was assessed via enzymatic assay.
Human Periosteum Derived Stem Cells (hPDSC) Culture
Human periosteum derived stem cells (passage between 10 and 14) were cultured in DMEM/high glucose in the presence of 10% FBS in the presence of 250 μM ascorbic acid 2 phosphate. They were seeded at a density a 5000 cells/cm2 (˜1700 cells per well) in 200˜μL of medium. The medium was changed every 2-3 days and the cell culture was done for 2 weeks.

Alkaline Phosphatase (ALP) Bioactivity

[0224] After a given number of days of culture, (3 days for C2C12, 3 for D1 cells and 14 for hPDSC) the growth medium was removed and the cells were fixed with 4% paraformaldehyde. They were stained for ALP activity fast blue RR salt in a 0.01% (w/v) naphthol AS-MX solution (Sigma Aldrich) according to the manufacturer's instructions. ALP enzymatic activity.

The culture medium was removed and the cells were washed with PBS and lysed by sonication over 5 s in 500 mL of 0.1% Triton-X100 in PBS. The ALP activity of these lysates was then quantified using standard protocol and normalized to the corresponding total protein content, which was determined using a bicinchoninic acid protein assay kit (Interchim, France).

Analysis of the Homogeneity of Bioactive Proteins Loaded in the Polyelectrolyte Films

[0225] In order to assess the homogeneity of BMP-2 loading in the (PLL/HA) films, BMP-2 labelled with carboxy fluorescein (BMP-2CF) was used (5% of the total BMP-2 concentration) and tile scans of the wells were performed in the Hepes-NaCl buffer after thorough rinsing of the films in order to get only matrix-bound BMP-2.

[0226] The bioactivity of the BMP proteins was assessed using BMP-responsive cells. To begin, we chose to work with films crosslinked with an EDC final concentration of 30 mg/mL (i.e. noted EDC30 since these films are known to be poorly adhesive for cells, unless they are presenting BMP-2 in a matrix-bound manner. The more heterogeneous the film is, the more differences in the cell response to matrix-bound BMPs is expected.

[0227] Since matrix-bond BMP-2 on EDC30 films drastically increases cell adhesion and spreading, we first assessed cell adhesion at 24 h. (FIG. 30).

[0228] For these experiments, the two “extreme conditions” of NT_10% and T_10% were selected and the Surface Wet condition.

[0229] Cells appeared to be round and poorly adherent on the NT_10% conditions while they were more numerous and also more spread in the T_10% condition.

[0230] BMP-2 bioactivity can be quickly assessed by staining for the expression of an early bone marker, the alkaline phosphatase (FIG. 31). All conditions lead to a BMP-2 dose-dependent and significant ALP expression, as can been seen after cell staining and corresponding quantifications.

High Throughput Screening of Stem Cell Adhesion and Fate

[0231] We selected the T_10% condition to further prove the versatility of the matrix-bound proteins to screen for cellular processes on stem cells at high throughput.

[0232] We first tested whether matrix-bound BMP-7 was bioactive toward murine D1 stem cells (FIG. 32). To this end, the cells were cultured for up to 9 days on the bioactive polyelectrolyte films. We found that cells selectively adhere on the matrix-bound BMP-7 and could growth for up to at least 9 days. Of note, cells detached in the absence of matrix-bound BMP-7 and formed nodules in its presence. Their ALP expression directly depended on the amount of matrix-bound BMP-7 and grow exponentially to a plateau value (fit in the graph of FIG. 32).

[0233] We further tested whether matrix-bound BMPs are bioactive toward murine C2C12 skeletal myoblasts (FIGS. 34 and 35).

[0234] To this end, we first verified that these BMPs could be effectively loaded in the biomimetic films (Table 4).

TABLE-US-00004 TABLE 4 Proportion and quantity of BMP loaded in the (PLL/HA) films. % incorporated Quantity (ng/cm.sup.2) SD BMP-9 86% 2325  65 BV-265 61% 1850  56 BMP-2 60% 1650 115 BMP-4 65% 1810  15 BMP-7 38% 1100  84

[0235] The cells were cultured for 3 days on the bioactive polyelectrolyte films (5 different BMP proteins and 4 different BMP loading concentrations). We found that cells selectively adhere on the matrix-bound BMPs and grow on this time period. Their ALP expression was assessed at high throughput using two different methods: first, ALP staining was visualized using a scanner and images of the whole microplate were taken, showing the ALP expression in each individual well (FIG. 34). Second, ALP staining was quantified at high throughput using a Tecan Infinite 1000 microplate reader, by quantifying the absorbance at 570 nm using multiple-read per well mode (76 different positions were measured in each individual microwell and the mean value of these 76 positions was taken). This quantification enables to plot the ALP as a function of the initial concentration of the BMPs in solution (FIG. 35), which clearly shows a dose-dependent ALP response: the ALP intensity depends on the type of BMPs (in the order BMP-9>BV265>BMP-2>BMP-4>BMP-7) and on the dose of BMPs (increased ALP expression with the increased BMP concentration). The corresponding exponential fits toward a plateau value (continuous and dashes lines) are also given for BMP-2, BMP-9, BV265, BMP-4 while the fit was linear for BMP-7 (continuous line).

Example 4: Automated (PLL/PGA) Film Buildup Using a Liquid Handling Machine and High Throughput Screening of Cell Adhesion and Spreading

[0236] The automated deposit method was also applied to another type of polyelectrolyte films, namely (PGA/PLL) films that we previously studied for cell adhesion (Picart et al, Adv. Funct Mat 2005).

[0237] For this study, we used films made only using the T_10% condition.

[0238] The (PGA/PLL) films were made of 5 layer pairs (eg (PGA/PLL) 5 films) and were either native (eg not crosslinked, CL 0) or crosslinked to different extents (EDC 5, EDC10, EDC30 named hereafter as CL5, CL10, CL30). So, in total, there were 4 different films conditions. They were finally rinsed with the Hepes-NaCl buffer using the liquid handling machine. On top of these films, a final layer was deposited. It is constituted of a mixture of PGA and PGA-RGD peptide (a RGD containing peptide grafted to the PGA) at fixed proportions. The PGA/PGA-RGD ratio used for the deposit of the final layer was varied in order to study 4 different conditions for the final layer: P0(3/0); P1 (2/1); P2(1/2); P3: 0/3). C2C12 myoblast C2C12 were seeded in the 96-well microplates at a density of 3500 cells/well (around 10 500 cells/cm2).

[0239] We first verified that the biomimetic films were homogeneous inside each well as observed using the tile scan option of the microscope (FIG. 36, showing the whole well of about 6 mm in diameter).

[0240] Cell adhesion and spreading was next quantified after 1H of cell culture on top of the different biomimetic films in the serum-free medium. To do so, their nucleus (stained with Hoechst) and cytoskeleton (stained with rhodamine phalloidin) were stained. Images were automatically acquired at high throughput at 20× objective in the two channels using an automated Zeiss fluorescence microscope. The number of adherent cells increases with the concentration of the RGD-peptide for the uncrosslinked films (CL 0) and films crosslinked at low extent (CL 5) but was peptide-independent for the more CL films (CL 10 and CL30) (FIG. 37). Regarding the cell spreading area (FIG. 38), a clear peptide-dependent cell spreading area was visible, with an enhanced myoblast spreading when the quantity of peptide increased. We can thus conclude that the (PGA/PLL) biomimetic films containing a peptide-grated layer can be used to do cell adhesion and spreading at high throughput.