FR3071359A1 - TUBULAR ORGANIC ELECTROCHEMICAL TRANSISTOR INTEGRATING BIOCOMPATIBLE MATRIX IN 3 DIMENSIONS - Google Patents

Abstract

The present invention relates to the field of microfluidic systems incorporating an organic electrochemical transistor. More particularly, the present invention relates to an organic electrochemical transistor configured in a tube and wherein the channel consists of a 3-dimensional biocompatible polymer matrix. This device makes it possible to cultivate cells within the matrix so as to reproduce a structure close to that of a tissue. This device can be used to test the physiological effect of substances, particularly in toxicity tests and evaluation of the therapeutic effect of drug substances in preclinical studies.

Description

TUBULAR ORGANIC ELECTROCHEMICAL TRANSISTOR INTEGRATING A BIOCOMPATIBLE MATRIX IN 3 DIMENSIONS

The present invention relates to the field of microfluidic systems incorporating an organic electrochemical transistor. More particularly, the present invention relates to an organic electrochemical transistor (TEO) configured in a tube and in which the channel consists of a matrix of biocompatible polymer in 3 dimensions. This device makes it possible to cultivate cells within the matrix so as to reproduce a structure close to that of a tissue. This device can be used to test the physiological effect of substances, in particular in toxicity tests and evaluation of the therapeutic effect of medicinal substances during preclinical studies.

State of the art

There has been a great deal of interest in electronic devices for monitoring cell functions and tissue formation for a long time. Indeed, electronic devices are very sensitive systems for the study of cell adhesion and the differentiation of many types of cells and tissues. Despite significant efforts, only two-dimensional in vitro systems have so far been proposed. These systems are not satisfactory when it is desired to know the cellular state and in particular the capacity to proliferate.

Different models of polymeric matrices are described in the literature (US 2011/0189764, US 9457128), some of which are conductive (US 2009/0171406 and W02017 / 011452) and act as bioreactors allowing the connection of cells with an electronic circuit. Among the bioreactors described, some have the shape of a tube US 2014/0273222 and W02010 / 115185.

The in situ electrical characterization of a cell culture can be carried out in a simple and inexpensive manner by measurement of transconductance using a TEO whose active channel consists of a biocompatible matrix. Such a system has been described by Wan etal (3D conducting polymer platforms for electrical control of protein conformation and cellular functions 2015, J. Mater. Chem. B).

A recent study describes a 3D matrix consisting of PEDOT: PSS whose properties are compatible with cell culture in 3 dimensions while being a final conductor et al, 2017, Advanced biosystems, 1700052). This article describes a tube feeding system allowing homogeneous growth of cells in the volume of the material. The development of cells in the material can be effectively characterized by electrical measurements using electrochemical impedance spectrometry.

Advantages of the invention

The inventors offer an innovative system to meet existing needs. It is a device comprising an organic electrochemical transistor configured in a tube and in which the channel consists of a matrix of 3-dimensional biocompatible polymer. The particular configuration of the elements constituting this device allows the growth of cells in a 3-dimensional matrix reproducing the tissue structure. In addition, different cell types can be co-cultivated within the matrix so as to reproduce the specific interactions existing within each tissue type. The sensitivity of the system based on an organic electrochemical transistor makes it possible to analyze precise cellular parameters such as cell adhesion, cell proliferation and cell state (good condition, integrity or damaged cells). Finally, this system can be integrated into a microfluidic system for supplying the electrolyte. Several systems can be switched on so that several cell or tissue types can be integrated within the same bioreactor.

DETAILED DESCRIPTION OF THE INVENTION

A first object of the invention relates to an organic electrochemical transistor (TEO) comprising:

(i) a channel consisting of a matrix of porous conductive polymer and biocompatible in three dimensions, (ii) a source electrode and a drain electrode each establishing electrical contact with said channel, and (iii) a gate electrode establishing a electrical contact with an electrolyte being in direct contact with said channel, characterized in that said channel is located inside a tube comprising at least 2 openings.

The TEO according to the invention can be used as a bioreactor. Indeed, the inventors have shown that cells can be cultivated within the matrix. The tubular shape creates a microfluidic system to supply the cells with nutrients and gases, in addition to allowing the supply of electrolyte. The configuration of the TEO according to the invention therefore makes it possible to measure in real time the evolution of a cell culture in 3D, the cell state being reflected by the measurement of the voltage, current or transconductance of the matrix hosting the cells.

By "conductive polymer" is meant in the sense of the invention an intrinsic conductive polymer (PCI) which has delocalized links allowing the circulation of an electric current carried by electrons or holes.

A transistor consists of a semiconductor film (channel) placed between two electrodes (source and drain) and a gate electrode separated from the channel by a dielectric layer (or an electrolyte). A voltage (drain voltage, Vd) applied between the source electrode and the drain electrode causes the current to flow through the channel (drain current, Id), which in turn is modulated by a voltage applied to the gate Vg. The current flow in the channel reflects the conductivity of the material of the channel. In the case of electrochemical transistors, the ions injected into the channel modify the doping state of the semiconductor polymer (that is to say of PEDOT: PSS), resulting in a significant variation of the drain current.

A suitable polymer to form a porous and biocompatible conductive matrix must be a water-based polymer, to allow the transition from the liquid state, state of the polymer when it is injected into the tube, to the solid state after freezing and lyophilization and therefore elimination of water which leaves voids (pores).

By “porous matrix” is meant within the meaning of the invention a permeable matrix, the diameter of the pores of which is greater than that of the cells so as to allow the passage of the cells through the matrix and the insertion of the cells as well as their growth inside the matrix. A porous matrix adapted to the TEO according to the invention may for example have pores whose average diameter is 100 μm. In a preferred embodiment, the pore diameter is between 50 and 100 µm.

The main characteristic of conductive polymers is the conjugate molecular structure of the main chain of the polymer. The conjugated polymers become conductive polymers after doping, which can be carried out by oxidation or chemical or electrochemical reduction. Oxidation of the polymer backbone is called p-doping and occurs when the main chain of the polymer loses an electron (forming a hole). In this case, the conductive polymer is of p type. When the polymer backbone is reduced, electrons are inserted into the polymer backbone and form negative charge carriers; this is called ndopage and an n-type conductive polymer. The vast majority of organic electronic devices and applications incorporating doped conjugated polymers are p-type due to the high stability and excellent conductive properties.

As examples, such polymers can be PEDOT-PSS, polypyrrole, or polyaninline, but any polymer having such properties can be used.

In a preferred embodiment, the porous and biocompatible three-dimensional conductive polymer is a sublimated PEDOT-PSS.

The internal diameter of the tube of a TEO according to the invention is between 0.5 and 5 mm. In a preferred embodiment, the inner diameter of the tube is 3 mm.

The diameter of the matrix is greater than 500 µm. In addition, it must be suitable to allow the gas exchange between the culture medium and the cells. In fact, if the diameter of the matrix is too large, the cells located in the center of the matrix will die for lack of food.

In a preferred embodiment, the diameter of the matrix is between 0.4 and 5 millimeters, preferably between 1.0 and 2.0 millimeters. In another particular embodiment, the diameter of the matrix is less than or equal to 300 micrometers, to allow the cells located in the center of the matrix to be fed. However, this limit can be overcome by arranging a channel in the center of the matrix, in the direction of the flow of the cellular medium to supply gases and nutrients to the cells located in the center. Thus, it is possible to arrange several parallel channels if it is desired to further increase the diameter of the microfluidic system.

The length of the matrix is generally between 2 and 5 millimeters.

In a preferred embodiment, the diameter of the matrix is between 1.0 and 2.0 millimeters and the length of the matrix is between 2 and 5 millimeters.

In another preferred embodiment, the matrix is associated with components such as synthetic polymers and proteins such as collagen, DBSA or a mixture of such components. In particular, collagen makes it possible to increase the rigidity and DBSA makes it possible to increase the conductivity of the final polymer (2017, Advanced biosystems, 1700052).

The TEO can be presented in different configurations.

Regarding the source electrode and the drain electrode, they can either be integrated into the wall of the tube (as illustrated in Figures IA and IB), or be inserted into an opening of the tube located in the upper position (like this is illustrated in Figures IC and 1D).

Regarding the gate electrode, it can come into contact with the electrolyte either through an opening located in the body of the tube (as illustrated in Figures IA and IB), or be embedded in the tubular system by one end of the tube (as illustrated in Figures IC and 1D).

Other configurations are possible from the moment when the electrodes are in contact with the channel (here the matrix) and when the gate electrode is in contact with the electrolyte (here the culture medium); those skilled in the art will know how to adapt the system so that the transistor operates in a tubular system.

When the tube has only 2 openings, the gate electrode can be integrated into the wall of the tube to ensure contact with the electrolyte, or embedded in the tubular system.

According to a particular embodiment of the invention, the tube comprises 3 openings, the third opening allowing said channel to come into contact with the gate electrode by means of the electrolyte, or the insertion of the source and drain electrodes , depending on the configuration of the TEO.

It is possible to provide additional openings which can be used for the supply of particular molecules so as to control the moment of this addition in the channel and to measure the effects, or else which can be used to carry out particular measurements either by introduction of '' a probe, either by taking samples.

The nature of the electrodes may vary. However, it is essential that they do not cause toxicity, especially when cell culture is continued for several days or even weeks. Thus, the electrodes can for example be in silver, if the culture time does not exceed 12 hours, beyond it is preferable to choose electrodes in gold, platinum, PEDOT-PSS, nanostructured graphene ... choice of electrodes will be made according to the culture time envisaged. Indeed, the silver or silver chloride electrodes are to be avoided for culture times greater than a few hours because they can release toxic ions for the cultured cells. For a culture up to 6 weeks, PEDOT-PSS will be preferred because it has good stability in liquid medium.

In a particular embodiment, the source and drain electrodes are made of gold or covered with gold.

In another preferred embodiment, the gate electrode is made of PEDOT-PSS.

In a preferred embodiment, the source and drain electrodes are made of gold or covered with gold and the gate electrode is made of PEDOT: PSS.

The tube used to prepare a device according to the invention will be made of a material capable of withstanding freezing and lyophilization (when the matrix is prepared directly inside the tube), but also at an extended temperature of 37 °. C and high humidity levels as is the case in a bioreactor, without this altering its structure and without it decomposing or releasing / diffusing toxic molecules into the culture medium.

For example, microfluidic tubes made of polyvinylidene fluoride (PVDFj, polyetheretherketone (PEEK), polytetrafluoroethylene (PTFEj Teflon ^R , Tygon ^R , fluorinated ethylene-propylene (FEP), ethylene tetrafluoroethylene (ETFE) or polypropylene (PP) can be used to make the transistor according to the invention.

In a preferred embodiment, the tube is made of polyvinylidene fluoride or polypropylene.

In a particular embodiment, the invention relates to an integrated system comprising at least two TEOs according to the invention switched on, either in parallel or in series.

This configuration can be implemented to produce “bioreactor” type chips comprising different TEOs hosting different cell types so as to reproduce complex biological systems. Such integrated systems constitute more relevant predictive models than animal models because they allow evaluations of the effect of molecules of interest directly on human cells reconstructing a 3D structure close to the tissue structure. They would respond to a high demand for real-time evaluation systems, in particular for carrying out preclinical studies, both for the evaluation of the toxicity and of the therapeutic effect of drug-candidate. These systems are also suitable for screening molecules.

In another particular embodiment, the invention therefore relates to the use of an integrated system comprising at least two TEOs as a bioreactor.

The TEO according to the invention constitutes a cell culture system physiologically suitable for cell culture in 3D (biocompatible matrix and flow due to the microfluidic system) at the same time as it integrates different methods of evaluation of cell morphology, of differentiation cell and cell integrity.

The TEO according to the invention is intended to be combined with cells, animal or human, cultivated within the matrix. Thus, depending on the cell state, the voltage, current and / or transconductance of the matrix are modified; their electrochemical measurements carried out by means of the transistor surrounding the matrix, provide information on the cellular state.

Thus, the invention also relates to a TEO or a set of TEO combined with cells cultured within the matrix.

A second object of the invention relates to a process for the preparation of an organic electrochemical transistor according to the invention.

In a first embodiment, a method for preparing an organic electrochemical transistor according to the invention consists in:

at. preparing a tube comprising at least two openings;

b. integrate two diametrically opposite conductive points in the wall of the tube to produce the source electrode and the drain electrode;

vs. inject a solution of liquid biocompatible conductive polymer into the tube through one of the openings;

d. freeze and then lyophilize said polymer solution so as to obtain a porous and biocompatible conductive matrix, in which the gate electrode is brought into contact with the electrolyte via one of the ends of the tube or via an opening made in the body of the tube.

In a particular embodiment, the tube prepared in step a. has only the two openings located at the ends of the tube and in which the gate electrode is integrated in the wall of the tube; alternatively, the gate electrode can be contacted with the electrolyte via one end of the tube (see Figures IC and 1D).

In another particular embodiment, the tube prepared in step a. has 3 openings, the third opening in the body of the tube allowing contact of said channel with the gate electrode via the electrolyte (see Figures IA and IB). In an alternative embodiment, the third opening allows the insertion of the source and drain electrodes (see Figures IC and 1D).

Thus, an alternative embodiment, a process for preparing an organic electrochemical transistor according to the invention consists in:

at. preparing a tube comprising at least three openings;

b. inject a solution of liquid biocompatible conductive polymer into the tube through one of the openings;

vs. freezing and then lyophilizing said polymer solution so as to obtain a porous and biocompatible conductive matrix;

in which the source and drain electrodes are inserted via the opening made in the body of the tube and the gate electrode is brought into contact with the electrolyte via one of the ends of the tube.

In a second embodiment, a method for preparing an organic electrochemical transistor according to the invention consists in:

at. prepare a tube comprising at least two openings and two diametrically opposite conductive points in the wall of the tube to produce the source electrode and the drain electrode,

b. preparing a porous and biocompatible conductive matrix by freezing and then lyophilizing a solution of liquid biocompatible conductive polymer,

vs. insert said matrix prepared in step b. in the tube prepared in step a in which the gate electrode is brought into contact with the electrolyte via one of the ends of the tube.

The conductive points integrated in the wall allow the electrical connection between the source and drain electrodes and the channel. In an alternative embodiment, the source and drain electrodes are not integrated into the wall of the tube but inserted through an opening of the tube located in the upper position (as illustrated in Figures IC and 1D).

Thus in such an alternative embodiment, a process for preparing an organic electrochemical transistor according to the invention consists in:

at. preparing a tube comprising at least three openings;

b. preparing a porous and biocompatible conductive matrix by freezing and then lyophilizing a solution of liquid biocompatible conductive polymer,

vs. insert said matrix prepared in step b. in the tube prepared in step a.

in which the source and drain electrodes are inserted via the opening made in the body of the tube and the gate electrode is brought into contact with the electrolyte via one of the ends of the tube.

When the matrix is prepared before being placed in the tube, its dimensions will be slightly smaller than that of the tube so that the matrix is in contact with the walls of the tube once inserted, or smaller so that the matrix, once inserted, is not in contact with the walls of the tube.

Whatever the embodiment, the preparation process according to the invention can also comprise the addition of synthetic polymers or proteins to the matrix either before freezing (and lyophilization) or after lyophilization.

In a preferred embodiment, lyophilization is a sublimation.

A third object of the invention relates to the use of a TEO according to the invention for the detection of a cellular state applicable in particular to cells in the course of adhesion, proliferation, or differentiation, apoptosis or of cell death. This state is correlated to the voltage, the current and the transconductance of the matrix measured thanks to the transistor. The use of the transistor therefore makes it possible to measure these parameters at a time t and to compare them with the measurements obtained at a time t + 1, to evaluate a change of state.

This system has the advantage of taking into account all of the cell's signals: biochemical signals, mechanical characteristics and electrical signals, which are amplified by the use of a transistor.

The TEO of the invention also has the advantage of allowing the co-culture of cells, for example fibroblasts and epithelial cells, or a combination of different types of cells reproducing tissue heterogeneity and the specificity of particular tissue ecosystems.

TEO is therefore a device for diagnosing and monitoring the development of cells in vitro, in particular human cells.

The device according to the invention can advantageously replace tests on animals since it reproduces tissue of human origin in 3D, while the relevance of animal models is often relative and disputed. It often happens that the interesting results obtained on an animal model are not confirmed during clinical studies. Likewise, negative results obtained in animal models lead to the exclusion of certain therapeutic approaches when they are in reality valid in humans. A device according to the invention is intended to respond to this type of problem.

Thus, the invention also relates to a method for evaluating the effect of a molecule or a combination of molecules on cells cultivated in a 3-dimensional matrix integrated in a device of the tubular transistor type according to the invention.

This method can be applied to many approaches such as:

- the evaluation of the functionality of differentiated cells from stem cells such as iPS, cells which can be used in screening and diagnostic tests and whose biological and physiological characteristics are important to verify;

- the measurement of a cellular state in real time and continuously in a culture in 3 dimensions;

- as an integrated microfluidic system on a chip, for example in order to reproduce a bioreactor involving tissues of different natures. To do this, the chip can include a set of TEOs put in circuit so as to reproduce a “multi-organ system”. Such an in vitro system can be used in numerous applications such as the evaluation of the metastatic potential of cancer cells, the evaluation of the toxicity of circulating molecules, of metabolites and their effect on distant tissues ...

Finally, it is possible to couple the measurements made using the transistor (voltage, current and transconductance) with other analysis methods. Indeed, it is possible to collect the medium which leaves the TEO and to analyze its composition, either directly or after concentration, to identify the metabolites of the cells or any other biomarker such as growth factors, cytokines ... On the other hand, it is possible to analyze the cell structure, the configuration of cells within the 3D matrix by imaging. This type of analysis can be carried out directly on a section because the matrix is transparent. These analyzes can reinforce or refine the result obtained thanks to the analysis of the signals collected thanks to the transistor. Due to the transparency of the matrix, it is also possible to perform optical and electrical analyzes in real time on cell cultures in 3D (it is not necessary to remove the matrix and therefore no cell manipulation is required) .

The inventors have also shown that the force of the flow (shear force) increases the speed of differentiation of cells, in particular stem cells (Curto, et al.,. Organic transistor platform with integrated microfluidics for in-line multiparametric in vitro cell monitoring . 2017, Nature Microsystems and Nanoengineering, 3: 17028 (2017) doi: 10.1038 / micronano.2017.28}. Thus, it is possible to adapt the speed of the flow to accelerate cell differentiation.

The present invention will be better understood in the light of the examples which follow, which have only an illustrative role and should not be considered as limiting the scope of the invention.

FIGURES

Figure 1: Schematic views of tubular devices comprising an organic electrochemical transistor integrated in a tube. In a first configuration, the source and drain electrodes (A and C) are integrated into the wall of the tube while the gate electrode (B) is in contact with the channel via an opening in the body of the tube, (IA) side view, (IB) cross section. In a second configuration, the source and drain electrodes (A and C) are inserted into an opening of the tube arranged in the wall of the body of the tube while the gate electrode (B) is embedded in the tubular system by the one end of the tube, (IC) side view, (1D) cross section.

Figure 2: Representation of the stages of manufacturing a tubular device according to the invention. (A) Preparation of a tube with 3 openings, (B) Insertion of conductive points for the source and drain electrodes, (C) Connection of the source and drain electrodes to the conductive points, (D) Injection of the PEDOT matrix: PSS in the tubular system (liquid solution), (E) Obtaining a PEDOT matrix: porous PSS by freeze-lyophilization.

Figure 3: (3A) Measurements of the output characteristics of the drain current Id as a function of the drain voltage, Vd for a gate voltage Vg varying from 0V to 0.6V (with a step of 0.1V) of a tubular device comprising a TEO integrating a matrix of the PEDOT: PSS type with a gate electrode of Ag / AgCl. (3B) shows the transfer curve of the transconductance g _m at Vd = 0.6V. The distance between the source and drain electrodes being equal to d = 800 μm.

Figure 4: (4A) Measurements of the output characteristics of the drain current Id as a function of the drain voltage, Vd for the gate voltage, Vg varying from 0V to 0.6V (with a step of 0.1V) d '' a tubular device comprising a TEO integrating a PEDOT: PSS type matrix with an Ag / AgCl gate electrode. (4B) shows the transconductance transfer curve g _m at Vd = 0.6V. The distance between the source and drain electrodes being equal to d = 250 μm.

Figure 5: Measurements of the output characteristics of the drain current Id as a function of the drain voltage, Vd for the gate voltage, Vg varying from 0V to 0.6V (with a step of 0.1V) of a device tubular comprising a PEDOT: PSS type TEO with (A) a PEDOT: PSS gate electrode and (B) a Pt gate electrode.

Figure 6: Representation of the drain and gate current as a function of time using PBS as an electrolyte (from 0 to about 585 seconds) then replacing the PBS with culture medium (from 775 to 920 seconds). Between the two time intervals, no electrolyte is present. A series of -0.3V pulses are applied to the grid, while the voltage is kept constant at -0.2V.

Figure 7: shows a comparison of the transient response to a pulse of Vg = 0.3V using PBS (dotted line), culture medium (solid line) as the electrolyte. The pulse level of the grid voltage is visible at the top of the figure (dotted line). The drain voltage is kept constant at -0.2V.

Figure 8: shows a comparison of the output characteristics of the tubular device comprising a TEO according to the invention before and after sterilization of the device in a 70% ethanol solution for 30 min.

Figure 9: shows a comparison of the output characteristics of the tubular device comprising a TEO operating in the presence of cells after 1, 3 or 5 days of culture. The voltage applied to the grid is equal to Vg = 0.6V.

Figure 10: shows an optical fluorescence image of MDCK11 cells inside a PEDOT: PSS matrix integrated into a tubular device comprising a TEO after 5 days of cell culture.

Figure 11: (11A) Comparison of the output curves, (11B) transfer curves and (11C) transconductance curve of the tubular device comprising a TEO before and after a 3-day culture of MDCK11 cells.

Figure 12: (12A) Comparison of the output curves, (12B) transfer curves and (12C) transconductance curve of the tubular device comprising a TEO before and after a 3-day fibroblast culture.

Figure 13: Histogram representative of the relative variations in the percentages of transconductance after a cell culture of 1, 3 or 5 days.

Figure 14: Response of the transient drain current to periodic pulses in slots (Vgs = 0.3V), before cell seeding and after 1 and 2 days of cell culture (MDCK11 cells).

EXPERIMENTAL PART

Preparation of the conductive matrix

A solution was obtained by mixing an aqueous solution of PEDOT: PSS (Clevios PH-100, Heraeus) with a concentration of 1.25% by weight with 3glycidoxypropyltrimethoxysilane (GOPS) as crosslinking agent at a concentration of 3% by weight to improve the mechanical properties and the stability of the matrix in water.

In addition, the PEDOT: PSS solution can be mixed with collagen (0.05% by weight) and / or with DBSA (0.5% by weight).

These different solutions have been used to prepare conductive matrices.

Tubes and electrodes

Commercially available tubes with 2 or 3 openings were used to make the conductive matrices and the corresponding TEO devices.

Tubes of various materials were tested in TEO devices such as (a) Polyvinylidene fluoride (PVDF), Polyetheretherketone (PEEK), (b) polytetrafluoroethylene (PTFE), (c) TygonR, (d) l fluorinated ethylene propylene (FEP), (e) ethylene tetrafluoroethylene (ETFE) or polypropylene (PP). The diameters of the tube tested varied between 1 millimeter and 5 millimeters, while the lengths varied between 10 millimeters and 50 millimeters. Various sizes of gold-coated electrode heads were used to prepare the source and drain electrodes.

Manufacture of TEO devices

The conductive matrices were prepared inside the tubular structures. Typically, a T-shaped tube with 3 openings is used to house the conductive matrix. The source-drain electrodes (pogo heads) are fixed in diametrically opposite directions inside the tube and at a distance of between 100 pm and 1 millimeter. The tubes are fixed inside the tube with a rubber strap and medical glue. The PEDOT: PSS solution was injected into the tubular system, ensuring that it was filled with the solution without forming bubbles or empty space. The filling volume depends on the desirable size of the conductive matrix. A volume is typically between 20 and 40 μl. After filling the tubes with the solution, the devices were placed in a freezer-dryer (Cryotec), where they were frozen from 5 ° C to -40 ° C at a controlled speed of -0.9 ° C. min1, point at which the ice is sublimated from the matrices. PEDOT: PSS matrix lyophilization methods are described in Wan et al. (J. Mater.B, 2015, 3, no.25, 5040-48)

The manufacturing process of the transistor is shown in Figure 2. This includes assembling the tube with the metal contact points used to connect the electrodes, then introducing the PEDOT: PSS liquid into the tube, followed by the in situ freeze drying using a freeze dryer. After sublimation, the matrices were placed in a vacuum oven at 60 ° C for 2 hours. Before use, the TEOs were rinsed several times with distilled water. They were then kept in distilled water for more than 24 hours to allow the diffusion of the low molecular weight components out of the structure. The matrices used for cell culture experiments were further treated with 70% ethanol for about 30 min to make them sterile.

Characterization of the matrix

The microstructure of the matrix was characterized by scanning electron microscopy (SEM). A SEM microscope (MEB ultra 55 - Cari Zeiss) was specifically used to evaluate the invasion of cells in the matrices. Briefly, the cells present in the matrix were fixed with 4% paraformaldehyde for 20 min under infusion at room temperature, then washed carefully with PBS. The matrix was detached from the electrodes and extracted from the tube by applying mechanical force. It was then dehydrated by passage through a series of increasing concentration ethanol baths. Finally, the sample was covered with a 10 nm layer of gold / paladium and analyzed by applying a current of 5 kilovolts. The compression capacity of the hydrated PEDOT matrix was measured using an Instron with a lkN dupiezoelectric pressure sensor. Each matrix was immersed for one hour in PBS and then examined. The elements were compressed at a speed of lmm / s. Young's modulus is calculated by considering the slope in the linear part of the tension force curve.

Cell culture experience

MDCK11 cells were grown in glucose-poor DMEM medium supplemented with 10% FCS (fetal calf serum), 2mM glutamine, 50 U.mL-1 penicillin and 50 pg.mL-1 streptomycin. The cells were labeled with fluorescent actin using T1F LifeAct according to the manufacturer's instructions (Ibidi, Gmbh) and as described for pCMVLifeAct-TagRFP. To maintain the expression of fluorescent actin, 500 pgmL-1 of Geneticin are added to the culture medium. 5 ml of 3 × 10 ⁶ MDCK11 cells in suspension were injected into the matrix using the same culture medium supplemented with HEPES 20 mM +/- geneticin, if necessary. Cells were seeded at the top of the matrix and then the medium was slowly removed until a thin layer of liquid was observed on the surface of the matrix. After seeding the cells, the matrices were incubated for 1 hour at 37 ° C to allow the cells to adhere, after which fresh medium was added to the system to remove non-adherent cells. A weak flow del, 6 pL.min1 of flow was applied to feed the cells for 1 to 5 days and the elements were incubated in a humidified atmosphere at 37 ° C, under 5% CO2.

For the trypsin treatment, the matrices were washed several times with PBS to remove the medium. Then the PBS solution was replaced by a solution containing 0.25% of trypsin and a constant flow rate 10 pL.min-1 was applied to help the dissociation of the cells for 1 h at 37 ° C. After incubation, several manual infusion / elimination cycles were applied to the matrices to remove the cells.

Immunofluorescent labeling

MDCKII cells were fixed in 4% paraformaldehyde for 20 min and then permeabilized using a solution of 0.1% Tritons X-100 for 10 min at room temperature. The matrices were thoroughly washed with PBS and incubated with a solution of rhodamine Phalloidin and DAPI (Invitrogen) for 30 min to label actin and nuclei, respectively. The matrix was examined under epifluorescence / confocal microscope (AxioObserver ZI LSM 800 ZEISS).

Electrical characterization of matrices

Three different grid electrodes were used to measure the characteristics of the transistors of the tubular systems, (a) commercially available Ag / AgCl electrode, (b) platinum mesh (45x25 mm) or (c) PEDOT matrix : PSS. As the electrolyte solution, either a buffered medium (PBS) or a cell culture medium were used. These media which were renewed before each measurement. The characteristics of the TEO were measured using a Keithley 2612A with the LabVIEW software adapted as needed.

Example 1: Configuration of a TEO

Figure 1 illustrates two embodiments of a TEO device according to the invention. Diagrams IA to ID show the positioning of the drain electrode (A), the gate electrode (B) and the source electrode (C) and their connection with the matrix (D) according to different configurations.

Example 2: Characterization of the device

at. In the absence of cells in the matrix

The operation of the transistor integrated in a tube and comprising a PEDOT: PSS matrix has been characterized. Figure 3 shows that the intensity of the drain current (Id) is correlated to the drain voltage (Vd) (Figure 3A). In addition, the intensity of the drain current decreases when the gate voltage increases and the transconductance is optimal when Vg is between 0.3 and 0.4 (Figure 3 B).

When the distance between the source and drain electrodes is decreased, the transconductance increases by about a factor of 10 (Figure 4B).

The voltage, current and transconductance characteristics of TEO are broadly identical whether a PEDOT: PSS or platinum gate electrode is used, as shown in Figure 5.

In addition, it has been shown that the TEO device works in an equivalent manner when a microfluidic flux solution is applied to the matrix, whether with a PBS (phosphate buffered saline) buffer solution or with culture medium, as illustrated. in Figures 6 and 7.

Figure 8 shows a comparison of the characteristics of the transistor before and after sterilization with ethanol. Sterilization does not affect the operation of the transistor. All of these data establish that the TEO in the proposed tubular configuration has all the characteristics required for its use as a transistor to be used to host a culture of cells in 3D to be studied.

b. In the presence of cells

The device was then tested after injection of cells inside the PEDOT matrix: PSS. The ability of cells to grow within the matrix depends on several factors, in particular the ability of cells to penetrate the heart of the matrix, adhere to the support and proliferate.

The graph presented in Figure 9 shows a gradual decrease in the drain current 1d over the time between the first day and the 5th day of culture. This difference reflects cell growth and the fact that the matrix is gradually colonized by cells.

has been shown on a culture of MDCK11 cells transfected with a fluorescent protein, that after 5 days of culture, the cells have effectively colonized the matrix, are viable and healthy (Figure 10).

These experimental data validate the fact that the PEDOT: PSS matrix integrated into the tubular TEO system is suitable for cell culture in 3D.

The functionality of the device was then tested in the presence of cells.

Figure 11 shows the results obtained with a MDCK11 kidney cell culture and Figure 12 the results obtained with a fibroblast culture. Similar results are obtained with the two cell types, namely that the presence of cells within the matrix significantly changes the electrical properties of the device. Indeed, the output voltage curves (Figures 11A and 12A), the transfer curves (Figures 11B and 12B) and the transconductance curves (Figures 11C and 12C) show a clear difference between a device comprising cells and a device without cell. Figure 13 illustrates the difference in transconductance observed for cell culture times of 1, 3 and 5 days. This figure shows that a cell quantification over time is possible and that it is thus possible to assess cell growth using the device according to the invention.

The sensitivity of the device is such that it is possible, after calibration, to assess cell growth but also cell functions (in particular cell integrity and proper functioning in the case of toxicity study) and to follow the training tissue.

EXAMPLE 3 Evaluation of Cell Growth Using TEO

MDCK 11 epithelial cells were seeded in the TEO and the system, with all of its electrical connections, was placed in an incubator for 3 days. After seeding, the system is continuously supplied with culture medium by a pump which maintains the flow. For this experiment, the TEO used is that shown in Figures IC and 1D, the source and drain electrodes of which are covered with gold and the platinum mesh gate electrode of which is embedded in the tubular system.

The high biocompatibility of the platinum electrodes made it possible to measure in situ, during cell culture, the electrical parameters of the system for an extended period without affecting the viability of the cells. As cells grow inside the conductive matrix (PEDOT: PSS), the presence of cells and the formation of tissue within the porous structure of the matrix induce significant changes in the electrical behavior (parameters) of the TEO transistor.

The results of this experiment are illustrated in Figure 14. The changes in the behavior of the transistor are accompanied by a slower response time of the current during the doping and dedoping phases, as it appears on graph 14B in conditions of normalized response. In addition, the transconductance values decrease significantly, from 1 day of culture, as shown in Figure IC. All of these changes can be attributed to an increase in resistance and capacitance of the matrix due to the presence of growing cells and then to tissue formation and the consequent decrease in ion transport. Observation under an optical microscope of the matrix on D2 shows the presence of cells covering the matrix both on the surface and inside the pores (data not shown); these results confirm the presence of cells within the matrix and the fact that these cells have the capacity to grow and colonize the matrix of PEDOT: PSS. Thus, the changes measured using TEO are correlated with a visual observation of cell growth within the matrix.

These results validate the fact that the TEO according to the invention can be used as an in situ detection tool for the real-time evaluation of the state of a cell culture.

Claims (12)

1. Organic electrochemical transistor comprising: (i) a channel consisting of a matrix of porous conductive polymer and biocompatible in three dimensions, (n) a source electrode and a drain electrode each establishing electrical contact with said channel, and (ni) a gate electrode establishing a electrical contact with an electrolyte being in direct contact with said channel, characterized in that said channel is located inside a tube comprising at least 2 openings. 2. Transistor according to claim 1 characterized in that said polymer matrix consists of PEDOT: PSS. 3. Transistor according to claim 2 characterized in that collagen and DBSA are associated with said matrix. 4. Transistor according to one of the preceding claims, characterized in that the source and drain electrodes are made of gold or covered with gold and the gate electrode is made of PEDOT: PSS. 5. Method for preparing an organic electrochemical transistor according to one of claims 1 to 4 comprising the following steps: at. preparing a tube comprising at least two openings; b. integrate two diametrically opposite conductive points in the wall of the tube to produce the source electrode and the drain electrode; vs. inject a solution of liquid biocompatible conductive polymer into the tube through one of the openings; d. freeze then lyophilize said polymer solution so as to obtain a porous and biocompatible conductive matrix, in which the gate electrode is brought into contact with the electrolyte via one of the ends of the tube or via a third opening made in the body of the tube. 6. Method for preparing an organic electrochemical transistor according to one of claims 1 to 4 comprising the following steps: at. preparing a tube comprising at least three openings; b. inject a solution of liquid biocompatible conductive polymer into the tube through one of the openings; vs. freezing and then lyophilizing said polymer solution so as to obtain a porous and biocompatible conductive matrix; in which the source and drain electrodes are inserted via the opening made in the body of the tube and the gate electrode is brought into contact with the electrolyte via one of the ends of the tube. 7. Process for the preparation of an organic electrochemical transistor according to one of claims 1 to 4 comprising the following steps: at. preparing a tube comprising at least three openings; b. preparing a porous and biocompatible conductive matrix by freezing and then lyophilizing a solution of liquid biocompatible conductive polymer, vs. insert said matrix prepared in step b. in the tube prepared in step a. in which the source and drain electrodes are inserted via the opening made in the body of the tube and the gate electrode is brought into contact with the electrolyte via one of the ends of the tube. 8. Method according to one of claims 5 to 7 characterized in that said lyophilization is a sublimation. 9. The preparation method according to claim 5, further comprising a step of adding synthetic polymers or proteins to the matrix, this step being either before freezing or after freeze-drying. 10. Use of a transistor according to one of claims 1 to 4 for the detection of a cellular state such as cells in the process of adhesion, proliferation, differentiation, apoptosis or cell death. 11. Integrated system claims 1 to 4. 12. Use of a s 5 bioreactor. comprising at least two transistors according to one of according to one of claims 10 or 11 as