Microfluidic devices and related methods for generation and/or culture and/or maturation of three-dimensional cells and/or tissue constructs

Abstract

A microfluidic device for controlled generation and/or culture and/or maturation of three-dimensional cells and/or tissue constructs that includes a culture chamber may include: a confinement apparatus configured to define at least one compartment configured to contain a cellular matrix and at least one compartment configured to contain a culture medium, the confinement apparatus being hydrophobic and pervious to the culture medium; and/or at least one counter element. The confinement apparatus and at least one counter element may be reciprocally mobile between resting and compression positions of the cellular matrix. A method for controlled generation and/or culture and/or maturation of three-dimensional cells and/or tissue constructs at a microscale may include: controlled compression of a cellular matrix for a predetermined period of time. The cellular matrix may be delimited by a confinement apparatus that is pervious to a culture medium.

Claims

1. A microfluidic device for controlled generation and/or culture and/or maturation of three-dimensional cells and/or tissue constructs, the microfluidic device comprising: a confinement means configured to define at least one first compartment configured to contain a cellular matrix and at least one second compartment configured to contain a culture medium; and at least one counter element; wherein the culture medium is liquid, wherein the confinement means is hydrophobic, wherein the confinement means and the at least one counter element are reciprocally mobile between a resting position of the cellular matrix and a compression position of the cellular matrix, and wherein the confinement means is pervious to the culture medium.

2. The device of claim 1, wherein in the compression position, the at least one counter element is in abutment with the confinement means.

3. The device of claim 1, wherein the confinement means comprises chemically inert material.

4. The device of claim 1, wherein the confinement means comprises a plurality of micropillars.

5. The device of claim 4, wherein the micropillars are positioned in two parallel rows so as to define a microfluidic channel.

6. The device of claim 1, wherein the at least one counter element comprises a mobile wall.

7. The device of claim 1, further comprising: an actuation chamber configured to cause movement of the at least one counter element toward the cellular matrix to compress the cellular matrix.

8. The device of claim 1, wherein the confinement means is made with one or more of silicon rubber, fluorinated rubber, polystyrene (PS), polymethyl methacrylate (PMMA), polycarbonate (PC), glass, silicon, or polyethyleneglycol (PEG).

9. A microfluidic device for controlled generation and/or culture and/or maturation of three-dimensional cells and/or tissue constructs, the microfluidic device comprising: a confinement means configured to define at least one first compartment configured to contain a cellular matrix and at least one second compartment configured to contain a culture medium; and at least one counter element; wherein the culture medium is liquid, wherein the confinement means is hydrophobic, wherein the confinement means and the at least one counter element are reciprocally mobile between a resting position of the cellular matrix and a compression position of the cellular matrix, and wherein the confinement means is pervious to the culture medium so that, in the compression position, the culture medium contacts the cellular matrix.

10. The device of claim 9, wherein in the compression position, the at least one counter element s in abutment with the confinement means.

11. The device of claim 9, wherein the confinement means comprises chemically inert material.

12. The device of claim 9, wherein the confinement means comprises a plurality of micropillars.

13. The device of claim 9, wherein the at least one counterelement comprises a. mobile wall.

14. The device of claim 9, further comprising: an actuation chamber configured to cause movement of the at least one counter element toward the cellular matrix to compress the cellular matrix.

15. A microfluidic device for controlled generation and/or culture and/or maturation of three-dimensional cells and/or tissue constructs, the microfluidic device comprising: a confinement means configured to define at least one first compartment configured to contain a cellular matrix and at least one second compartment configured to contain a culture medium; and at least one counter element; wherein the culture medium is liquid, wherein the confinement means is hydrophobic, wherein the confinement means and the at least one counter element are reciprocally mobile between a resting position of the cellular matrix and a compression position of the cellular matrix, and wherein the confinement means is pervious to the culture medium so that, in the compression position, a portion of the culture medium passes from the at least one second compartment into the at least one first compartment.

16. The device of claim 15, wherein in the compression position, the at least one counter element is in abutment with the confinement means.

17. The device of claim 15, wherein the confinement means comprises chemically inert material.

18. The device of claim 15, wherein the confinement means comprises a plurality of micropillars.

19. The device of claim 15, wherein the at least one counter element comprises a mobile wall.

20. The device of claim 15, further comprising: an actuation chamber configured to cause movement of the at least one counter element toward the cellular matrix to compress the cellular matrix.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows a schematic representation of the device according to a preferred embodiment of the present invention in a resting position (a) and a compression position (b).

(2) FIG. 2 shows a schematic representation of a detail of the culture chamber of a device according to a preferred embodiment of the present invention in a resting position (a) and a compression position (b).

(3) FIG. 3 shows an exploded view of the device of the invention according to a preferred embodiment (FIG. 3a) and a representation, according to a preferred embodiment of the present invention (FIG. 3b), of the culture chamber and actuation chamber and their respective inlet and outlet openings for the cells and for the culture medium.

(4) FIG. 4 shows a schematic representation of the parts (a-c) that compose the device of the invention according to a preferred embodiment.

(5) FIG. 5 shows a representation of the device when functioning according to a preferred embodiment of the invention illustrated through the sequence of steps (i) to (vi).

(6) FIG. 6 shows the device of the invention in an alternative embodiment of the invention in a resting position (a) and a compression position (b).

DETAILED DESCRIPTION OF THE INVENTION

(7) With reference to the figures, in particular FIG. 1, according to a preferred embodiment of the present invention, the microfluidic device, indicated as a whole with the numeral 1, is composed of a culture chamber 4 and an actuation chamber 7 below the culture chamber.

(8) The confinement means 2 are situated, in this embodiment, on the upper wall 8 of the culture chamber 4 and consist of a series of micropillars (in this case, the micropillars are prisms with an hexagonal cross section) positioned in two parallel rows between each other so as to form a microfluidic channel, that is rectangular-shaped. The perimeter of the microfluidic channel is therefore defined, on two opposite sides, by the two parallel rows of micropillars 2 and on the remaining opposite sides by the walls of the same culture chamber itself (not shown herein).

(9) In other embodiments, the micropillars can be positioned so as to have any other geometrical form. It is not necessary that the walls of the culture chamber form two of the opposite walls that define the perimeter of the cellular matrix, as the micropillars can define the whole perimeter of the polymeric matrix. For a better control of the compression, it is preferable for the perimeter to define a regular geometrical form, for example, a circumference, a square, a hexagon, etc.

(10) In other embodiments, the micropillars can be positioned so as to form a plurality of microfluidic channels, for example parallel or in series with respect to each other, functionally isolated from each other.

(11) In this way, in fact, in the application of the device for the development, for example, of a drug, various tests can be carried out in parallel, thus optimizing the time-scale and the space required, in addition to the reliability of the same test.

(12) According to a further embodiment of the invention, the confinement means 2 can be produced so as to be suitable for containing various layers of cellular matrices, in order to form composite cellular matrices. This further embodiment can be implemented, for example, by the introduction of one or more additional layers of cell culture overlying (or underlying) the main layer, and separated from the adjacent layers by means of a pervious membrane.

(13) It is not essential that the microfluidic channel is positioned centrally with respect to the width of the upper wall 8. This however is the preferred embodiment as it allows an even perfusion of the culture medium (CM) on the two sides.

(14) The confinement means, in particular the micropillars, are preferably made of a hydrophobic material, for example silicon rubber, for example polydimethylsiloxane (PDMS), fluorinated rubber, polystyrene (PS), and combinations thereof, more preferably silicon rubber.

(15) Such means can also be made of another material, such as, for example, polymethyl methacrylate (PMMA), polycarbonate (PC), polyethyleneglycol (PEG), glass, silicon, and combinations thereof, and be provided with a hydrophobic nature, for example by means of chemical and/or physical surface treatment.

(16) It is important for the confinement means 2 to have hydrophobic or low hydrophilicity characteristics, as the device of the present invention exploits the surface tension characteristics of the cellular matrix, in particular when it is introduced in liquid or fluid form into the compartment suitable for containing the cellular matrix. This allows the cellular matrix to be confined within the perimeter defined by the confinement means, in this case micropillars, and within the thickness delimited by the proportions between the height of the confinement means and the height of the culture chamber, and at the same time an efficient cell culture is obtained thanks to the passage of the culture medium (CM) through the spaces separating adjacent micropillars.

(17) Furthermore, as explained in greater detail hereunder with reference to FIG. 1b, the spaces between adjacent micropillars allow the cellular matrix to extend during the compression step, occupying the above spaces, in the compression position.

(18) The confinement means, in particular the micropillars, therefore exert four functions: they contain the cellular matrix, they allow the extension of the cellular matrix, they allow the passage of the culture medium, and they possibly also act as a run-end for stopping the counter element (which will be discussed in further detail hereunder).

(19) In order to obtain the desired result through the device of the invention, in fact, it is essential that the culture medium be able to optimally reach all the culture cells, even the innermost cells in the cellular matrix.

(20) This aspect becomes particularly important as the cell culture becomes gradually transformed into a three-dimensional tissue construct, more preferably mature, wherein there are possibly a larger number of cells and which are more compact.

(21) In the specific case of micropillars, if, on the one hand, it is advantageous to have a relatively large distance between adjacent micropillars for the reasons indicated above, said distance must, however, be sufficiently small as to allow the surface tension characteristics of the cellular matrix to be exploited during its injection into the device, in order to be able to confine the cellular matrix.

(22) In this embodiment represented in FIG. 1, the counter element 3 is the lower wall of the culture chamber 4.

(23) Other solutions are of course possible, such as, for example, the fact that the counter element can form only a part of the wall.

(24) In a preferred embodiment, the counter element has a behaviour which includes an elastic component, in particular it is a flexible membrane.

(25) In a preferred embodiment, the counter element 3, or the flexible membrane, is made of silicon rubber (for example polydimethylsiloxane (PDMS), but fluorinated rubber, polystyrene (PS), polymethyl methacrylate (PMMA), polycarbonate (PC), glass, silicon, polyethyleneglycol (PEG), and combinations thereof, are equally valid.

(26) In the embodiment of FIG. 1, it can be seen that the cellular matrix 6 rests on the counter element 3 in the resting position without undergoing compression.

(27) In the same way, the cellular matrix can be in contact with the counter element and can additionally be subjected to compression, which however is always lower than that at which the same is subjected when in a compression position.

(28) The cellular matrix may also not be in contact with the counter element in a resting position (embodiment not shown).

(29) In the embodiment shown in FIG. 1, the actuation chamber 7 has the function of generating a mechanical stimulus suitable for causing the movement of the membrane 3 in the direction of the cellular matrix 6, compressing it.

(30) Other embodiments can of course also envisage different relative positions of the various elements, wherein, for example, the confinement means are on the lower wall of the culture chamber and the counter element is on the upper wall, and the actuation chamber is above the culture chamber.

(31) As mentioned above, the actuation chamber is not essential, as the compression step of the cellular matrix can be realized with other expedients known to skilled person in the field (actuation of the type chosen from the pneumatic, hydraulic, mechanical, electrical, magnetic, thermal types, and combinations thereof). A piston mechanical actuation can be used, for example, or an electromechanical actuation using a piezoelectric element.

(32) Furthermore, the mechanical stimulus does not have to be created by the counter element 3, as it is also possible for the wall comprising the confinement means 8 to undergo a movement towards the counter element 3 which is stationary.

(33) Similarly, both the wall comprising the confinement means 2 and the counter element 3 can undergo movement towards each other.

(34) In order to obtain the compression of the cellular matrix in the compression step, the confinement means and the at least one counter element must in fact undergo a movement of at least one of these towards each other.

(35) In the embodiment shown in FIG. 1, the actuation chamber 7 comprises, on its upper wall, the counter element 3, in particular a flexible membrane.

(36) Preferably, supporting means 5, for example two rows of micropillars, are positioned between the counter element and the lower wall 9 of the actuation chamber 7, whose function is to support the membrane when the device is in the resting position. Their presence, however, is not compulsory.

(37) The actuation chamber 7 can comprise a saline solution in its interior.

(38) In order to generate the movement of the membrane 3 towards the cellular matrix, the interior of the actuation chamber 7 is subjected to a pressure increase which leads to the upward flexion or deflection of the membrane 3 which thus compresses the cellular matrix (as illustrated in FIG. 1b).

(39) In a preferred embodiment, the pressure inside the actuation chamber 3 in resting position (FIG. 1a) is atmospheric pressure and in order to generate the movement of the membrane 3 towards the cellular matrix, the interior of the actuation chamber 7 is subjected to a positive pressure which causes the upward flexion of the membrane 3 which thus compresses the cellular matrix.

(40) In an alternative embodiment (shown in FIG. 6), the pressure inside the actuation chamber 3 in resting position (a) is negative and in order to generate the movement of the membrane 3 towards the cellular matrix, the interior of the actuation chamber 7 is subjected to a pressure increase which causes the upward deflection of the membrane 3 which thus compresses the cellular matrix in compression position (b).

(41) The width of the counter element is preferably sufficiently high so that the flexion induced by its activation results in a low curvature in the central part, so that the cellular matrix perceives this flexion mainly as a shifting of the counter element, thus creating a substantially homogeneous compression of the cellular matrix with respect to the whole of its surface in contact with the counter element.

(42) Thanks to the controlled pressure regulation, a controlled compression can be exerted on the cellular matrix, which can be regulated according to requirements. Alternatively, as shown in FIG. 1b, the compression control can be accurately obtained with the use of the confinement means as run-end for the counter element. In this way, the regulation of the compression can be obtained by controlling the relative geometric ratios (height of the confinement means, distance between the confinement means and the counter element and the distance between the same confinement means).

(43) The quantity of pressure applied and its application frequency depend on the cell culture and the type of construct to be formed.

(44) In a preferred embodiment, the construct to be obtained is a beating myocardial tissue construct and consequently, after introducing cardiac cells into the device, pressure is exerted cyclically with a frequency of about 1 Hz, as explained in greater detail hereunder.

(45) As shown in FIG. 1b, the upward bending of the membrane in a compression position leads to the compression of the cellular matrix which consequently extends in the direction of the width of the microfluidic channel, i.e. causing a uniaxial extension. As already mentioned, in fact, as two opposite ends are delimited by the very walls of the culture chamber, it is not possible for the cellular matrix to extend in the direction of the length.

(46) As indicated above, there is a possible embodiment in which the microfluidic channel is not delimited by the walls of the culture chamber in the direction of the length but, on the contrary, it is delimited by a further two rows of micropillars. In this case, there is a biaxial extension of the cellular matrix.

(47) The extension of the cellular matrix in the direction of the width of the microfluidic channel is enabled by the spaces present between adjacent micropillars.

(48) As can be seen from FIG. 2, in fact, which shows a schematic representation of a detail of the culture chamber, according to a preferred embodiment, in resting position (a) and in compression position (b), with particular reference to the resting step, the cellular matrix 6 rests on the flexible membrane 3. The micropillars 2 are adjacent to the cellular matrix on two of its opposite sides. There is no mechanical stimulus.

(49) In the compression position (b), the membrane 3 bends upwards thus compressing the cellular matrix which in turn extends along the direction of the width of the microfluidic channel, thus slipping into the spaces between adjacent micropillars (not shown). It can be seen from FIG. 2b that the thickness of the cellular matrix 6 is reduced with respect to FIG. 2a and, at the same time, the width is increased, thus occupying the areas around the micropillars 2 which, in FIG. 2a, were free.

(50) The compression exerted on the cellular matrix is controlled, i.e. it is of a known entity, as it can be regulated as desired by a skilled person in the field, and is reproducible. This feature is obtained, for example, thanks to the action of the actuation chamber which allows a well-defined pressure to be exerted in its interior, which in turn results in a well-defined compression on the cellular matrix. Furthermore, thanks to the geometrical regularity characterizing the positioning of the micropillars, and the homogeneity of the distances between adjacent micropillars, the extension of the cellular matrix takes place uniformly along the whole length of the microfluidic channel. Finally, the fact that, according to the preferred embodiment represented in FIG. 1 and in FIG. 2, the microfluidic channel is delimited on two opposite walls by the walls of the culture chamber itself, means that the extension can take place in only one direction, and is therefore mainly uniaxial.

(51) The compression of the cellular matrix causes a reduction in the thickness of the cellular matrix comprised between 1 and 90% with respect to the thickness of the same in resting position and/or in resting step and/or before the compression step, more preferably comprised between 15 and 50%, even more preferably about 35%.

(52) The extension of a dimension of the cellular matrix in compression position (or step) is preferably comprised between 1 and 100% with respect to the same dimension of the cellular matrix in resting position (or step) and/or before the compression step, more preferably comprised between 5 and 20%, even more preferably about 10%.

(53) On the basis of the result to be obtained, a skilled person in the field is able to select the optimal compression degree.

(54) In the same way, the minimum distance between the confinement means and the counter element can be selected so that it is as optimal as possible on the basis of the result to be obtained. In the compression position, in fact, the micropillars can form run-end elements of the counter element, as already mentioned above. In other words, when, in the compression position, the counter element and the confinement means come into abutment with each other, this defines the maximum compression of the cellular matrix that can be obtained in that device. By suitably selecting the reciprocal distances between the confinement means and the counter element, and also the thickness of the polymeric matrix, the desired reproducible compression can therefore be reliably obtained.

(55) A skilled person in the field is naturally able to calibrate the minimum distance between the confinement means and the counter element, the thickness of the cellular matrix and the distance between adjacent micropillars and between rows of micropillars, for obtaining the desired result.

(56) Once the compression has been exerted on the cellular matrix, in the compression step, the device is then restored to resting position, exploiting the elastic recoil of the material of the counter element. Analogously, thanks to its behaviour which includes an elastic component, the cellular matrix tends to return to its original thickness once restored to resting position.

(57) If bent during the compression step, the confinement means also return to their original resting position. The duration of the compression, in the compression step, is extremely variable. For the generation of certain three-dimensional cell and/or tissue constructs, a compression can in fact be applied that has a duration even up to the completion of the generation of a mature tissue construct, therefore requiring a few days.

(58) The duration of a compression step preferably ranges from 0.01 to 10 seconds, more preferably from 0.1 to 1 second.

(59) In the compression step, the compression can be applied once only, or it can be repeated multiple times.

(60) Even more preferably, the compression step is carried out cyclically interspersed by a resting step (or position) which is maintained for a time comprised between 0.01 and 10 seconds, more preferably between 0.1 and 1 second.

(61) Even more preferably, the compression step is carried out cyclically with a frequency comprised between 0.05 and 50 Hz, more preferably comprised between 0.5 and 5 Hz, most preferably about 1 Hz.

(62) FIG. 3a illustrates an exploded view of the device of the invention according to a preferred embodiment and shows the three main parts forming it: the upper wall 8 associated with the confinement means 2 in the form of micropillars; the wall comprising the counter element 3; and the lower wall 9 of the actuation chamber 7, also associated with supporting means 5, in the form of micropillars.

(63) FIG. 3b shows a representation of the culture chamber and actuation chamber and their respective inlet 10 and outlet 11 openings for the cellular matrix and inlet and outlet for the culture medium 12. The inlet of the pressure line 13 is also shown.

(64) It is evident that whereas the inlets and outlets for the cellular matrix 10, 11 and for the culture medium 12 are compulsory, the inlet of the pressure line 13 is optional, depending on the embodiment to be implemented.

(65) It should be noted that the number of inlets and outlets shown in the figure is not compulsory, but the number of the above can vary as desired.

(66) Furthermore, it is not compulsory to provide housings for electrodes. The electrodes can in any case be inserted directly inside the access gates already present (for example, the wells for the inlet and outlet of the culture medium).

(67) In the production of the device of the invention through microfabrication techniques, for example lithographic techniques, various parts can be produced separately, which are subsequently joined together according to techniques known in the field for forming the device of the invention. There can be three of these parts corresponding to those illustrated in FIG. 3a. Alternatively, two parts can be obtained with microfabrication techniques, as illustrated in FIG. 4, in which the two parts (a,b) above, which together define the culture chamber and part of the actuation chamber, are produced with the above techniques, whereas the lower part (c) can be produced with different techniques, as it is a simple plane which will form the floor of the actuation chamber.

(68) With reference now to FIG. 5, the method of the present invention is described hereunder according to a preferred embodiment, in its various steps represented by numbers (i) to (vi) for the formation of a three-dimensional cardiac tissue construct. According to a preferred embodiment, therefore, as represented in FIG. 5(i), the device of the invention is provided according to a preferred embodiment which corresponds to that represented in FIG. 1. The device is previously sterilized and a quantity of cellular matrix is inserted in its interior through the opening for the cellular matrix 10, which is sufficient for occupying the specific microfluidic channel (FIG. 5(ii)). This corresponds to a quantity of about 0.5 l. This quantity is naturally variable depending on the volume to be occupied by the cellular matrix.

(69) The cellular matrix preferably comprises a polymer, in particular fibrin and is in fluid form. The isolation of the cells, for example cardiac cells extracted from hearts of neonatal rats to be seeded, and their treatment before mixing with the polymer and their introduction into the microfluidic device is effected according to procedures known and consolidated in the field, described in detail in the Examples.

(70) As explained above, other types of cells which are equally suitable for forming three-dimensional cell and/or tissue constructs, preferably mature three-dimensional tissue constructs can of course be used with the device of the present invention.

(71) The polymers that can be adopted in the cellular matrix can be in the liquid or semi-solid state to enable them to be injected into the device and be capable of polymerizing after injection to form a gel.

(72) Aggregates of cells can also be injected into the device in a small amount of a fluid (normally the culture medium), in the absence of a polymer. The possible presence of natural polymers generated by the cells themselves can in fact be sufficient for providing the cellular matrix thus formed, with the elastic characteristics necessary for the proper functioning of the method and device of the present invention.

(73) The concentration of the cardiac cells in the cellular matrix is preferably 110.sup.5 cells/l. This concentration can naturally vary in relation to the cells involved and the type of cell construct desired from 110.sup.2 to 110.sup.6 cells/l.

(74) The device is then incubated at 37 C. for 5 minutes to allow the cellular matrix to polymerize. At this point, the cellular matrix is in the form of a semisolid gel (cell construct), in particular, as represented in FIG. 5(ii), in the form of a microfluidic channel.

(75) The polymerization conditions can naturally vary in relation to the materials used.

(76) The culture medium (liquid) is then injected, as shown in FIG. 5(iii) through one of the inlets 12 and goes into the culture chamber in the compartments suitable for containing the culture medium (CM). In this way, in practice, and if the volume of culture medium allows this, the culture medium occupies the whole space remaining available in the culture chamber upon polymerization of the cellular matrix.

(77) Thanks to the spaces between the micropillars, the medium is in contact with the cellular matrix in many points along the length of the microfluidic channel thus being able to efficiently feed the cells of the cellular matrix.

(78) A fluid is then introduced into the actuation chamber 7 by means of the pressure line 13.

(79) In the embodiment represented in FIG. 6, a negative pressure is created inside the actuation chamber, with the expulsion of air from the actuation chamber, so as to cause a downward flexion of the counter element. The air to be expelled can be air previously and specifically introduced.

(80) After creating a negative pressure, the cellular matrix is introduced and goes onto the supporting element (in this case a membrane).

(81) In this embodiment, the compression of the cellular matrix is obtained with an increase in the pressure inside the actuation chamber, which causes an upward deflection of the membrane.

(82) The device thus prepared, according to FIG. 5, is then incubated at a temperature of 37 C. for 7 days with the application of a pressure at the inlet 13 of 0.5 atm at a frequency of 1 Hz.

(83) Although the above parameters are variable, it is clearly not possible to deviate much from the physiological temperature of 37 C. in order to have the best results, as this corresponds to the temperature of a healthy human body.

(84) If the tissue construct to be generated is of an animal origin, whose healthy body temperature is different from 37 C., the incubation temperature can naturally be adjusted to the temperature of the healthy body of the animal in question.

(85) The pressure is regulated, as shown in FIGS. 5(iv) and 5(v), to 0.5 atm by a precision pressure regulator, and the cyclic stimulus is obtained, for example, by means of an electronically controlled electrovalve destined for alternating the pressure in the actuation chamber between environmental pressure to an overpressure of 0.5 atm.

(86) The frequency of the compression step is variable depending on the type of cells used.

(87) As shown in FIG. 5(iv), the confinement means and the counter element 3 (in this case a membrane) are in a compression position, wherein the membrane is flexed towards the micropillars 2, whereas in FIG. 5(v), they are in a resting position, and there is no mechanical stimulus.

(88) During the incubation period, it may be necessary to substitute the culture medium (CM), which is changed through the access openings 12.

(89) A fresh culture medium can be introduced still through the inlets 12 at a desired time of the incubation period.

(90) During the incubation period, it can be observed that the cellular matrix, initially consisting of cells separate from each other, is transformed into a more compact three-dimensional construct in which the cells are functionally bound to each other and capable of responding to stimuli as a syncytium. In particular, the three-dimensional cell construct has movements of contraction and relaxation, in which the cells have a synchronous behaviour with respect to each other, until the formation of a mature tissue construct.

(91) With reference to FIG. 5(vi), this figure illustrates a possible method for performing electrical pacing analysis of a mature beating cardiac three-dimensional tissue construct in synchronous contraction, generated according to the method of the present invention.

(92) As mentioned above, in fact, although the method of the present invention does not require electrical stimuli for its functioning, the latter can be used once a mature cardiac construct has been obtained, for example, at first to verify that a beating construct has been obtained, and subsequently to impose a certain beating pace. A construct thus regulated is particularly useful in those tests which imply the heart as target organ.

(93) In this respect, it should be noted that this latter aspect can only be applied to muscle constructs and not to other types of constructs (for example bone or cartilage).

(94) In order to carry out tests on a substance, preferably a drug or active ingredient, a molecule or a nanoparticle, using the construct obtained with the method of the invention, the device of the invention is preferably used. At the end of the generation of a mature three-dimensional tissue construct inside the device of the invention, the substance to be tested can in fact be introduced directly into the culture medium, for example in the form of a solution.

(95) The amount or concentration of the substance to be tested, as well as other experimental factors such as the temperature and duration of the incubation of the device, is naturally variable and depends on numerous factors known to skilled persons in the field.

(96) The present invention is now further illustrated by means of embodiment examples as provided hereunder.

Example 1

(97) Description of the Device

(98) A multilayer microfluidic device in silicon polydimethylsiloxane (PDMS) was manufactured. The device obtained is composed of two compartments separated by means of a membrane (counter element): an upper chamber (culture chamber, dimensions: 1600 m in width, 3 mm in length and 150 m in height), for obtaining three-dimensional tissue constructs, and a lower chamber (actuation chamber), used for providing the cyclic mechanical stimulation, by means of pneumatic actuation. The culture chamber (height 150 m) consists of a central microfluidic channel having a width of 300 m delimited by two rows of hexagonal micropillars (height 100 m, side 28 m), wherein adjacent micropillars are equidistant from each other (by 50 m). The micropillars were produced attached only to the upper wall of the compartment, so as to be left suspended and separated from the membrane (which represents the floor of the culture chamber) at a distance of 50 m. The central channel is surrounded by two side channels (width 600 m each), dedicated to containing and transporting the culture medium for feeding the cells.

(99) Construction of the Device

(100) The device previously defined was manufactured with the use of photo- and soft-lithographic techniques. The layout of the device was produced through computer aided design (CAD) and is composed of three layers (see FIG. 3a): the first two layers that form the culture chamber, respectively 100 m (layer containing the micropillars) and 50 m in height (confinement tank that defines the minimum distance between the micropillars and floor of the culture chamber); and the third layer containing the mechanical actuation chamber, 50 m in height. The layout of each layer was printed at high resolution (32,000 dpi) on a photomask and subsequently transferred onto a silicon wafer by means of photolithography. For each layer, a film of photosensitive polymer (fotoresist negative, SU-8 2050), having a thickness equal to the design-based height of said layer, was deposited on a silicon wafer having a diameter of 4 (about 10 cm). Said film was then exposed to collimated ultraviolet light (A=365 nm), filtered by the corresponding photomask, so as to induce the crosslinking of the polymer in correspondence with the exposed areas alone. Once the excess of non-polymerized photoresist had been eliminated, the three microstructured silicon wafers obtained (i.e. having the layouts of the layers in bas-relief) were used as moulds for obtaining three blocks of PDMS. In particular, a solution of PDMS in liquid phase was prepared by mixing its two components, pre-polymer and crosslinking agent, in a ratio of 10:1 and put under vacuum for at least 15 minutes, so as to eliminate the presence of residual air. The solution was then poured onto the moulds and left to crosslink at 65 C. for 3 hours. The volume of PDMS to be poured onto each mould was established so as to obtain the desired height of the block: more specifically, 5 mm on the mould containing the micropillars, 300 m on the mould of the confinement tank and 300 m on the mould of the actuation chamber. The inlets to the various compartments were obtained by perforating the PDMS blocks with biopsy punchers having adequate dimensions in correspondence with the access channels designed. In particular, two inlets were produced, having a diameter of 500 m for the injection of the cellular matrix, an inlet having a diameter of 500 m for the pressurizing of the actuation, 4 inlets having a diameter of 5 mm for the injection of the culture medium. Finally, the assembly of the device was effected by chemically gluing the three blocks of PDMS, after alignment, and after plasma activation treatment.

Example 2

(101) Preparation of the Device

(102) The device thus obtained in Example 1 was sterilized by means of an autoclave (20 minute cycle, temperature of 120 C. and pressure of 2 bar) and left in an oven at 80 C. for 24 hours to restore the natural hydrophobicity of the PDMS. The device was then used for the generation and maturation of 3D cardiac constructs, produced by charging a solution of fibrin hydrogel with cardiac cells extracted from hearts of neonatal rats. Before use, the actuation chamber was completely filled (1 l) with a buffer solution (saline phosphate buffer, or PBS 1). More specifically, the actuation chamber was connected to a flexible rubber tube (internal diameter 0.5 mm), half filled with PBS and half with air, to a compressed air line through a pressure regulator. A constant pressure of 0.3 atm was applied for the filling of the actuation chamber with PBS, for 45 minutes, so as to impose the outflow of air contained in the chamber through the same walls of PDMS and its substitution with PBS.

(103) Preparation of the Cells

(104) The cells of interest were isolated using the following process. The cardiac tissue removed from the ventricles of neonatal rats was digested in trypsin (0.6 mg/ml, in a Hank balanced saline solution, HBSS) for 18 hours. The digestion was then blocked by adding 10 ml of culture medium (Dulbecco Modified Eagle's Medium, DMEM) containing: 10% of fetal bovine serum, 1% of penicillin-streptomycin, 1% of HEPES and 1% of L-Glutamine. The isolation of the cells from the matrix was then obtained with five passages in a solution of collagenase (1 mg/ml in HBSS) at 37 C. for 5 minutes. The cell suspension thus obtained was centrifuged at 700 rpm for 10 minutes, re-suspended in 25 ml of culture medium and seeded in a culture flask to isolate the fraction of cardiomyocytes (non-adherent).

(105) After an hour, the fraction of non-adhering cells (consisting of about 85% of cardiomyocytes and 15% of cardiac fibroblasts, was collected and centrifuged at 1,200 rpm for 5 minutes, in order to estimate the number of cells obtained.

(106) Generation of the Mature Cardiac Tissue Construct

(107) For seeding inside the device, the cardiac cells thus isolated were diluted in a solution of fibrin gel at a cell concentration of 110.sup.5 cells/l. More specifically, for the seeding of 6 devices, 20 l of fibrin gel were prepared by mixing 2010.sup.5 cells (estimated volume of 8.2 l), 4 l of calcium chloride, 4 l of fibrinogen (20 mg/ml in sodium chloride), 2.8 l of aprotinin (16 TIU/ml in distilled water) and 1 l of thrombin (5 U/ml in calcium chloride). The solution composed of fibrin gel and cells was sucked by means of a 1 ml syringe into a flexible rubber tube (internal diameter of 0.5) terminating with a metallic tube (standard measurement 23 G and length 12.5 mm). The cell seeding in each device was effected by directly inserting the metallic terminal tube into the inlet of the device destined for the injection of the cellular matrix, and injecting a volume of 0.5 l of solution, by activating the plunger of the syringe, into the central channel of the culture chamber. In order to obtain a complete polymerization of the fibrin gel, the devices were inserted in an environmental incubator (T=37 C., CO.sub.2=5%) for 5 minutes and a culture medium containing a fraction of aprotinin (1.15 TIU/ml) was subsequently injected into the side channels, until the 4 compartments for the culture medium had been completely filled (aprotinin inhibits the digestion of the fibrin gel on the part of the cells). The culture medium was changed manually once a day during the whole incubation period (7 days). Immediately after the polymerization of the fibrin gel, the cell constructs were subjected to cyclic uniaxial mechanical stimulation (extension of the dimension of the width of the microfluidic channel equal to about 10%, frequency 1 Hz), obtained by pressurizing the saline solution contained in the actuation chamber, through an electronically controlled electrovalve (destined for alternating the pressure in the chamber between atmospheric pressure and an overpressure of 0.5 atm).

Example 3

(108) Analysis of the Constructs: Stains

(109) The effect of the cyclic mechanical stimulation on the maturation of the 3D cardiac tissue constructs was analyzed and the results were compared with those obtained in analogous non-stimulated devices used as negative controls. In particular, the effect of the mechanical stimulation on the cell viability was evaluated after 3 days by means of LIVE/DEAD assay showing a statistically lower mortality in the presence of cyclic mechanical stimulation. More specifically, 85.39%0.58% of viable cells were quantified in the mechanically stimulated cell constructs with respect to 66.87%1.84% in the non-stimulated control constructs, after three days of culture. The morphology, organization, maturation, and formation of synergic structures between the cells were then examined through optical microscopy and immunofluorescence techniques. More specifically, the transparency of the PDMS allowed staining in immunofluorescence to be effected directly inside the devices, sequentially perfusing the solutions required in relation to the desired stain, and visualizing the constructs through confocal microscopy, consequently without there being the necessity of extraction from the device. The analysis of the stains for connexin 43 and cardiac troponin showed a greater formation of synergic structures between the cells in the presence of stimulus. The total area of positive construct for connexin 43, normalized for the total number of cardiomyocytes (positive for cardiac troponin) proved in fact to be significantly higher (69.56%9.05%) for the stimulated constructs with respect to the controls (52.98%4.03%). This result was then confirmed by the onset of a synchronous beat in the stimulated constructs already 3 days after the beginning of the mechanical stimulation.

(110) Analysis of the Constructs: Pacing

(111) The level of maturation of the cell constructs thus obtained was examined by means of electric pacing after 7 days of culture. The cell constructs were positioned under an inverted microscope to monitor their contractile response during the whole duration of the electric pacing. A temperature of 37 C. and an atmosphere of 5% of CO.sub.2 were maintained through an integrated environmental incubation chamber. Two stimulation electrodes were inserted in two of the openings for the entry of the culture medium, respectively opposed with respect to the channel containing the cell construct. Said electrodes were connected to an electrostimulator and used for transferring an electrical stimulus having a controlled form and duration, to the cell construct.

(112) The parameters considered for defining the level of maturation of the construct were the excitation threshold and maximum capture rate.

(113) The excitation threshold was defined as the minimum amplitude of the pacing signal necessary for sustaining the synchronous contraction of the cell constructs. Said excitation threshold, measured by gradually increasing the signal amplitude and keeping the frequency fixed at 1 Hz, proved to be lower for the mechanically stimulated constructs (468 mV/cm) with respect to the control constructs (675 mV/cm).

(114) The maximum capture rate was defined as the highest electrical stimulation frequency at which the constructs are capable of following the electrical stimulus signal with a synchronous contraction. This parameter, measured by gradually increasing the frequency and keeping the signal amplitude fixed, proved to be greater for the stimulated constructs (7 Hz) with respect to the control constructs (4 Hz), showing a higher synergy level.

Example 4

(115) Use of the Device for Drug Screening

(116) The mature cardiac construct generated inside the device, as described in Examples 2 and 3, can be used as in vitro model for drug screening. Once the maturation of the constructs had been verified by electric pacing, as described in Example 3, the same experimental set-up was used for verifying the effect of a drug on the electric response of cardiac constructs. More specifically, the effect of the drug lidocaine, an antiarrhythmic drug, was verified. The drug was diluted in two different concentrations (specifically 1 and 2 g/ml) in a culture medium and these dilutions were injected in sequence into the device from one of the openings used as entry for the culture medium, and their effect was evaluated by direct observation. A control was obtained by maintaining a cardiac construct in the culture medium without the addition of the drug. The spontaneous contraction frequency is quantified and used as parameter for determining the response of the cardiac construct to the drug, and compared with that obtained for the control cardiac construct. The construct stimulated with increasing concentrations of drug shows a decrease in the contraction frequency with respect to the control. The quantity of drug suspension used for each replicate is equal to 50 l. The response of the constructs is evaluated 60 seconds after the injection of the drug, keeping the construct at a constant temperature of 37 C.

BIBLIOGRAPHY

(117) Agarwal, A., Goss, J. A., Cho, A, McCain, M. L., Parker, K. K. (2013) Microfluidic heart on a chip for higher throughput pharmacological studies. Lab Chip, 13, 3599-3808. Huang, P. C., Lu, J., Seon, H.; Lee, A. P., Flanagan, L. A., Kim, H., Putnam, A. J., Jeon, N. L. (2009) Engineering microscale cellular niches for three-dimensional multicellular co-cultures. Lab on a chip, 9, 1740-1748. Mathur, A., Loskill, P., Shao, K., Huebsch, N., Hong, S G., Marcus, S. G., Marks, N., Mandegar, M., Conklin, B. R., Lee, L. P., Healy, K. E. (2015) Human iPSC-based cardiac microphysiological system for drug screening applications. Scientific Reports, 5:8883, 1-7.