WO2018122760A1

WO2018122760A1 - Three-dimensional multilayer constructs in microchannels

Info

Publication number: WO2018122760A1
Application number: PCT/IB2017/058458
Authority: WO
Inventors: Giovanni Stefano UGOLINI; Roberta VISONE; Alberto Redaelli; Matteo Moretti; Marco Rasponi
Original assignee: Politecnico Di Milano; Istituto Ortopedico Galeazzi S.P.A.
Priority date: 2016-12-28
Filing date: 2017-12-28
Publication date: 2018-07-05
Also published as: IT201600131735A1

Abstract

The present invention relates to a kit comprising a matrix and a punch, and to a method for obtaining 3D cellular constructs in microfluidic systems.

Description

"Three-dimensional multilayer constructs in microchannels"

Background art

3D cell cultures are a powerful and promising tool for the study of in-vitro biological models [M. Ravi, V. Paramesh, S. R. Kaviya, E. Anuradha, F. D. Paul Solomon, J. Cell. Physiol. 2015, 230, 16] . To date, hydrogel-based approaches, in which cells are trapped in biopolymers, typically of collagen or fibrin, are particularly promising, being capable of mimicking the extracellular matrix [M. W. Tibbitt, K. S. Anseth, Biotechnol. Bioeng. 2009, 103, 655; A. Khademhosseini , R. Langer, Biomaterials 2007, 28, 5087] . 3D cellular constructs obtained inside microfluidic channels have been developed in the vascular [J. S. Jeon, S. Bersini, M. Gilardi, G. Dubini, J. L. Charest, M. Moretti, R. D. Kamm, Proc. Natl. Acad. Sci. U. S. A. 2014, 112, 214], cardiac [A. Marsano, C. Conficconi, M. Lemme, P. Occhetta, E. Gaudiello, E. Votta, G. Cerino, A. Redaelli, M. Rasponi, Lab Chip 2016, 16, 599], hepatic [Y.-C. Toh, T. C. Lim, D. Tai, G. Xiao, D. van Noort, H. Yu, Lab Chip 2009, 9, 2026], and central nervous system [J. D. Wang, E. S. Khafagy, K. Khanafer, S. Takayama, M. E. H. Elsayed, Mol. Pharm. 2016, 13, 895] fields.

However, many physiological structures have a compartmentalized morphology, characterized by organized architectures of different cell types or ECM material particularly difficult to be modelled in vitro [K. Yum, S. G. Hong, K. E. Healy, L. P. Lee, Biotechnol. J. 2014, 9, 16; D. Huh, Y. Torisawa, G. a. Hamilton, H. J. Kim, D. E. Ingber, Lab Chip 2012, 12, 2156] . 3D bio-molding is a promising strategy for the control of the fine geometry of in-vitro biological constructs, but requires expensive equipment and shows considerable limitations especially related to cell viability and compatibility with available bio-inks [S. V Murphy, A. Atala, Nat. Biotechnol . 2014, 32, 773; L. Yang, S. V. Shridhar, M. Gerwitz, P. Soman, Biofabrication 2016, 8, 35015]. The greater limitation of all the techniques listed above lies in the impossibility of microfluidically controlling the cellular constructs. In fact, microfluidic techniques available to date do not allow a fine control of 3D cellular constructs. For example, multiple hydrogel constructs enclosed in a single channel have been obtained by injecting hydrogel into neighbouring structures, separated, for example, by partitions [C. P. Huang, J. Lu, H. Seon, A. P. Lee, L. a Flanagan, H.-Y. Kim, A. J. Putnam, N. L. Jeon, Lab Chip 2009, 9, 1740] or by simultaneously injecting a controlled flow of different hydrogels [A. P. Wong, R. Perez- Castillejos, J. C. Love, G. M. Whitesides, Biomaterials 2008, 29, 1853] . Furthermore, by means of molding techniques, hydrogels traversed by microchannels for microfluidic control have been obtained [M.P. Cuchiara, A.C.B. Allen, T.M. Chen, J.S. Miller, J.L. West, Biomaterials 2010, 31, 5491-5497] . The presence of confinement structures, such as the partitions, has the limitation of producing composite biological constructs which comprise artificial dividing structures, the controlled flow is very complex to obtain and is not practically feasible. The solution suggested by Cuchiara et al . is not very flexible, exclusively allowing for the formation of a hydrogel structure traversed by a channel. Because of these strong limitations, the most advanced models describing a tissue interface/tissue are still based on 2D cell cultures [D. Huh, B. D. Matthews, A. Mammoto, M. Montoya- Zavala, H. Y. Hsin, D. E. Ingber, Science 2010, 328, 1662; H. J. Kim, D. Huh, G. Hamilton, D. E. Ingber, Lab Chip 2012, 12, 2165] . It is the object of the present invention to provide a kit and method for obtaining versatile 3D cell cultures which overcome the limitations of currently available solutions. 3D cell cultures, obtained according to procedures described herein, are also the subject of the present invention.

Description of the invention

The present invention relates to a kit comprising a matrix and a punch, and to a method based on the use of said kit for obtaining complex 3D cellular structures, in which a fine microfluidic control is possible. 3D cellular constructs obtained according to method of the present invention and micro devices ready for use for obtaining said constructs are also the subject of the present invention.

Description of the drawings

Figure 1: diagrammatic perspective depiction of the components of the kit A) matrix; B) intermediate mould; C) closing element.

Figure 2: diagram of the seeding method according to an embodiment of the present invention.

Figure 3: A) diagrammatic depiction of an embodiment of the kit according to the present invention: matrix, intermediate mould and assembling kit; B) i) fluorescence and ii) phase-contrast top view of the reticulum; C) i) phase-contrast top view and ii) fluorescence view and iii) fluorescence side sectional view of the cells in the reticulum on day 0; D) i) phase-contrast top view, ii) fluorescence top view and iii) fluorescence side sectional view of the cells in the reticulum on day 3.

Figure 4: A) diagrammatic depiction of a further embodiment of the kit according to the present invention: matrix, intermediate mould and assembling kit; B) i) fluorescence and ii) phase-contrast top view of the reticulum; C) i) phase-contrast top view and ii) fluorescence view and iii) fluorescence side sectional view of the cells in the reticulum on day 0; D) i) phase-contrast top view, ii) fluorescence top view and iii) fluorescence side sectional view of the cells in the reticulum on day 3.

Figure 5: A) diagrammatic depiction of a further embodiment of the kit according to the present invention: matrix, intermediate mould and assembling kit; B) phase-contrast top view of the reticulum; C) i) phase-contrast top view, ii) fluorescence top view and iii) fluorescence side sectional view of the empty perfusion channel; D) i) fluorescence perspective view and ii) fluorescence longitudinal section view of the endothelialised channel.

Figure 6: A) diagrammatic depiction of a further embodiment of the kit according to the present invention: matrix, intermediate mould and assembling kit; B) phase-contrast top view of the reticulum; C) i) phase-contrast top view, ii) fluorescence top view of the endothelialised construct, iii) fluorescence perspective view and iv) fluorescence 3D reconstruction of the endothelial wall. Figure 7: further diagrammatic top view of the embodiments of Figure 3 (panel A) and Figure 4 (panel B) according to the present invention. Gray line: matrix profile. Black line: intermediate mould profile.

Figure 8: diagrammatic preparation procedure (A-D panels) and product obtained (E panel), which is the micro device ready for use according to an embodiment of the present invention.

Figure 9: 3D collagen-fibrin construct obtained according to the method of the present invention. A) phase-contrast top view; B) fluorescence top view; C) fluorescence side sectional view.

Figure 10: 3D constructs comprising three different cell populations obtained using intermediate moulds having the same height of the cavity. In B, a patent lumen is obtained which traverses the central cell population.

Detailed description of the invention:

For the purposes of the present invention, the following terms have the meaning hereby indicated.

Matrix: a female half-mould which has an upper face and a lower face, in which on the upper face at least one opening (door) is present, which connects the external environment with a cavity which is inside the matrix itself, said cavity being open on said lower face.

Punch: a male half-mould which cooperates with said matrix by partially entering into said cavity. The present invention provides for two alternative embodiments for the punch: i.e., a single half-mould for the different steps of the method, in which said single half-mould is capable of assuming different shapes in the different steps of the method, or a punch comprising an intermediate mould, to be used in a first step of the method, and a closing element, to be used at a later step.

Semi-solid matrix: solid substance comprising a liquid phase dispersed inside it. Typically, it is a gel prepared by cooling a colloidal solution or by means of a rapid reaction with a high concentration of reactants in the liquid phase.

Sacrificial material: solid or semi-solid substance which, by means of melting or otherwise, turns into liquid form, used as a temporary filler of a certain volume.

The present invention relates to a kit (1) for obtaining 3D cellular constructs in microfluidic channels, in which said kit (1), with reference to Figures 1 and 2, comprises: a matrix (2) and at least one punch (19) . Said matrix (2) has an upper face (40) and a lower face (41) and on said upper face (40) at least one inlet door (10) and/or at least one outlet door (11) is present, which leads into in a cavity (5) inside said matrix (2), said cavity (5) being open on said lower face (41) . Said punch (19) partially enters said cavity (5) in said matrix (2), leaving a first empty volume (12) . Said punch (19) either is a single half-mould assuming variable shapes, for example it consists of an inflatable element which, when necessary, is deflated and possibly re-inflated in a different geometry, or is made up of at least two elements, by way of example, a fixed geometry intermediate mould (3) and a closing element (4) (Figure 1) . In an embodiment, with reference to Figure 8, said intermediate mould (3) has a temporary fixed geometry, being said intermediate mould (3) of sacrificial material, i.e. made of a material which, when necessary is, for example, melted and removed by said matrix, for example a sugar, a low melting metal, or jelly.

In a preferred embodiment, said kit comprises a matrix (2) and a punch (19) which is made up of a fixed geometry intermediate mould (3) and of a closing element (4) . Said closing element (4) is a plane element, or it too has portions which at least partially enter into said cavity (5) . By way of example, said closing element (4) is a coverslip.

In a particularly preferred embodiment, said matrix (2) also comprises at least two side channels (6) facing said cavity (5) and open towards the outside of said matrix (2) by means of an inlet (15) and an outlet (16) for each of said channels. Said side channels are in fluidic connection with said cavity (5), for example, as shown in Figure 3, they run alongside, for a part or for the entire length, said cavity (5), a channel per side and, in the portion running alongside said cavity (5) are delimited from the same, exclusively by means of partitions (14) . In an alternative embodiment, said partitions are not present.

The smaller dimension of said cavity (5), typically represented by the height, has a size comprised between Ιμπι and 10mm, preferably comprised between ΙΟμπι and 1mm, even more preferably it is about 200μπι.

In a preferred embodiment, said kit is made of PDMS (Polydimethylsiloxane) and, preferably, the punch and the intermediate moulds are suitably coated so as to prevent the adhesion of the material injected therein, typically are coated with 3% bovine serum albumin (Sigma, Italy) .

The possibility of using said kit (1) for subsequent steps allows for a compartmentalization of the cavity (5) such to ensure the formation of different empty volumes therein with the succession of the steps and the consequent obtainment of complex 3D cellular constructs on a micrometric scale. In particular, the use of said kit (1) comprises:

a) the formation of a first empty volume (12) when said punch (19) penetrates said matrix (2);

b) the formation of a second empty volume (13) when, upon filling said first empty volume (12) with a semi-solid matrix, said punch (19) modifies the geometry thereof or, as soon as said punch (19) is made up of an intermediate mould (3) and of a closing element (4), when said intermediate mould (3) is extracted and replaced by said closing element (4) .

By means of said inlet doors (10) present on said matrix (2), in said at least first empty volume (12) and second empty volume (13), at least two fluids are fed, therefore in subsequent steps, of which at least one capable of creating a semi-solid matrix, in which at least one of said fluids comprises one or more cell population .

Advantageously, the kit of the present invention allows to obtain complex 3D cellular constructs, in which at least one cell population is supplemented with one or more semi-solid matrices and/or in a liquid matrix, in which said at least two matrices face one another. Said semi-solid and/or liquid matrices, formed in adjacent spaces inside the overall empty volume (20) of said cavity (5), therefore create neighbouring structures, not separated by any artificial structure. By virtue of the kit and of the method according to the present invention, it is therefore possible for the first time to obtain 3D cellular constructs on a micrometric scale with maximum versatility in terms of shapes and usable substances and without the limitations imposed by artificial structures, which the methods known in the background art impose inside the constructs themselves.

In a particularly preferred embodiment, where said matrix (2) also comprises, in addition to said cavity (5), said side channels (6) in fluidic connection with the cavity (5) itself, the filling method for subsequent steps described above allows for the formation of semi-solid matrix structures flanked by channels which make the fine control of the microfluidics of the system possible. For example, they allow to inject a culture medium and make it flow therein, which medium allows for the optimal conditions of the cell culture present in the 3D structure formed inside said cavity (5) .

In an embodiment, diagrammatically shown in Figure 7, the assembled matrix (2) and punch (19) leave a gap between the contact surfaces, typically of about 10 microns, highlighted between the two arrows in Figure 7. This distance favours the penetration and facilitates the matrix/punch alignment process, without, however, allowing the release in the gap itself of fluid injected into the first empty volume (12) from the inlet door (10) and without compromising the injection.

Said matrix (2) and punch (19) may also comprise further channels and emergencies in accordance with what described above. Some particularly preferred embodiments are described further on, without however limiting the scope of the present invention.

The present invention further relates to a method for obtaining 3D cell cultures. Said method comprises:

a) Providing a kit (1) comprising a matrix (2) and a punch (19) as described above;

b) Providing primary, immortalized and/or tumour cells in culture ;

c) Providing at least one fluid that creates a semi-solid matrix, in which said fluid optionally comprises one or more cell populations;

d) Assembling said matrix (2) with said punch (19);

e) Injecting said fluid through said at least one inlet door

(10) to occupy said first empty volume (12) between said matrix (2) and said punch (19);

f ) Solidifying said fluid;

g) Obtaining a second empty volume (13) in said cavity (5), for example modifying the geometry of said punch (19), or, where said punch (19) comprises an intermediate mould (3) and a closing element (4), extracting said intermediate mould (3) and replacing it with said closing element (4); h) Injecting a further fluid, optionally capable of creating a semi-solid matrix, optionally loaded with one or more cell populations, through said at least one inlet door (10) to occupy said second empty volume (13);

i ) Optionally, solidifying said further fluid.

The method may comprise further injection and solidification passages, where more punches (19) are provided, which penetrate said matrix (2) to give further empty volumes to be filled.

In a particularly preferred embodiment, the present invention relates to a method comprising:

a) Providing a kit (1) comprising a matrix (2) and a punch comprising an intermediate mould (3) and a closing element (4) as described above;

b) Providing at least one fluid which creates a semi-solid matrix or a solid which is to be intended as sacrificial material ;

c) Assembling said matrix (2) with said intermediate mould

(3) ;

d) Injecting said fluid through said at least one inlet door

(10) to occupy said first empty volume (12) between said matrix (2) and said intermediate mould (3);

e) Solidifying said fluid; f) Extracting said intermediate mould (3) and replacing it with said closing element (4) .

A micro device (30) is thus obtained, diagrammatically shown in Figure 8, comprising the matrix (2) and said closing element (4), in which the cavity (5) of said matrix is partially occupied by said sacrificial material (31) which leaves said second empty volume (13) . Said micro device (30) may be produced under sterile conditions and stored ready for use for subsequent employments. Said micro device is a further aspect of the present invention. At the moment of use, a fluid, optionally loaded with cells, is injected in said second empty volume (13) in said micro device, through said inlet door (10) and said fluid is solidified. Said sacrificial material is therefore eliminated from the device by means of said outlet door (11) thus freeing said first empty volume (12), before occupied by the sacrificial material (31) itself, in which a further fluid is injected, optionally loaded with cells.

Advantageously, the device described and claimed herein allows to obtain complex 3D cellular constructs in an absolutely simplified manner, excluding assembly and opening operations of the kit and the need to have punches available, where said device is provided assembled and ready for use. Those skilled in the art know the sacrificial material which can be advantageously employed for the purpose .

The kit according to the present invention, therefore, allows to obtain complex 3D cellular constructs which are maintained in said kit, typically in said closed matrix (2) and said closing element (4) . Said 3D cellular constructs may be kept in culture for several days, as highlighted by the following examples, by virtue of the fact that the micro device described and claimed herein advantageously gives the possibility of a fine microfluidic control. In fact, said 3D cellular constructs are fed by means of the culture medium injected through said inlets (15) in said side channels (6), where present. The outlets (16) ensure the possibility of maintaining the desired flow of the culture medium. In the embodiment shown in Figure 3 and in Figure 6, with a vertical section, i.e. in which two different cell cultures are arranged on the two sides of the cavity (5), each population faces a side channel (6), thus allowing for a selection of the culture medium to flow into the channel according to the cell culture present in the portion of cavity (5) which said channel faces. In an alternative embodiment, in the absence of said side channels (6), said microfluidic control is made possible by the fact that a portion of said overall empty volume (20) of said cavity (5) is occupied by a semi-solid matrix comprising a cell population, the remaining portion is fed by means of a culture medium, possibly containing a further cell population.

Particularly preferred embodiments are described hereinbelow.

With reference to Figure 3, the kit comprises a matrix (2), an intermediate mould (3) and a closing element (4) . The matrix (2) comprises a cavity (5) and two side channels (6) facing along the length of said cavity (5) and which are open towards the outside with an inlet (15) and an outlet (16) . Said matrix also comprises two inlet doors (10) and two outlet doors (11) for said cavity (5) . Said intermediate mould (3) occupies, for the entire height, half of said cavity (5), where the portion which said intermediate mould (3) occupies separates in a longitudinal direction said cavity (5) . By means of the process described above and by using the kit diagrammatically shown in Figure 3, two semi-solid matrices, facing one another, are obtained, as diagrammatically shown in the insert in Figure 3, panel A, each comprising a cell population, each open along the entire length on a channel (6), a channel in which is possible to make a culture medium flow (media channel) . Figure 3B shows a top view of the culture thus obtained, in which it is possible to identify the longitudinal boundary line between the two matrices and the two cell populations. Figures 3C and 3D show the cultures on day 0 and on day 3 after the seeding. It is apparent how the two matrices, and consequently the two cell populations contained therein, face one another without anything artificial being interposed, allowing to elegantly reproduce the physiology of biological systems in vitro, also by virtue of the fact of being able to provide the cultures with the necessary nutrients by means of said media channels capable of spreading to said matrices .

With reference to Figure 4, the kit comprises a matrix (2), an intermediate mould (3) and a closing element (4) . The matrix (2) comprises a cavity (5) and two side channels (6) facing along the length of said cavity (5) and which are open towards the outside with an inlet (15) and an outlet (16) . Said matrix also comprises one inlet door (10) and two outlet doors (11) for said cavity (5) . Said intermediate mould (3) occupies the entire surface of said cavity (5), for a height equal to about half of the height of said cavity (5) . With the process described above, two semi-solid matrices are obtained, as highlighted in the insert of Figure 4A. The two matrices overlap one another, each one comprising a cell population, flanked by channels in which the culture medium can flow. Figure 4B shows a top view of the culture. Figures 4C and 4D show the cultures on day 0 and on day 3 after the seeding. The considerations made for the embodiment of Figure 3 are also easily applicable here.

With reference to Figure 5, the kit comprises a matrix (2), an intermediate mould (3) and a closing element (4) . The matrix (2) comprises a cavity (5) and two side channels (6) facing along the length of said cavity (5) and which are open towards the outside with an inlet (15) and an outlet (16), in addition to two inlet doors (10) and two outlet doors (11) . Said intermediate mould (3) occupies a portion of said cavity (5), for a width lower than that of said cavity (5) . With the process described above, a semi-solid matrix is obtained, possibly comprising a cell population, containing a channel with a rectangular base interfacing it on at least two sides. Figure 5B shows a top view of the culture. Figures 5C and 5D show top views of cultures on day 3, following the seeding, of the sole cavity filled with matrix and cells and in section (5C) . In 5D the side views are shown and reconstructed with the construct made up of both hydrogel with cells and endothelial cells seeded in liquid medium into the channel resulting from the removal of the punch. Figure 10, panels A and B, show alternative configurations obtainable with intermediate moulds having the same height as the cavity. The channel left patent may be filled with a liquid or semi-solid matrix, possibly containing cells.

Figure 6 shows an alternative configuration, which reflects the embodiment of Figure 4 by adding partitions in said cavity (5) .

In a further aspect, the present invention describes a complex 3D cellular structure, in which different cell populations grow trapped in at least one of two semi-solid matrices equal to or different from one another, facing one another, or grow in a liquid matrix, facing a semi-solid matrix which in turn contains or does not contain cells. Said cell populations, therefore, constitute 3D structures facing one another and not separated by any artificial structure, reproducing common physiological situations of interaction between different cell populations.

The following examples exemplify the embodiments of the kit and method of the present invention and of 3D cellular constructs obtained .

Examples

Example 1: Design of micro devices and manufacture of matrices and punches

The design of the micro devices was developed by means of standard CAD software (AutoCAD, Autodesk Inc., USA) . Transparency masks were printed at high resolution (64000 dpi) and used as photomasks for manufacturing matrices and punches by means of photolithography SU-8 (SU8-2100, SU8-2050, MicroChem, USA) . The height features were set as follows: 200 μπι for the upper culture channels (cavity 5 in the matrix 2) of all the micro device configurations and for the reliefs of the punch (19) or of the intermediate mould (3) of the devices with a vertical interface (Figure 3) and of the devices with side endothelium (Figure 6); 100 μπι for the reliefs of the punch (19) or of the intermediate mould (3), for the other micro device configurations (Figure 4, 5) . The designs of the punch or of the intermediate mould were developed to obtain relief features slightly smaller than the upper culture channels (cavity 5) . To this end, a 10 micron side distance was employed between the features of the intermediate mould and the upper culture channels, a distance that does not release the hydrogel and does not alter the injection. Figures 7A and 7B highlight the side gaps for two layouts of representative devices .

PDMS moulds are manufactured by means of PDMS replication molding on support moulds (Sylgard 184, Dow Corning, Germany. Mixing ratio 10:1) . Inlet doors (10) and outlet doors (11) of the cavity (5) were created with a 1 mm punching, inlet (15) and outlet (16) of the side channels (6) were created with a 5 mm punching by means of holes on the matrix.

Example 2: cell extraction and culture

Primary human BM-MSC cells were isolated from bone marrow aspirates obtained from patients during routine orthopaedic surgical procedures, after obtaining written informed consent. The cells were expanded and cultivated in a 37 °C humidified incubator with 5% CO2 at all stages. The culture medium employed had the following composition: Eagle alpha-modified medium supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin and 300 g/ml L-glutamine ( ThermoFisher, Italy) , supplemented with 5 ng/ml fibroblast-2 growth factor (Peprotech, UK) . At the sixth passage, two cell populations were incubated with two different Vybrant labelling solutions (ThermoFisher, Italy. DiO and Dil dyes) and then detached from the culture plates for seeding experiments.

GFP expressing HUVECs were purchased from Lonza and cultivated in EGM-2 Bullet-kit medium (Lonza, Italy) . The cells were used for experiments at the third passage.

Example 3: assembly of the micro device and seeding procedure

Matrices and intermediate PDMS moulds were sterilized by UV irradiation. The intermediate moulds were coated with 3% bovine serum albumin (Sigma, Italy) , to prevent the adhesion of the hydrogel, and then carefully aligned and assembled with the matrices. Fibrin gels were obtained by mixing fibrinogen and thrombin solutions (Sigma, Italy) to obtain the following final concentrations: 20 mg/ml fibrinogen, 2U/ml thrombin, 10⁷ cells/ml. Type I rat tail collagen (Sigma, Italy) was used for preparing collagen gels with final concentrations of 3 mg/ml of collagen neutralized with 1M NaOH at pH 7.2-7.4 and incorporated with 10⁷ cells/ml. Cell-loaded hydrogels were injected through the inlet doors (10) in said matrices (2) assembled with said intermediate moulds (3) and cross-linking was performed in humidified chambers placed in standard incubators for 3' (fibrin gel) or 30' (collagen gel) . Following the reticulation, the intermediate moulds were carefully detached from the matrices, and the matrices were quickly brought into contact with a sterile closing element and a second hydrogel was injected, incorporated with cells labelled with a different dye. Following the reticulation, the culture medium was injected into the media channels. For the endothelialisation experiments, GFP-HUVECs were suspended in EGM-2 medium at a concentration of 5x10^s cells/ml. Following the assembly of the matrices with the closing elements, the cell solution is directly injected into the perfusion channel (Figure 5) or pipetted into the pockets of the media channels (layout 6) pre^¬ loaded with 30 μΐ of EGM-2 medium.

Example 4: Image acquisition

Micro devices were set for 20' with 4% paraformaldehyde immediately after the seeding (Figure 3, 4) and after 3 days of culture (Figure 3, 4, 5, 6) . Image acquisition was performed with an Olympus FluoView FVlOi confocal microscope. The images were obtained at a lOx or 63x magnification, with an approximate resolution of the z-axis of 2 μπι and 0.6μπι, respectively. Image processing and 3D reconstructions were performed using the ImageJ software (NIH, USA) .

Claims

1. A kit (1) for obtaining 3D cellular constructs in micrometric scale, wherein said kit (1) comprises a matrix (2) and a punch (19), where said matrix (2) comprises at least one cavity (5), at least one inlet door (10) and/or at least one outlet door (11) and said punch (19) at least partially penetrates said cavity (5), leaving a first empty volume (12) .

2. The kit according to claim 1, wherein said punch (19) is made up of an intermediate mould (3) and a closing element (4) .

3. The kit according to one of claims 1 to 2, wherein the smaller dimension of said cavity (5), typically represented by its height, is comprised between Ιμπι and 10mm, preferably comprised between ΙΟμπι and 1mm, even more preferably it is about 200μπι.

4. The kit according to one of claims 1 to 3, wherein said matrix (2) also comprises at least two side channels (6) facing said cavity (5) and open towards the outside of said matrix with an inlet (15) and an outlet (16), characterised in that said side channels run alongside a part or the entire length of said cavity (5), one channel per side, and are on it.

5. A method for obtaining 3D cellular constructs wherein said method comprises:

a) Providing a kit according to one of claims 1 to 4 ;

b) Providing primary, immortalised and/or tumour cells in culture ;

c) Providing at least one fluid that creates a semi-solid matrix, wherein said fluid optionally comprises one or more of said cell populations;

d) Assembling said matrix (2) with said punch (19), wherein said assembly leaves a first empty volume (12) in said cavity (5) comprised in said matrix (2);

e) Injecting said fluid through said at least one inlet door

(10) to occupy said first empty volume (12) between said matrix (2) and said punch (19); f ) Solidifying said fluid;

i ) Optionally, solidifying said further fluid.

6. A method for preparing micro devices (30) ready for use for obtaining 3D cellular constructs wherein said method comprises :

a) Providing a kit according to one of claims 1 to 4, wherein said punch (19) is made up of an intermediate mould (3) and a closing element (4);

b) Providing at least one fluid that, by solidifying, creates a sacrificial material;

c) Assembling said matrix (2) with said intermediate mould (3) creating said first empty volume (12);

d) Injecting said fluid through said at least one inlet door

(10) to occupy said first empty volume (12);

e) Solidifying said fluid;

f) Extracting said intermediate mould (3) and replacing it with said closing element (4) .

7. A micro device, preferably sterile, ready for use for obtaining 3D cellular constructs that comprises:

-a matrix (2) that comprises a cavity (5) open towards the outside with an inlet door (10) and an outlet door (11);

-a closing element (4);

-sacrificial material,

wherein said matrix is assembled with said closing element and said cavity (5) is made up of a first empty volume (12) and a second empty volume (13) and said sacrificial material occupies said first empty volume (12) in said cavity (5) .

8. A method for obtaining 3D cellular constructs in micrometric channels that comprises:

a) Providing a micro device (30) ready for use according to claim 7 ;

b) Injecting, through said inlet door (10), a fluid capable of creating a semi-solid matrix, optionally loaded with cells, in said second empty volume (13) in said micro device ( 30 ) ;

c) Solidifying said fluid;

d) Eliminating said sacrificial material from the micro device through said outlet door (11) thus freeing said first empty volume (12);

e) Injecting a further fluid, optionally loaded with cells, optionally capable of solidifying, through said inlet door (10) to occupy said first empty volume (12) thus obtaining a complex 3D cellular construct.

9. The method according to one of claims 5, 6 or 8 wherein said fluid that is capable of creating a semi-solid matrix is of natural or synthetic origin, or a combination thereof, and is preferably selected in the group comprising: polyethylene glycol solutions, fibrinogen solutions, thrombin solutions, collagen solutions, hyaluronic acid solutions, elastin solutions, fibroin, agarose, chitosan, alginate solutions, and similar, and mixtures thereof, even more preferably it is a solution that comprises about 20 mg/ml of fibrinogen and about 2 U/ml thrombin.

10. The method according to one of claims 5 or 8, wherein said cell population is selected in the group that comprises stem or somatic cells, or a combination thereof, wherein said stem cells are selected in the group that comprises embryonic and adult stem cells, preferably pluripotent cells and induced pluripotent cells, even more preferably obtained from placenta, umbilical cord, adipose tissue, nerve tissue, muscle tissue, heart tissue, parenchyma tissues, epidermal tissue, and bone marrow, and combinations thereof; and said somatic cells are selected in the group that comprises cells derived from mesoderm, endoderm and ectoderm, preferably endothelial cells, bone cells, cartilage cells, nerve cells, adipose cells, epithelial cells, fibroblasts, myofibroblasts, interstitial cells, hepatocytes, pancreatic cells, blood cells, muscle cells, and combinations thereof.

11. A three-dimensional construct of cells obtained according to the method in accordance with one of claims 6 or 8-10.