Cell culture system

Abstract

The present invention pertains to the field of tissue or cell culture device Especially, the invention relates to a cell culture patch comprising pores and its use. The present invention also relates to a method for manufacturing said cell culture patch, a method of cell growth and cell differentiation using said cell culture patch, and a cell culture system which may include said cell culture patch.

Description

[0001] CELL CULTURE SYSTEM

[0002] FIELD OF THE INVENTION

[0003] The present invention pertains to the field of tissue or cell culture device. Especially, the invention relates to a cell culture patch comprising pores and its use. The present invention also relates to a method for manufacturing said cell culture patch, a method of cell growth and cell differentiation using said cell culture patch, and a cell culture system which may include said cell culture patch.

[0004] PRIOR ART

[0005] The considerable development of organ-on-a-chip technologies generates the need to vascularise tissue models, which remains a challenge. Although the microfabrication of channels several hundred pms in diameter is possible for perfusion, for example in skin equivalents [Abaci et al. Lab Chip 2015 15, 882; Wufuer et al. SciRep 2016 6, 37471; Kwak et al. Biotechnol Bioeng 2020 117, 1853], the distribution and diameters of these artificial vessels are not physiologically relevant by an order of magnitude. Another approach is to rely on the self-assembly of endothelial cells to generate microvascular networks, but these lack hierarchical organisation, and perfusion remains largely unfeasible [Jones et al. Front BioengBiotechnol 2022 10, 915702], Indeed, the main problem remains connecting the microfabricated channels to the microvascular networks for continuous perfusion. This technical challenge has been met by dedicated microfluidic vascular chips, which do not yet provide adequate flow control [Van Duinen et al. Angiogenesis 2019 22, 157], These chips are commercially available (Mimetas), but they do not allow flow control or the creation of sprouting models, and do not allow vessels to be studied [Van Duinen et al. IntJMol Sci. 202021, 4804],

[0006] In addition, there is currently no standardised device for independently monitoring the key stages of angiogenesis: sprouting and anastomosis. The absence of a controlled standardised device prevents the quantification of these vascularisation processes in physiological contexts such as development, but also including pathological contexts such as cardiovascular disease. In addition, given the impact of shear forces associated with flow in vascular physiological contexts, it is necessary to be able to incorporate controlled flow into these devices.

[0007] BRIEF DESCRIPTION

[0008] In order to overcome these drawbacks, the inventors have conceived a new 3D cell culture patch (1) for the 3D quantification of angiogenesis that enables both precise engineering and control of the microvascular network, and thus offers unprecedented flexibility, resulting in unique features that extend its use to other fields. A first subject matter of the invention is therefore a cell culture patch (1) and its use. A second subject matter of the invention concerns processes of manufacture of the cell culture patch (1) according to the invention. Further subject matters of the invention relate to an apparatus (6) for handling a cell culture device, such as the cell culture patch (1) according to the invention, and processes of manufacture of the apparatus (6) according to the invention. Another subject matter of the invention concerns the processes of seeding the cell culture patch (1) according to the invention using, in particular, the apparatus (6) according to the invention.

[0009] DETAILED DESCRIPTION

[0010] According to a first aspect and as illustrated on Figures 1 and 7, a subject matter of the invention relates to a cell culture patch (1) having an upward opening comprising a compartment unit for cell culture cut into the thickness of said cell culture patch (1) wherein said compartment unit comprises: a lower case including a bottom surface (3) of at least 28,27 mm2and a sidewall (2) vertically extending from 100 pm to 500 pm upward from an edge of the bottom surface (3) which corresponds to the lower part of said cell culture patch (1); said bottom surface (3) being perforated by pores (4), said pores (4): being arranged so as to pass through the thickness of said bottom surface (3) ( / .e. the thickness of said cell culture patch (1)); having a symmetrical shape and / or an asymmetrical shape, the surface of which is comprised from 1 256.6 pm2to 7 853.98 pm2; being spaced apart by a distance comprised from 200 pm to 1 000 pm; and having a density comprised from 50 pores / cm2to 250 pores / cm2.

[0011] Described more simply, the cell culture patch (1) of the invention (see e.g. Figures 1 and 7) corresponds to a disc-shaped patch (Figure 1) or a rectangular-shaped patch (Figure 7), the rims of which form a ring or a rectangle, the width of which makes it possible to define a compartment unit for cell culture. Said compartment unit for cell culture is open to the outside at its upper extremity and is "closed" at its lower extremity by the cell culture patch (1) itself. The term "closed" is used here in inverted commas because the base of the compartment unit (or the base of the cell culture patch (1) defining the base of the compartment unit) is perforated by symmetric and / or asymmetric pores (4).

[0012] By "a lower case including a bottom surface (3) of at least 28,27 mm2", it means that the surface of said bottom surface (3) is comprised from 28,27 mm2to 150 mm2, from 28,27 mm2to 125 mm2, from 28,27 mm2to 100 mm2, from 28,27 mm2to 75 mm2, from 30 mm2to 150 mm2, from 55 mm2to 150 mm2, from 80 mm2to 150 mm2, from 105 mm2to 150 mm2, from 130 mm2to 150 mm2or from 55 mm2to 100 mm2. It also means that the surface of said bottom surface (3) is of at least 30 mm2, at least 40 mm2, at least 50 mm2, at least 60 mm2, at least 70 mm2, at least 80 mm2, at least 90 mm2, at least 100 mm2, at least 110 mm2, at least 120 mm2, at least 130 mm2or at least 140 mm2. In particular, the surface of said bottom surface (3) is of 63.62 mm2. According to another embodiment, a subject matter of the invention thus concerns the cell culture patch (1) as described above, wherein said bottom surface (3) is of 63.62 mm2or 75 mm2.

[0013] By "a sidewall (2) vertically extending from 100 pm to 500 pm upward from an edge of the bottom surface (3)", it means that the height of said sidewall (2) is comprised from 100 pm to 500 pm, from 150 pm to 500 pm, from 200 pm to 500 pm, from 250 pm to 500 pm, from 300 pm to 500 pm, from 350 pm to 500 pm, from 400 pm to 500 pm, from 450 pm to 500 pm, from 100 pm to 450 pm, from 100 pm to 400 pm, from 100 pm to 350 pm from 100 pm to 300 pm from 100 pm to 250 pm from 100 pm to 200 pm from 100 pm to 150 pm, from 100 pm to 300 pm, from 200 pm to 400 pm or from 300 pm to 500 pm. It means that the height of said sidewall (2) is of 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm or 500 pm. In particular, the height of said sidewall (2) is of 300 pm.

[0014] According to another embodiment, a subject matter of the invention thus concerns the cell culture patch (1) as described above, wherein said sidewall (2) vertically extends 300 pm upward from an edge of the bottom surface (3).

[0015] By "having a symmetrical shape and / or an asymmetrical shape, the surface of which is comprised from 1 256.6 pm2to 7 853.98 pm2", it means that the surface of said pores (4) is comprised from 1 256.6 pm2to 7 853.98 pm2, from 1 256.6 pm2to 7 000 pm2, from 1 256.6 pm2to 6 000 pm2, from 1 256.6 pm2to 5 000 pm2, from 1 256.6 pm2to 4 000 pm2, from 1 256.6 pm2to 3 000 pm2, from 1 256.6 pm2to 2 000 pm2, from 2 000 pm2to 7 853.98 pm2, from 3 000 pm2to 7 853.98 pm2, from 4 000 pm2to 7 853.98 pm2, from 5 000 pm2to 7 853.98 pm2, from 6 000 pm2to 7 853.98 pm2, from 7 000 pm2to 7 853.98 pm2, from 2 000 pm2to 7 000 pm2, from 2 000 pm2to 4 000 pm2, from 3 000 pm2to 5 000 pm2or , from 1 500 pm2to 3 000 pm2. It also means that the surface of said pores (4) is of 1 256.6 pm2, 1 500 pm2, 2 000 pm2, 2 500 pm2, 3 000 pm2, 3 500 pm2, 4 000 pm2, 4 500 pm2, 5 000 pm2, 5 500 pm2, 6 000 pm2, 6 500 pm2, 7 000 pm2, 7 500 pm2or 7 853.98 pm2. In particular, the surface of said pores (4) is of 1 963.5 pm2.

[0016] According to another embodiment, a subject matter of the invention thus concerns the cell culture patch (1) as described above, wherein the surface of said pores (4) is of 1 963.5 pm2.

[0017] By "being spaced apart by a distance comprised from 200 pm to 1 000 pm", it means that the distance between each pore (4) is comprised from 200 pm to 1 000 pm, from 200 pm to 900 pm, from 200 pm to 800 pm, from 200 pm to 700 pm, from 200 pm to 600 pm, from 200 pm to 500 pm, from 200 pm to 400 pm, from 200 pm to 300 pm, from 200 pm to 1 000 pm, from 300 pm to 1 000 pm, from 400 pm to 1 000 pm, from 500 pm to 1 000 pm, from 600 pm to 1 000 pm, from 700 pm to 1 000 pm, from 800 pm to 1 000 pm, from 900 pm to 1 000 pm, from 400 pm to 800 pm or from 300 pm to 600 pm. It also means that the distance between each pore (4) is of 200 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm or 1 000 pm. In particular, the distance between each pore (4) is comprised from 300 pm to 600 pm.

[0018] According to another embodiment, a subject matter of the invention thus concerns the cell culture patch (1) as described above, wherein the distance between each pore (4) is comprised from 300 pm to 600 pm.

[0019] By "having a density comprised from 50 pores / cm2to 250 pores / cm2", it means that the density of said pores (4) on said bottom surface (3) is comprised from 50 pores / cm2to 250 pores / cm2, from 50 pores / cm2to 200 pores / cm2, from 50 pores / cm2to 150 pores / cm2, from 50 pores / cm2to 100 pores / cm2, from 100 pores / cm2to 250 pores / cm2, from 150 pores / cm2to 250 pores / cm2, from 200 pores / cm2to 250 pores / cm2, from 150 pores / cm2to 200 pores / cm2or from 100 pores / cm2to 200 pores / cm2. It also means that the density of said pores (4) on said bottom surface (3) is of 50 pores / cm2, 100 pores / cm2, 150 pores / cm2, 200 pores / cm2or 250 pores / cm2. In particular, the density of said pores (4) on said bottom surface (3) is of 150 pores / cm2.

[0020] According to another embodiment, a subject matter of the invention thus concerns the cell culture patch (1) as described above, wherein said pores (4) have a density of 150 pores / cm2.

[0021] According to another embodiment, a subject matter of the invention concerns the cell culture patch (1) as described above, wherein said bottom surface (3) which corresponds to the lower part of said cell culture patch (1) has a thickness of 50 pm (Figure 7).

[0022] According to another embodiment, a subject matter of the invention concerns the cell culture patch (1) as described above, said cell culture patch (1) being in the form of a dish shape (Figure 1) or in the form of a rectangle shape (Figure 7).

[0023] According to another embodiment and as illustrated on Figure 1, a subject matter of the invention relates to a cell culture patch (1) as described above, in the form of a dish shape, having an upward opening comprising a compartment unit for cell culture cut into the thickness of said cell culture patch (1) wherein said compartment unit comprises: a lower case including a bottom surface (3) of at least 28,27 mm2and a sidewall (2) vertically extending from 100 pm to 500 pm upward from an edge of the bottom surface (3) which corresponds to the lower part of said cell culture patch (1); said bottom surface (3) being perforated by pores (4), said pores (4): being arranged so as to pass through the thickness of said bottom surface (3) ( / .e. the thickness of said cell culture patch (1)); having a symmetrical shape and / or an asymmetrical shape, the surface of which is comprised from 1 256.6 pm2to 7 853.98 pm2; being spaced apart by a distance comprised from 200 pm to 1 000 pm; and having a density comprised from 50 pores / cm2to 250 pores / cm2.

[0024] According to another embodiment, a subject matter of the invention relates to the cell culture patch (1) as described above, wherein: said cell culture patch (1) is in the form of a disc, the diameter of which being in particular of 13 mm; said compartment unit is in the form of a disc, the diameter of which being in particular of 9 mm; and said sidewall (2) is in the form of a ring, the width of which being in particular of 2 mm.

[0025] According to another embodiment, a subject matter of the invention relates to the cell culture patch (1) as described above, wherein: said cell culture patch (1) is in the form of a disc, the diameter of which being of 13 mm; said compartment unit is in the form of a disc, the diameter of which being of 9 mm; and said sidewall (2) is in the form of a ring, the thickness of which being of 2 mm.

[0026] According to another embodiment and as illustrated on Figure 7, a subject matter of the invention relates to a cell culture patch (1) as described above, in the form of a rectangular shape, having an upward opening comprising a compartment unit for cell culture cut into the thickness of said cell culture patch (1) wherein said compartment unit comprises: a lower case including a bottom surface (3) of at least 28,27 mm2and a sidewall (2) vertically extending from 100 pm to 500 pm upward from an edge of the bottom surface (3) which corresponds to the lower part of said cell culture patch (1); said bottom surface (3) being perforated by pores (4), said pores (4): being arranged so as to pass through the thickness of said bottom surface (3) ( / .e. the thickness of said cell culture patch (1)); having a symmetrical shape and / or an asymmetrical shape, the surface of which is comprised from 1 256.6 pm2to 7 853.98 pm2; being spaced apart by a distance comprised from 200 pm to 1 000 pm; and having a density comprised from 50 pores / cm2to 250 pores / cm2.

[0027] According to another embodiment, a subject matter of the invention relates to the cell culture patch (1) as described above, wherein: said cell culture patch (1) is in the form of a rectangle, the length of which being in particular of 19 mm and the width of which being in particular of 9 mm; said compartment unit is in the form of a rectangle, the length of which being in particular of 15.0 mm and the width of which being in particular of 5.0 mm; and said sidewall (2) is in the form of a rectangle, the width of which being in particular of 2 mm.

[0028] According to another embodiment, a subject matter of the invention relates to the cell culture patch (1) as described above, wherein: said cell culture patch (1) is in the form of a rectangle, the length of which being of 19 mm and the width of which being of 9 mm; said compartment unit is in the form of a rectangle, the length of which being of 15.0 mm and the width of which being of 5.0 mm; and said sidewall (2) is in the form of a rectangle, the width of which being of 2 mm.

[0029] According to another embodiment, a subject matter of the invention relates to the cell culture patch (1) as described above, wherein said pores (4) have:

[0030] - a symmetric center, such as a hexagon or a circle (see Figure 2); or

[0031] - a symmetric axis, such as an arrow (see Figure 2).

[0032] A pore has a symmetric center if its image by central symmetry of its centre is the image itself. Such examples are: parallelogram, rhombus, rectangle, square, hexagon or circle. According to another embodiment, a subject matter of the invention thus concerns the cell culture patch (1) as described above, wherein said pores (4) have a symmetric center and the shape of which is selected from: parallelogram, rhombus, rectangle, square, hexagon and circle. In particular, a subject matter of the invention concerns the cell culture patch (1) as described above, wherein said pores (4) have a symmetric center and the shape of which is a hexagon or a circle.

[0033] A pore has a symmetric axis when the pore is divided by a straight line into two superimposable parts. Such examples are: drop (an angle at one end and a semi-circle at the other), trapeze or arrow. According to another embodiment, a subject matter of the invention thus concerns the cell culture patch (1) as described above, wherein said pores (4) have a symmetric axis and the shape of which is selected from: drop (an angle at one end and a semi-circle at the other), trapeze and arrow. In particular, a subject matter of the invention concerns the cell culture patch (1) as described above, wherein said pores (4) have a symmetric axis and the shape of which is an arrow. Interestingly, a subject matter of the invention concerns the cell culture patch (1) as described above, wherein certain pores (4) have a symmetric center and the shape of which is selected from: parallelogram, rhombus, rectangle, square, hexagon and circle; and other pores (4) have a symmetric axis and the shape of which is selected from: drop (an angle at one end and a semi-circle at the other), trapeze and arrow.

[0034] According to another embodiment and as illustrated on Figure 2, a subject matter of the invention relates to the cell culture patch (1) as described above, wherein said pores (4) are capable of being arranged so as to define at least two repeating patterns (5), each pattern (5) being spaced from the others by a distance comprised from 600 pm to 1000 pm.

[0035] By "said pores (4) are capable of being arranged so as to define at least two repeating patterns (5)", it means that a set of pores ( / .e. at least 2 pores) defines a pattern that is (symmetrically) repeated over the bottom surface (3). As illustrated, said pattern may be a line, a column or a square.

[0036] By "each pattern (5) being spaced from the others by a distance comprised from 600 pm to 1 000 pm", it means that the distance between each pattern is comprised from 600 pm to 1000 pm, from 600 pm to 900 pm, from 600 pm to 800 pm, from 600 pm to 700 pm, from 700 pm to 1000 pm, from 800 pm to 1 000 pm, from 900 pm to 1000 pm or from 700 pm to 900 pm. It also means that the distance between each pattern is of 600 pm, 700 pm, 800 pm, 900 pm or 1 000 pm.

[0037] According to another embodiment, a subject matter of the invention relates to the cell culture patch (1) as described above, said cell culture patch (1) being made with a polymer material, said material having at least the following characteristic:

[0038] - microresistant to visible and near-UV light, water or any polar or apolar solvent, at low and high temperatures;

[0039] - transparent;

[0040] - non-porous;

[0041] - non-rough / smooth; and

[0042] - biocompatible, said cell culture patch (1) being in particular made from Polyethylene Glycol Diacrylate (PEGDA) or a biocompatible photo-crosslinkable polymer resin, said biocompatible photo-crosslinkable polymer resin being in particular OrmoStamp® (UV curable resin from micro resist technology GmbH, Berlin).

[0043] According to another embodiment, a subject matter of the invention concerns the cell culture patch

[0044] (1) as described above, wherein said cell culture patch (1) is made from PEGDA or a biocompatible photo-crosslinkable polymer resin, said biocompatible photo-crosslinkable polymer resin being in particular OrmoStamp® (UV curable resin from micro resist technology GmbH, Berlin).

[0045] According to another embodiment, a subject matter of the invention concerns the cell culture patch (1) as described above, wherein said cell culture patch (1) is made from PEGDA or OrmoStamp® (UV curable resin from micro resist technology GmbH, Berlin).

[0046] According to another embodiment, a subject matter of the invention relates to the cell culture patch (1) as described above, wherein the backside of said bottom surface (3) ( / .e. the outside of said compartment unit for cell culture) comprises:

[0047] - a first coating with a 10 nm thick conductive and biocompatible material; and

[0048] - optionally a second coating with nanofibers having a diameter comprised from 350 nm to 550 nm, said nanofibers being in particular gelatin nanofibers.

[0049] According to another embodiment, a subject matter of the invention concerns the cell culture patch (1) as described above, wherein the backside of said bottom surface (3) comprises:

[0050] - a first coating with a 10 nm thick conductive and biocompatible material; and

[0051] - a second coating with nanofibers having a diameter comprised from 350 nm to 550 nm, said nanofibers being in particular gelatin nanofibers.

[0052] According to another embodiment, a subject matter of the invention concerns the cell culture patch (1) as described above, wherein the conductive and biocompatible material is gold.

[0053] By "nanofibers having a diameter comprised from 350 nm to 550 nm", it means that the diameter of said nanofibers is comprised from 350 nm to 550 nm, from 400 nm to 550 nm, from 450 nm to 550 nm, from 500 nm to 550 nm, from 350 nm to 500 nm, from 350 nm to 450 nm, from 350 nm to 400 nm or from 400 nm to 500 nm. It also means that the diameter of said nanofibers is at least of 350 nm, at least of 400 nm, at least of 450 nm or at least of 500 nm. In particular, the diameter of said nanofibers is 448 ± 89 nm.

[0054] According to another embodiment, a subject matter of the invention thus concerns the cell culture patch (1) as described above, wherein said nanofibers have a diameter of 448 ± 89 nm ( / .e. from 359 nm to 537 nm).

[0055] According to another embodiment, a subject matter of the invention concerns the cell culture patch (1) as described above, wherein said nanofibers are gelatin nanofibers. According to another embodiment, a subject matter of the invention relates to the cell culture patch (1) as described above, wherein said cell culture patch (1) comprises a coating with polydopamine (PDA).

[0056] According to another embodiment and as illustrated on Figure 5, a subject matter of the invention relates to the cell culture patch (1) as described above, wherein said backside of said bottom surface (3) is seeded with "patch-cells", in particular primary endothelial cells, iPS-derived cells or circulating progenitor cells, and the inside of said compartment unit is filled with hydrogel, said hydrogel be liable to contain cells, hormones, growth factors, drugs.

[0057] “Patch-cells" refers to eucaryotic cells which are seeded on the backside of said bottom surface (3), which corresponds to the backside of the cell culture patch (1) of the invention. This particular expression "patch-cells" is used here to differentiate the cells seeded under the cell culture patch (1) from those which may be present in the hydrogel.

[0058] “Patch-cells" refers in particular to primary endothelial cells, iPS-derived cells or circulating progenitor cells. “Patch-cells" also refers in particular to human eukaryotic cells. According to another embodiment, a subject matter of the invention thus concerns the cell culture patch (1) as described above, wherein said "patch-cells" are selected from: human primary endothelial cells, human iPS- derived cells and human circulating progenitor cells.

[0059] "Hydrogel" refers to a non-fluid polymer network that is expanded throughout its whole volume by water. As previously mentioned, this hydrogel may contain cells, hormones, growth factors, drugs. According to another embodiment, a subject matter of the invention thus concerns the cell culture patch (1) as described above, wherein said hydrogel contains (eukaryotic) cells, hormones, growth factors and / or drugs.

[0060] According to another embodiment, a subject matter of the invention relates to the cell culture patch (1) as described above, wherein said backside of said bottom surface (3) is seeded at confluency with a density comprised from lxlO4cells per cm2to 3xl06cells per cm2.

[0061] By "density comprised from lxlO4cells per cm2to 3xl06cells per cm2", it means that said "patch-cells" are seeded with a density comprised from lxlO4cells per cm2to 3xl06cells per cm2, from 5xio4cells per cm2to 3xl06cells per cm2, from lxlO5cells per cm2to 3x10scells per cm2, from 5xl05cells per cm2to 3x10scells per cm2, from 1x10scells per cm2to 3x10scells per cm2, from 2x10scells per cm2to 3x10scells per cm2, from lxlO4cells per cm2to 2x10scells per cm2, from lxlO4cells per cm2to 1x10s cells per cm2, from lxlO4cells per cm2to 5xl05cells per cm2, from lxlO4cells per cm2to lxlO5cells per cm2, from lxlO4cells per cm2to 5xl04cells per cm2, from lxlO5cells per cm2to lxlO6cells per cm2, from 5xl05cells per cm2to 2xio6cells per cm2or from 1.5xl04cells per cm2to 5xio5cells per cm2. It also means that said "patch-cells" are seeded with a density of lxlO4cells per cm2, 1.5xl04cells per cm2, 5xl04cells per cm2, lxlO5cells per cm2, 5xl05cells per cm2, 1x10scells per cm2, 2x10scells per cm2or 3x10scells per cm2. In particular, said "patch-cells" are seeded with a density of lxlO5cells per cm2.

[0062] According to another embodiment, a subject matter of the invention thus concerns the cell culture patch (1) as described above, wherein said backside of said bottom surface (3) is seeded at confluency with a density comprised from 1.5xl04cells per cm2to 5xl05cells per cm2.

[0063] According to a second aspect, a subject matter of the invention relates to use of a cell culture patch (1) as described above for implementing cellular biology analysis methods, in particular for analysing angiogenesis mechanisms, said methods being optionally performed in a microfluidic array (10) suitable for receiving the cell culture patch (1), said microfluidic array (10) being in particular perfused so that:

[0064] - said backside of said bottom surface (3) is fed with first medium culture; and

[0065] - said inside of said compartment unit is fed with second medium culture which can be identical or different to the first medium culture, in particular said second medium culture being different to the first medium culture.

[0066] According to another embodiment, a subject matter of the invention concerns the use of a cell culture patch (1) as described above for analysing angiogenesis mechanisms. Indeed, an important field of application is the 3D quantification of angiogenesis under static and perfusion conditions, but not only. The cell culture patch (1) of the invention also provides a unique and versatile platform for drug screening in vascular models, enabling:

[0067] - quantification of the impact of secreted factors

[0068] - quantification of the impact of mechanical stresses

[0069] - quantification of the impact of flow-induced shear stress.

[0070] The cell culture patch (1) of the invention can be used for applications in personalized medicine. This includes the use of endothelial cells from patients with vascular diseases, as well as the exposure of healthy endothelial cells to patient serum. In this way, pathologies affecting vascular flow and integrity can be studied: cancer, ischemic cardiovascular disease, inflammatory cell trafficking or pathogens specific to the human endothelium. The fields of application also extend to the development of invasion assays, including cancer cells and metastases. To this end, the same technical specifications are used for cell seeding and the formation of a 3D tissue model by replacing endothelial cells with cancer cells, first of all lineage cells and then patient-derived cells.

[0071] "Microfluidic array (10)" is illustrated on Figure 4 and may be manufactured by MesoBioTech® (mesobiotech.com). Microfluidic arrays (10) are formed with two plastic plates, each having an elastomeric thin film to ensure good sealing when clamped. They are typically used for patch integration, such as the cell culture patch (1) of the invention, for which micro-chambers and microchannels are predefined in both plastic plates. Additionally, inlet and outlet chambers may be patterned in both plates. Thus when a patch, such as the cell culture patch (1) of the invention, is placed between the two plates, culture media containing different factors can flow in the upper and lower chambers separated by the patch. Efficient sealing is achievable with magnetic bonding thanks to small magnets at each of the four corners of the two plastic plates and a loose-leaf clamp or screw enhanced assembling. After fluidic processing with the array, the integrated patch can be released for other operations.

[0072] According to another embodiment, a subject matter of the invention concerns the use of a cell culture patch (1) as described above, wherein said methods are performed in a microfluidic array (10) suitable for receiving the cell culture patch (1).

[0073] According to another embodiment, a subject matter of the invention concerns the use of a cell culture patch (1) as described above, wherein said microfluidic array (10) is perfused so that:

[0074] - said backside of said bottom surface (3) is fed with first medium culture; and

[0075] - said inside of said compartment unit is fed with second medium culture which can be identical to the first medium culture.

[0076] According to another embodiment, a subject matter of the invention concerns the use of a cell culture patch (1) as described above, wherein said microfluidic array (10) is perfused so that:

[0077] - said backside of said bottom surface (3) is fed with first medium culture; and

[0078] - said inside of said compartment unit is fed with second medium culture which can be different to the first medium culture.

[0079] According to a third aspect, a subject matter of the invention relates to a method of manufacture of a cell culture patch (1) as described above comprising at least the following steps of: 1. dropping a polymers solution in the center of a mold, the print of which has the form of said cell culture patch (1), to obtain a mold coated with said polymers solution;

[0080] 2. exposing to UV light, in particular at 9.1 mW / cm2for 85 s, said mold coated with said polymers solution to cure polymers and to obtain a mold coated with a cured polymer; and

[0081] 3. taking off said cured polymers from said mold to obtain said cell culture patch (1), wherein said polymers have at least the following characteristic:

[0082] - microresistant to visible and near-UV light, water or any polar or apolar solvent, at low and high temperatures;

[0083] - transparent;

[0084] - non-porous;

[0085] - non-rough / smooth; and

[0086] - biocompatible, said polymers being in particular PEGDA or a biocompatible photo-crosslinkable polymer resin, said biocompatible photo-crosslinkable polymer resin being in particular OrmoStamp® (UV curable resin from micro resist technology GmbH, Berlin), and wherein said mold is made in particular from PDMS.

[0087] According to another embodiment, a subject matter of the invention concerns the method of manufacture as described above of a cell culture patch (1) of the invention, wherein the step 2. of UV light exposing is performed at 9.1 mW / cm2for 85 s.

[0088] According to another embodiment, a subject matter of the invention concerns the method of manufacture as described above of a cell culture patch (1) of the invention, wherein said polymers is PEGDA or a biocompatible photo-crosslinkable polymer resin, said biocompatible photo-crosslinkable polymer resin being in particular OrmoStamp® (UV curable resin from micro resist technology GmbH, Berlin).

[0089] According to another embodiment, a subject matter of the invention concerns the method of manufacture as described above of a cell culture patch (1) of the invention, wherein said polymers is PEGDA or OrmoStamp® (UV curable resin from micro resist technology GmbH, Berlin).

[0090] According to another embodiment, a subject matter of the invention concerns the method of manufacture as described above of a cell culture patch (1) of the invention, wherein said mold is made from PDMS. According to another embodiment, a subject matter of the invention concerns the method of manufacture as described above of a cell culture patch (1) of the invention, which further comprises a preliminary mold manufacturing step.

[0091] According to another embodiment, a subject matter of the invention concerns the method of manufacture as described above of a cell culture patch (1) of the invention, which further comprises a preparation step of said cell culture patch (1) wherein at least the following steps are implemented:

[0092] 1. firstly coating the backside of the bottom surface (3) with a 10 nm thick conductive and biocompatible material;

[0093] 2. secondly coating said backside of the bottom surface (3) with nanofibers having a diameter comprised from 350 nm to 550 nm; and

[0094] 3. coating said cell culture patch (1) with polydopamine (PDA).

[0095] For instance, and in details the method of manufacture as described above of a cell culture patch (1) of the invention is as follows:

[0096] The cell culture patch (1) of the invention frame made of PEGDA or a biocompatible photo- crosslinkable polymer resin, said biocompatible photo-crosslinkable polymer resin being in particular OrmoStamp® (UV curable resin from micro resist technology GmbH, Berlin), is fabricated by UV assisted molding using a polydimethylsiloxane (PDMS) mold. First, two chromium masks are produced using a micro-pattern generator (pPG 101, Heidelberg Instruments, Germany). The first one is the pore pattern with the desired shape / size / interdistance / distribution of the pores. The second one is the external ring of the cell culture patch (1) of the invention. The two photomasks are used to replicate the desired pattern by photolithography on a silicon wafer with the desired polymer thickness / wall size obtained by dry film photoresist-based microfabrication (lamination).

[0097] For that, a commercial laminator is used to laminate SUEX dry film sheets. SUEX dry films with 50 to 500 pm thickness covered with two polyester (PET) films are used. The PET sheet of one side is removed before laminating the photoresist against the substrate. The lamination temperature of the roller depends on the thickness of the dry film sheet. The second PET sheet kept during lamination is removed before exposing the laminated layer. Laminator temperature and speed are 100°C and 0.2 m / min, respectively for the 50 pm SUEX sheet. The laminated sheet is exposed to UV with the first mask of the inner pattern of the cell culture patch (1) of the invention. The exposure energy of 95 mJ / cm2is used to crosslink the 50 pm thick SUEX film. Then, a post exposure bake at 95°C for 5 min is carried out on a hot plate to complete the crosslinking and ensure proper adhesion of the patterned layer to the substrate. The inner part of the cell culture patch (1) of the invention is developed in cyclohexanone for 7 min with agitation. Next, a second round of lamination is performed to fabricate the outer ring with the desired wall thickness. Lamination temperature and speed are 80°C and 0.2 m / min respectively for the 500 pm SUEX sheet. The laminated sheet is exposed to 2500 mJ / cm2UV light with the second mask of the outer ring of the cell culture patch (1) of the invention. After this, a post lamination bake is carried out in two steps, first for 5 min at 65°C, followed by 20 min at 85°C to relieve stresses and ensure complete curing and adhesion. At the end, the outer ring of the cell culture patch (1) of the invention is developed in PGMEA for 20 min under agitation. The drying and polymer relaxation process are carried out by a hard bake at 200°C for 2 min.

[0098] After development, the master mold with photoresist patterns on the silicon wafer is exposed to a vapor of trimethylchlorosilane (TMCS, Sigma, France). A mixture of the PDMS pre-polymer and the cross-linker (GE RTV 615, GE, France) is prepared at a ratio of 10:1 and poured on the resist layer. After curing at 72°C for 4 h, the PDMS layer is peeled off and placed on a glass slide after punching holes (1.5 mm in diameter) at the four extremities and in the center. The PDMS-glass assembly is placed under vacuum for degassing for 15 min to ensure proper contact between PDMS mold and glass and to remove air bubbles. A PEGDA or a biocompatible photo-crosslinkable polymer resin (e.g. OrmoStamp®) commercial solution as photo-initiator is dropped in the center of the PDMS mold to fill the cavity of the PDMS-glass assembly by diffusion. Then, a cured frame of PEGDA or a biocompatible photo- crosslinkable polymer resin (e.g. OrmoStamp®) is formed after UV exposure at 9.1 mW / cm2for 85 s and peeling-off the PDMS mold. At the end, the PEGDA or OrmoStamp® frame is gently taken off using a scalpel.

[0099] To favor cell adhesion on the culture patch (1) of the invention, gelatin nanofibers are produced by electrospinning. 10 wt% gelatin powder (G2625, Sigma, France) are dissolved in a mixture of acetic acid, ethyl acetate and distilled water with a volume ratio of 21:14:10. To collect the electrospun nanofibers more evenly, the backside (3) of the supporting PEGDA or a biocompatible photo- crosslinkable polymer resin (e.g. OrmoStamp®) frame is sputtered with 10 nm thick gold (125 mA for 30 seconds). Then, the PEGDA or a biocompatible photo-crosslinkable polymer resin (e.g. OrmoStamp®) substrate with a golden surface on top is taped onto an aluminum foil as a conductive substrate. After loading into a syringe, the gelatin solution is ejected to the collector at a distance of 10 cm, using a syringe pump (KD Scientific) at 0.2 mL.h1pumping speed through a stainless steel 23- gauge needle for 7 min. The spinneret is connected to the anode of high potential power supply (TechDempaz, Japan) with a voltage of 11 kV and the collector is connected to the cathode of the power supply.

[0100] When cells were placed within a fiber-based gel environment, such as a collagen gel, they tend to pull on the fibers, which often leads to gel contraction and subsequent detachment of the gel from the walls. To prevent this, applying polydopamine (PDA) coating is performed to maintain the gel adhesion to the culture patch (1) of the invention: a PDA solution is prepared at a concentration of 1.5 mg / mL in 10 mM TRIS-HCI (Tris(hydroxymethyl)aminomethane hydrochloride) buffer. The cell culture patches (1) of the invention are incubated inside PDA solution at room temperature (from 20°C to 25°C) overnight (at least lOhrs). Then, the cell culture patches (1) of the invention are rinsed three times with PBS for 5 minutes each to remove unbound PDA. The cell culture patches (1) of the invention are then allowed to dry completely before use.

[0101] According to another aspect and as illustrated on Figures 3 and 8, a subject matter of the invention relates to an apparatus (6) for handling a cell culture device, said apparatus (6) comprising a bottomchamber (7) and a top-chamber (8) separated by a transition space (9), the surface of which:

[0102] - is greater than the one of said bottom-chamber (7); and

[0103] - is smaller than the one of said top-chamber (8), wherein said a transition space (9) is suitable for accommodating said cell culture device so that both bottom-chamber (7) and top-chamber (8), when said cell culture device is present, define two independent spaces (allowing precise control of the environment inside each chamber (7, 8)), said cell culture device being in particular a cell culture patch (1) as described above.

[0104] The "cell culture device" refers to patch similar to the one of the invention. Such patches may be manufactured by MesoBioTech® (mesobiotech.com). According to another embodiment, a subject matter of the invention thus concerns the apparatus (6) as described above for handling a cell culture patch (1), in particular the cell culture patch (1) as described above.

[0105] "Bottom-chamber (7)", "top-chamber (8)" and "transition space (9)" refer to three separate compartments stacked one on top of the other. The bottom-chamber (7) is used to contain a culture medium. The transition space (9) is used to support a cell culture device, such as the cell culture patch (1) of the invention, which, when it is present, closes off the bottom-chamber (7) and isolates it from the top-chamber (8). The top-chamber (8) is used, once a cell culture device, such as the cell culture patch (1) of the invention, is accommodated in the transition space (9), either to seed the backside of said cell culture device, to fill the compartment unit with hydrogel or to contain a culture medium. Importantly, when said cell culture device is placed in said transition space (9) a biocompatible polymer ring (e.g. PDMS) is used to secure the cell culture device in place and minimize media mixing between the bottom-chamber (7) and the top-chamber (8).

[0106] According to another embodiment, a subject matter of the invention relates to the apparatus (6) as described above, said apparatus (6) being in the form of a hollow cylinder (Figure 3) or in the form of a hollow parallelepiped (Figure 8) According to another embodiment and as illustrated on Figure 3, a subject matter of the invention relates to the apparatus (6) as described above in the form of a hollow cylinder for handling a cell culture device, said apparatus (6) comprising a bottom-chamber (7) and a top-chamber (8) separated by a transition space (9), the surface of which:

[0107] - is greater than the one of said bottom-chamber (7); and

[0108] - is smaller than the one of said top-chamber (8), wherein said a transition space (9) is suitable for accommodating said cell culture device so that both bottom-chamber (7) and top-chamber (8), when said cell culture device is present, define two independent spaces (allowing precise control of the environment inside each chamber (7, 8)), said cell culture device being in particular a cell culture patch (1) as described above.

[0109] According to another embodiment, a subject matter of the invention relates to the apparatus (6) as described above, said apparatus (6) being in the form of a hollow cylinder having a diameter of 25 mm and a height of 13 mm wherein: said bottom-chamber (7) has a diameter of 12 mm and a height of 5,9 mm; said top-chamber (8) has a diameter of 19 mm and a height of 3,5 mm; and said transition space (9) has a diameter of 14 mm and a height of 0,6 mm, said cell culture device (in particular said cell culture patch (1) as described above) being in the form of a disc having a diameter of 13 mm and a height of 0,5 mm.

[0110] According to another embodiment and as illustrated on Figure 8, a subject matter of the invention relates to the apparatus (6) as described above in the form of a hollow parallelepiped for handling a cell culture device, said apparatus (6) comprising a bottom-chamber (7) and a top-chamber (8) separated by a transition space (9), the surface of which:

[0111] - is greater than the one of said bottom-chamber (7); and

[0112] - is smaller than the one of said top-chamber (8), wherein said a transition space (9) is suitable for accommodating said cell culture device so that both bottom-chamber (7) and top-chamber (8), when said cell culture device is present, define two independent spaces (allowing precise control of the environment inside each chamber (7, 8)), said cell culture device being in particular a cell culture patch (1) as described above.

[0113] According to another embodiment, a subject matter of the invention relates to the apparatus (6) as described above, said apparatus (6) being in the form of a hollow parallelepiped having a length of 31 mm, a width of 21 mm and a height of 13 mm wherein: said bottom-chamber (7) has a length of 18.0 mm, a width of 8 mm and a height of 5.9 mm; said top-chamber (8) has a length of 25 mm, a width of 15 mm and a height of 3.5 mm; and said transition space (9) has a length of 20 mm, a width of 10 mm and a height of 0.6 mm, said cell culture device (in particular said cell culture patch (1) as described above) being in the form of a rectangle having a length of 19 mm and a width of 9 mm.

[0114] According to another embodiment, a subject matter of the invention relates to the apparatus (6) as described above, said apparatus (6) being made from PDMS.

[0115] According to another aspect, a subject matter of the invention relates to a method of manufacture of an apparatus (6) as described above comprising at least the following steps of:

[0116] 1. pouring a biocompatible pre-polymer and cross-linker mixture, in particular pre-polymer and cross-linker at a 10:1 ratio, over a negative mold, the print of which has the form of said apparatus (6), to obtain a negative mold coated with said biocompatible prepolymer and cross-linker mixture;

[0117] 2. exposing to heat, in particular at 75°C for 4 hours, said negative mold coated with said biocompatible pre-polymer and cross-linker mixture for curing the biocompatible prepolymer to obtain a negative mold coated with cured biocompatible polymer; and

[0118] 3. taking off said cured biocompatible polymer from said negative mold and optionally trimming off any excess of biocompatible polymer to obtain said apparatus (6), said biocompatible (pre-)polymer being in particular PDMS.

[0119] According to another embodiment, a subject matter of the invention concerns the method of manufacture as described above of an apparatus (6) as described above, wherein step 2. of heat exposure is performed at 75°C for 4 hours.

[0120] According to another embodiment, a subject matter of the invention concerns the method of manufacture as described above of an apparatus (6) as described above, wherein said biocompatible (pre-)polymer is PDMS.

[0121] According to another embodiment, a subject matter of the invention concerns the method of manufacture as described above of an apparatus (6) as described above, which further comprises a preliminary aluminum negative mold manufacturing step. For instance, and in details the method of manufacture as described above of an apparatus (6) as described above comprises:

[0122] To create the PDMS structure, a negative mold is first carved out of a suitable sized aluminum block using computer numerical control (CNC) machining. The mold is then placed in a Petri dish, with its larger-diameter cylinder facing downward and the smaller one upward (see Chamber dimensions), and the PDMS pre-polymer and cross-linker mixture (GE RTV 615, GE, France) at a 10:1 ratio are poured over it, fully immersing the mold (negative mold dipped 3 mm deep from the surface to provide 3 mm thickness to the bottom base). After curing at 75°C for 4 hours, the mold is removed, and any excess of PDMS is trimmed off to obtain the final chamber.

[0123] According to another aspect, a subject matter of the invention relates to a method of seeding of a cell culture patch (1) as described above comprising at least the following steps of:

[0124] 1. casting and polymerizing a hydrogel inside the compartment unit of said cell culture patch (1); and

[0125] 2. seeding "patch-cells" on the backside of the bottom surface (3) of said compartment unit.

[0126] According to another embodiment, a subject matter of the invention relates to the method of seeding as described above of a cell culture patch (1) of the invention comprising at least the following steps of:

[0127] 1. placing the cell culture patch (1) in the transition space (9) of an apparatus (6) as described above, so that the compartment unit of said cell culture patch (1) is facing the top-chamber (8) of said apparatus (6);

[0128] 2. casting and polymerizing a hydrogel inside said compartment unit; and

[0129] 3. turning for the first time said cell culture patch (1) upside down so that said compartment unit filled with polymerised hydrogel is facing the bottom-chamber (7) of said apparatus (6);

[0130] 4. seeding "patch-cells" on the backside of the bottom surface (3) of said compartment unit which is facing the top-chamber (8); and

[0131] 5. turning for the second time said cell culture patch (1) upside down so that:

[0132] - said compartment unit filled with polymerised hydrogel is facing again the top-chamber (8) of said apparatus (6); and

[0133] - said backside of said bottom surface (3) seeded with "patch-cells" is facing the bottom-chamber (7) of said apparatus (6). According to another embodiment, a subject matter of the invention relates to the method of seeding as described above of a cell culture patch (1) of the invention, wherein a biocompatible polymer ring is used when said cell culture patch (1) is placed in said transition space (9) to secure the cell culture patch (1) in place and minimize media mixing, said biocompatible polymer ring having in particular a 12 mm inside diameter, said biocompatible polymer being in particular PDMS.

[0134] According to another embodiment, a subject matter of the invention concerns the method of seeding as described above of a cell culture patch (1) of the invention, wherein said biocompatible polymer ring has a 12 mm inside diameter.

[0135] According to another embodiment, a subject matter of the invention concerns the method of seeding as described above of a cell culture patch (1) of the invention, wherein said biocompatible polymer ring is made from PDMS.

[0136] According to another embodiment, a subject matter of the invention concerns the method of seeding as described above of a cell culture patch (1) of the invention, wherein: said polymerized hydrogel contains cells, hormones, growth factors, drugs; and / or said "patch-cells" are selected from the group: primary endothelial cells, iPS-derived cells and circulating progenitor cells.

[0137] According to another embodiment, a subject matter of the invention concerns the method of seeding as described above of a cell culture patch (1) of the invention, wherein said patch-cells" are human cells.

[0138] According to another aspect, a subject matter of the invention relates to a kit comprising at least:

[0139] - a cell culture patch (1) as described above; and

[0140] - an apparatus (6) as described above, and / or a microfluidic array (10), said microfluidic array (10) being in particular perfused.

[0141] According to another embodiment, a subject matter of the invention concerns the kit as described above, said kit comprising at least a cell culture patch (1) as described above and an apparatus (6) as described above.

[0142] According to another embodiment, a subject matter of the invention concerns the kit as described above, said kit comprising at least a cell culture patch (1) as described above and an apparatus (6) as described above and a biocompatible polymer ring. According to another embodiment, a subject matter of the invention concerns the kit as described above, said kit comprising at least a cell culture patch (1) as described above and a microfluidic array (10), said microfluidic array (10) being in particular perfused. According to another embodiment, a subject matter of the invention concerns the kit as described above, said kit comprising at least a cell culture patch (1) as described above and a perfused microfluidic array (10).

[0143] According to another embodiment, a subject matter of the invention concerns the kit as described above, said kit comprising at least a cell culture patch (1) as described above, an apparatus (6) as described above, optionally a biocompatible polymer ring, and a microfluidic array (10), said microfluidic array (10) being in particular perfused.

[0144] In any event, it should be noted that the various aspects of the invention, as well as the various embodiments thereof, are interdependent. These can therefore be combined with each other to obtain preferred aspects and / or embodiments of the invention not explicitly described. This is also true for the set of definitions provided in this description, which applies to all aspects of the invention and its embodiments.

[0145] Furthermore, the present invention is illustrated by, but not limited to, the following Figures and Examples.

[0146] LIST OF FIGURES

[0147] Figure 1. Schematic and dimensions of one embodiment of the cell culture patch (1) of the invention. Figure 2. Microfabricated support with adjustable pattern (no cell), pore size / shape / distribution.

[0148] Figure 3. Top view, side view, bottom view and isometric projection of one embodiment of the apparatus (6) of the invention.

[0149] Figure 4. Schematic and dimensions of the top and bottom parts of one example of a microfluidic chip. Figure 5. Schematic of the setup. Immunofluorescence of sprouting structures (top left image) and anastomosis (top right image) (top side of the device) and endothelial monolayer (bottom image & bottom side of the device).

[0150] Figure 6. Immunofluorescence of sprouting and anastomosis with time, after 24, 48 and 72 h of culture on the patch (from left to right).

[0151] Figure 7. Schematic and dimensions of another embodiment of the cell culture patch (1) of the invention. Figure 8. Top view, side view, bottom view and isometric projection of another embodiment of the apparatus (6) of the invention.

[0152] EXAMPLES

[0153] EXAMPLE 1 - Manufacturing of the cell culture patch (1) and the apparatus (6)

[0154] MATERIALS & METHODS

[0155] 1. Microfabrication of the cell culture patch (1)

[0156] 1.1. Micropattern generation

[0157] The patch frame made of PEGDA or OrmoStamp® (UV curable resin from micro resist technology GmbH, Berlin) was fabricated by UV assisted molding using a polydimethylsiloxane (PDMS) mold. First, two chromium masks were produced using a micro-pattern generator (pPG 101, Heidelberg Instruments, Germany). The first one was the pore pattern with the desired shape / size / interdistance / distribution of the pore. The second one was the external ring of the cell culture patch (1). The two photomasks were used to replicate the desired pattern by photolithography on a silicon wafer with the desired polymer thickness / wall size obtained by dry film photoresist-based microfabrication (lamination).

[0158] 1.2. Dry film photoresist-based microfabrication

[0159] A commercial laminator was used to laminate SUEX dry film sheets. SUEX dry films with 50 to 500 pm thickness covered with two polyester (PET) films were used. The PET sheet of one side was removed before laminating the photoresist against the substrate. The lamination temperature of the roller depends on the thickness of the dry film sheet. The second PET sheet kept during lamination was removed before exposing the laminated layer.

[0160] Laminator temperature and speed were 100°C and 0.2 m / min, respectively for the 50 pm SUEX sheet. The laminated sheet was exposed to UV with the first mask of the inner pattern of the cell culture patch (1). The exposure energy of 95 mJ / cm2 was used to crosslink the 50 pm thick SUEX film. Then, a post exposure bake at 95°C for 5 min was carried out on a hot plate to complete the crosslinking and ensure proper adhesion of the patterned layer to the substrate. The inner part of the cell culture patch (1) was developed in cyclohexanone for 7 min with agitation. Next, a second round of lamination was performed to fabricate the outer ring with the desired wall thickness. Lamination temperature and speed were 80°C and 0.2 m / min respectively for the 500 pm SUEX sheet. The laminated sheet was exposed to 2500 mJ / cm2UV light with the second mask of the outer ring of the cell culture patch (1). After this, a post lamination bake was carried out in two steps, first for 5 min at 65°C, followed by 20 min at 85°C to relieve stresses and ensure complete curing and adhesion. At the end, the outer ring of the cell culture patch (1) was developed in PGMEA for 20 min under agitation. The drying and polymer relaxation process were carried out by a hard bake at 200°C for 2 min.

[0161] 1.3. Fabrication of the PDMS mold

[0162] After development, the master mold with photoresist patterns on the silicon wafer was exposed to a vapor of trimethylchlorosilane (TMCS, Sigma, France). A mixture of the PDMS pre-polymer and the cross-linker (GE RTV 615, GE, France) was prepared at a ratio of 10:1 and poured on the resist layer. After curing at 72°C for 4 h, the PDMS layer was peeled off and placed on a glass slide after punching holes (1.5 mm in diameter) at the four extremities and in the center. The PDMS-glass assembly was placed under vacuum for degassing for 15 min to ensure proper contact between PDMS mold and glass and to remove air bubbles. A PEGDA or OrmoStamp® commercial solution as photo-initiator was dropped in the center of the PDMS mold to fill the cavity of the PDMS-glass assembly by diffusion. Then, a cured frame of PEGDA or OrmoStamp® was formed after UV exposure at 9.1 mW / cm2for 85 s and peeling-off the PDMS mold. At the end, the PEGDA or OrmoStamp® frame was gently taken off using a scalpel.

[0163] 1.4.1. Cell culture patch (1) dimensions - Figures 1-2

[0164] The cell culture patch (1) was a disc with 13 mm outer diameter and 9 mm inner diameter 100 to 500 pm wall thickness

[0165] Pore shape: symetrical (circle, hexagon) or asymetrical (arrow)

[0166] Pore diameter: 20 to 100 pm, typically 50 pm

[0167] Pore interdistance: 300 to 600 pm

[0168] Minimum pore density: 100 pores. cm-2

[0169] 1.4.2. Cell culture patch (1) dimensions - Figure 7

[0170] The cell culture patch (1) was a a rectangle with a length of 19 mm and a width of 9 mm 100 to 500 pm wall thickness

[0171] Pore shape: symetrical (circle, hexagon) or asymetrical (arrow)

[0172] Pore diameter: 20 to 100 pm, typically 50 pm

[0173] Pore interdistance: 300 to 600 pm

[0174] Minimum pore density: 100 pores. cm-2

[0175] 2. 3D printing and fabrication of the apparatus (6)

[0176] To seed cells and cast gels, as well as to allow working with the cell culture patch (1) under static culture conditions, a two-compartment device, referred to as apparatus (6) was fabricated. Since the material used in this apparatus (6) comes into direct contact with the cells, it must support cell viability, leading to the choice of PDMS. To create the PDMS structure, a negative mold was first carved out of a suitable sized aluminum block using computer numerical control (CNC) machining. The mold was then placed in a Petri dish, with its larger-diameter cylinder facing downward and the smaller one upward (see Chamber dimensions), and the PDMS pre-polymer and cross-linker mixture (GE RTV 615, GE, France) at a 10:1 ratio were poured over it, fully immersing the mold (negative mold dipped 3 mm deep from the surface to provide 3 mm thickness to the bottom base). After curing at 75°C for 4 hours, the mold was removed, and any excess of PDMS was trimmed off to obtain the final chamber.

[0177] 2.1.1. Apparatus (6) dimensions - Figure 3

[0178] The PDMS based apparatus (6) featured a hollow cylinder with an outer diameter of 25 mm. The topchamber (8) had an internal diameter of 19 mm, which transitions to the transition zone (9) separating two chambers to accommodate a cell culture patch (1). The entire structure had a height of 13 mm, with a top-chamber (8) measuring 3.5 mm, bottom-chamber (7) 5.9 mm, and a transition zone (9) of 0.6 mm (to allow for cell culture patch (1) placement). These compartments were designed to ensure that the two chambers remain separated while allowing for precise control over the environment within each compartment.

[0179] 2.1.2. Apparatus (6) dimensions - Figure 8

[0180] The PDMS based apparatus (6) featured a hollow parallelepiped with a length of 31 mm and a width of 21 mm. The top-chamber (8) had a length of 25 mm and a width of 15 mm , which transitions to the transition zone (9) separating two chambers to accommodate a cell culture patch (1). The entire structure had a height of 13 mm, with a top-chamber (8) measuring 3.5 mm, bottom-chamber (7) 5.9 mm, and a transition zone (9) of 0.6 mm (to allow for cell culture patch (1) placement). These compartments were designed to ensure that the two chambers remain separated while allowing for precise control over the environment within each compartment.

[0181] EXAMPLE 2 - Angiogenesis analysis

[0182] MATERIALS & METHODS

[0183] 1. Implementation of the cell culture patch (1) for angiogenesis study

[0184] 1.1. Cell culture patch (1) coating with a gelatin nanofibers

[0185] To favor cell adhesion on the culture patch (1), gelatin nanofibers were produced by electrospinning. 10 wt% gelatin powder (G2625, Sigma, France) were dissolved in a mixture of acetic acid, ethyl acetate and distilled water with a volume ratio of 21:14:10. To collect the electrospun nanofibers more evenly, the backside (3) of the supporting PEGDA or OrmoStamp® frame was sputtered with 10 nm thick gold (125 mA for 30 seconds). Then, the PEGDA or OrmoStamp® substrate with a golden surface on top was taped onto an aluminum foil as a conductive substrate. After loading into a syringe, the gelatin solution was ejected to the collector at a distance of 10 cm, using a syringe pump (KD Scientific) at 0.2 mL.h1pumping speed through a stainless steel 23-gauge needle for 7 min. The spinneret was connected to the anode of high potential power supply (TechDempaz, Japan) with a voltage of 11 kV and the collector was connected to the cathode of the power supply.

[0186] 1.2. Polydopamine (PDA) coating and sterilization of the cell culture patch (1)

[0187] When cells were placed within a fiber-based gel environment, such as a collagen gel, they tend to pull on the fibers, which often leads to gel contraction and subsequent detachment of the gel from the walls. To prevent this, applying polydopamine (PDA) coating was essential to maintain the gel adhesion to the culture patch (1). A PDA solution was prepared at a concentration of 1.5 mg / mL in 10 mM TRIS- HCI (Tris(hydroxymethyl)aminomethane hydrochloride) buffer. The cell culture patches (1) were incubated inside PDA solution at room temperature (from 20°C to 25°C) overnight (at least lOhrs). Then, cell culture patches (1) were rinsed three times with PBS for 5 minutes each to remove unbound PDA. The cell culture patches (1) were then allowed to dry completely before use.

[0188] 1.3. Collagen gel preparation and loading

[0189] The hydrogel was prepared on ice: rat tail type I collagen (Corning, Bedford, USA, ref 354236) was first diluted in 20 mM acetic acid. NaHCOs was then added to achieve final concentrations of 2.15 mg / mL collagen and 30 mM NaHCOs in M199 culture medium. The cell culture patch (1) was placed in the apparatus (6) the transition zone (9) with the compartment unit facing up. 40 pL of hydrogel was casted on the cell culture patch (1). The hydrogels were allowed to polymerize for 15 minutes at room temperature.

[0190] 1.4. Cell seeding

[0191] Endothelial Cells (ECs) were seeded with a density of 105cells per cm2. Mesenchymal stem cells- or fibroblast-conditioned medium (CM) was filled into the bottom-chamber (7) of the apparatus (6). The cell culture patch (1) was then placed with the hydrogel facing down, and a PDMS ring (12 mm inside diameter) was used to secure the cell culture patches (1) in place and minimize media mixing. Cells were seeded in the backside of the bottom surface (3) of the cell culture patch (1) using Endothelial Cell Growth Medium 2 (ECMG2). Both CM and ECGM2 were supplemented with primocin (InvivoGen) at a final concentration of 50 pg / mL. The apparatus (6) was incubated for up to 72 h. During the first 4 h, an endothelial cell monolayer formed on the surface of the cell culture patch (1), after which the cells began to migrate in 3D in the hydrogel. Endothelial monolayer formation and cell-cell interactions were assessed by immunofluorescent staining of CD31, VE-Cadherin and cytoskeleton staining with phalloidin.

[0192] 2. Characterization of cells - Immunofluorescence

[0193] Cell culture patches (1) with the monolayer and 3D gel were fixed for 30 minutes in 4% paraformaldehyde in PBS, then rinsed and washed three times with PBS for 15 minutes. Cells were permeabilized with 0.5% Triton-X-100 in PBS for 30 minutes, then washed with 0.1% Triton-X-100 in PBS. Samples were incubated overnight (at least lOhrs) at 4°C on a shaker with primary antibody anti- VE-cadherin-Alexa488 (Affymetrics - eBioscience #15570197 clone 16B1 - 1 / 100) in 0,1% Triton-X-100 in PBS. Samples were then rinsed and washed six times for 30 minutes with 0,1% Triton-X-100 in PBS, and then one more time 10 to 30 minutes in PBS. Nuclei are stained with 4' ,6-diamidino-2- phenylindole (DAPI) (Invitrogen).

[0194] RESULTS

[0195] 3D microfabricated cell culture patches (1) hosted a collagen hydrogel and culture medium stimulating angiogenesis. On the backside of the bottom surface (3) of the cell culture patch (1), a monolayer of endothelial cells was grown. On the compartment unit of the cell culture patch (1) a collagen hydrogel loaded with vascular endothelial growth factor was hosted. Endothelial cells from the bottom monolayer migrated through the pores and invaded the hydrogel as sprouting structures to form de novo vascular structures by anastomosis forming a luminized vascular network within 72 hours (Figures 5-6).

Claims

CLAIMS1. Cell culture patch (1) having an upward opening comprising a compartment unit for cell culture cut into the thickness of said cell culture patch (1) wherein said compartment unit comprises: a lower case including a bottom surface (3) of at least 28,27 mm2and a sidewall (2) vertically extending from 100 pm to 500 pm upward from an edge of the bottom surface (3) which corresponds to the lower part of said cell culture patch (1); said bottom surface (3) being perforated by pores (4), said pores (4): being arranged so as to pass through the thickness of said bottom surface (3); having a symmetrical shape and / or an asymmetrical shape, the surface of which is comprised from 1 256.6 pm2to 7 853.98 pm2; being spaced apart by a distance comprised from 200 pm to 1 000 pm; and having a density comprised from 50 pores / cm2to 250 pores / cm2.

2. Cell culture patch (1) according to claim 1, wherein: said cell culture patch (1) is in the form of a disc, the diameter of which being in particular of 13 mm; said compartment unit is in the form of a disc, the diameter of which being in particular of 9 mm; and said sidewall (2) is in the form of a ring, the width of which being in particular of 2 mm.

3. Cell culture patch (1) according to claim 1 or 2, wherein said pores (4) have:- a symmetric center, such as a hexagon or a circle; or- a symmetric axis, such as an arrow.

4. Cell culture patch (1) according to any of claims 1 to 3, wherein said pores (4) are capable of being arranged so as to define at least two repeating patterns (5), each pattern (5) being spaced from the others by a distance comprised from 600 pm to 1 000 pm.

5. Cell culture patch (1) according to any of claims 1 to 4, said cell culture patch (1) being made with a polymer material, said material having at least the following characteristic:- microresistant to visible and near-UV light, water or any polar or apolar solvent, at low and high temperatures;- transparent;- non-porous;- non-rough / smooth; and- biocompatible, said cell culture patch (1) being in particular made from Polyethylene Glycol Diacrylate (PEGDA) or a biocompatible photo-crosslinkable polymer resin.

6. Cell culture patch (1) according to any of claims 1 to 5, wherein the backside of said bottom surface (3) comprises:- a first coating with a 10 nm thick conductive and biocompatible material; and- optionally a second coating with nanofibers having a diameter comprised from 350 nm to 550 nm, said nanofibers being in particular gelatin nanofibers.

7. Cell culture patch (1) according to any of claims 1 to 6, wherein said cell culture patch (1) comprises a coating with polydopamine (PDA).

8. Cell culture patch (1) according to any of claims 1 to 7, wherein said backside of said bottom surface (3) is seeded with "patch-cells", in particular primary endothelial cells, iPS-derived cells or circulating progenitor cells, and the inside of said compartment unit is filled with hydrogel, said hydrogel be liable to contain cells, hormones, growth factors, drugs.

9. Use of a cell culture patch (1) according to any of claims 1 to 8 for implementing cellular biology analysis methods, in particular for analysing angiogenesis mechanisms, said methods being optionally performed in a microfluidic array (10) suitable for receiving the cell culture patch (1), said microfluidic array (10) being in particular perfused so that:said backside of said bottom surface (3) is fed with first medium culture; and said inside of said compartment unit is fed with second medium culture which can be identical or different to the first medium culture, in particular said second medium culture being different to the first medium culture.

10. Method of manufacture of a cell culture patch (1) according to any of claims 1 to 8 comprising at least the following steps of:

1. dropping a polymers solution in the center of a mold, the print of which has the form of said cell culture patch (1), to obtain a mold coated with said polymers solution;2. exposing to UV light, in particular at 9.1 mW / cm2for 85 s, said mold coated with said polymers solution to cure polymers and to obtain a mold coated with a cured polymer; and3. taking off said cured polymers from said mold to obtain said cell culture patch (1), wherein said polymers have at least the following characteristic:- microresistant to visible and near-UV light, water or any polar or apolar solvent, at low and high temperatures;- transparent;- non-porous;- non-rough / smooth; and- biocompatible, said polymers being in particular PEGDA or a biocompatible photo-crosslinkable polymer resin, and wherein said mold is made in particular from PDMS.

11. Apparatus (6) for handling a cell culture device, said apparatus (6) comprising a bottom-chamber(7) and a top-chamber (8) separated by a transition space (9), the surface of which:- is greater than the one of said bottom-chamber (7); and- is smaller than the one of said top-chamber (8), wherein said a transition space (9) is suitable for accommodating said cell culture device so that both bottom-chamber (7) and top-chamber (8), when said cell culture device is present, define two independent spaces, said cell culture device being in particular a cell culture patch (1) according to any of claims 1 to 8.

12. Apparatus (6) according to claim 11, said apparatus (6) being made from PDMS.

13. Method of manufacture of an apparatus (6) according to claim 11 or 12 comprising at least the following steps of:

1. pouring a biocompatible pre-polymer and cross-linker mixture, in particular pre-polymer and cross-linker at a 10:1 ratio, over a negative mold, the print of which has the form of said apparatus (6), to obtain a negative mold coated with said biocompatible prepolymer and cross-linker mixture;2. exposing to heat, in particular at 75°C for 4 hours, said negative mold coated with said biocompatible pre-polymer and cross-linker mixture for curing the biocompatible prepolymer to obtain a negative mold coated with cured biocompatible polymer; and3. taking off said cured biocompatible polymer from said negative mold and optionally trimming off any excess of biocompatible polymer to obtain said apparatus (6), said biocompatible (pre-)polymer being in particular PDMS.

14. Method of seeding of a cell culture patch (1) according to any of claims 1 to 8 comprising at least the following steps of:

1. casting and polymerizing a hydrogel inside the compartment unit of said cell culture patch (1); and2. seeding "patch-cells" on the backside of the bottom surface (3) of said compartment unit.

15. Kit comprising at least:- a cell culture patch (1) according to any of claims 1 to 8; and- an apparatus (6) according to claim 10 or 11, and / or a microfluidic array (10), said microfluidic array (10) being in particular perfused.

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