# US 2019/0168216 A1 · Cell co-culture chip and process for the production thereof

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- Number: US 2019/0168216 A1
- Document ID: US20190168216A1
- Status: Published application
- Kind: A1
- Application number: 16/300,504
- Filed: 2017-05-11
- Published: 2019-06-06
- Earliest claimed: 2016-05-11
- Assignees: Universite Grenoble Alpes; Commissariat A L'energie Atomique Et Aux Energies Alternatives
- Applicants: Universite Grenoble Alpes; Commissariat A L'energie Atomique Et Aux Energies Alternatives
- Inventors: Donald Keith MARTIN; Nathalie PICOLLET-D'HAHAN
- Web page: https://patentgrep.com/patent/US20190168216A1

## CPC

- B01L 3/502707: {characterised by the manufacture of the container or its components}
- C12M 21/08: {for producing artificial tissue or for ex-vivo cultivation of tissue (prostheses A61F2/00, grafts A61L27/00)}
- C12M 23/16: {Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093)}
- C12M 25/00: {Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings}
- C12M 25/04: {in combination with well or multiwell plates, i.e. culture inserts}
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}

## Abstract

A microfluidic cell culture chip which contains a central module comprising a central unit, which contains a support consisting of a non-resorbable membrane, a 3D nanostructured porous membrane, comprising at least one protuberance, and the 3D nanostructured porous membrane and that at least one protuberance being composed of materials suitable for the culture of two distinct cell types; a base, and the central unit being integrated in the base, and forming a whole with the base.

## Claims

### Claim 1

A microfluidic cell culture chip which contains a central module comprising:
a central unit, which contains
a support consisting of a non-resorbable membrane, comprising an upper face and a lower face, perforated by at least one perforation, said support being composed, in particular of bioprinted plastic, polycarbonate, tissue culture plastic, glass or SU8,
a 3D nanostructured porous membrane, comprising an upper face and a lower face, and comprising at least one protuberance, said at least one protuberance comprising an outer face and an inner face and forming a relief structure on the side of the upper face of the 3D nanostructured membrane, said protuberance being, in particular in the shape of a hollow dome having a circular base,
said lower face of said 3D nanostructured porous membrane being positioned secured to said upper face of said support,
or said upper face of the 3D nanostructured membrane being positioned secured to said lower face of said support,
and said 3D nanostructured porous membrane and the at least one protuberance being composed of materials, suitable for the culture of two distinct cell types;
a base,
said central unit being integrated in said base, and forming a whole with said base.

### Claim 2 (depends on 1)

The microfluidic cell culture chip according to claim 1 which contains a central module comprising:
a central unit, which contains
a support consisting of a non-resorbable membrane, comprising an upper face and a lower face, perforated by at least one perforation, said support being composed, in particular of bioprinted plastic, polycarbonate, tissue culture plastic, glass or SU8,
a 3D nanostructured porous membrane, comprising an upper face and a lower face, and comprising at least one protuberance, said at least one protuberance comprising an outer face and an inner face and forming a relief structure on the side of the upper face of the 3D nanostructured membrane, said protuberance being in particular in the shape of a hollow dome having a circular base,
said lower face of said 3D nanostructured porous membrane being positioned secured to said upper face of said support,
and said 3D nanostructured porous membrane and the at least one protuberance being composed of materials, suitable for the culture of two distinct cell types;
a base,
said central unit being integrated in said base, and forming a whole with said base.

### Claim 3 (depends on 1)

The microfluidic cell culture chip according to claim 1 which contains a central module comprising:
a central unit, which contains
a support consisting of a non-resorbable membrane, comprising an upper face and a lower face, perforated by at least one perforation, said support being composed, in particular of bioprinted plastic, polycarbonate, tissue culture plastic, glass or SU8,
a 3D nanostructured porous membrane, comprising an upper face and a lower face, and comprising at least one protuberance, said at least one protuberance comprising an outer face and an inner face and forming a relief structure on the side of the upper face of the 3D nanostructured membrane, said protuberance being, in particular in the shape of a hollow dome having a circular base,
said upper face of the 3D nanostructured membrane being positioned secured to said lower face of said support,
and said 3D nanostructured porous membrane and the at least one protuberance being composed of materials, suitable for the culture of two distinct cell types;
a base,
said central unit being integrated in said base, and forming a whole with said base.

### Claim 4 (depends on 1)

The microfluidic cell culture chip according to claim 1, wherein said at least one perforation of the support has
a circular-shaped section at the upper face of the support (upper section of the perforation) having a diameter d1 (upper diameter d1 of the perforation) and an axis (x) passing through the centre of said upper section of the perforation and perpendicular to said support,
and a circular-shaped section at the lower face of the support (lower section of the perforation) having a diameter d2 (lower diameter d2 of the perforation) and an axis (w) passing through the centre of said lower section of the perforation and perpendicular to said support,
wherein said axes (x) and (w) are combined,
the diameter d1 being from a value, greater than or equal to 10 μm to a value, less than or equal to 500 μm, and the diameter d2 being from a value, greater than or equal to 10 μm to a value, less than or equal to 500 μm,
such that the value of the diameter d2 is greater than or equal to the value of the diameter d1,
and wherein said circular base of said protuberance has a diameter d3 (diameter d3 of the circular base of the protuberance), said diameter d3 of the protuberance being from value of 10 μm to a value of 500 μm, and being from a value, less than, equal to or greater than d1, and said protuberance has a height of 1 to 600 μm, more specifically from 1 to 300 μm, more specifically, from 50 to 300 μm, more specifically of 50 μm.

### Claim 5 (depends on 2)

The microfluidic cell culture chip according to claim 2, wherein said inner face of said at least one protuberance is in whole, or partially facing said at least one perforation of said support,
and in particular in whole, facing said at least one perforation of said support:
when the value of the upper diameter d1 of said at least one perforation is greater than or equal to the value of the diameter d3 of said at least one protuberance, and the axis (y), passing through the centre of said circular base and perpendicular to said support, and the axis (x), passing through the centre of the upper section of said perforation and perpendicular to said support, are combined; or
when the value of the upper diameter d1 of said at least one perforation is greater than the value of the diameter d3 of said at least one protuberance, and the axis (y), passing through the centre of said circular base and perpendicular to said support, and the axis (x), passing through the centre of the upper section of said perforation and perpendicular to said support, are distinct from one another by a distance of a value, less than or equal to [(value of d1−value of d3)/2].

### Claim 6 (depends on 5)

The microfluidic cell culture chip according to claim 5, wherein the outer face of said protuberance supports at least one adherent cell, said at least one adherent cell being, in particular a stromal cell, and wherein the inner face of said protuberance supports at least one adherent cell, said at least one adherent cell being, in particular an epithelial cell,
and more specifically, said outer face of said protuberance supports a first set of adherent cells at the stage of the confluence, said first set being a set of stromal cells, and said inner face of said protuberance supports a second set of adherent cells at the stage of the confluence, said second set being a set of epithelial cells.

### Claim 7 (depends on 6)

The microfluidic cell culture chip according to claim 6, comprising at least two protuberances and wherein the distance between two contiguous protuberances is of a value 1, said value 1 being from 10 to 100 μm, more specifically, from 50 to 100 μm,
and a maximum number of protuberances for a surface of the central unit of 1 cm2 equal to the formula
π
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d
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3
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2
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2
(
l
+
d
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π
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d
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2
where 1 is the distance between two contiguous protuberances, said value 1 being from 10 to 100 μm, more specifically, from 50 to 100 μm,
and where d3 is the diameter of the circular base of said protuberance.

### Claim 8 (depends on 1)

The microfluidic cell culture chip according to claim 1, wherein the 3D nanostructured porous membrane consists of at least one layer of a polyelectrolyte selected from among poly(sodium 4-styrenesulphonate) (PSS), poly(ethyleneimine), poly(diallyldimethylammonium chloride), poly(acrylamide-co-diallyldimethylammonium chloride), diallyldimethylammonium chloride, poly(allylamine hydrochloride) (PAH), polyanetholesulfonic acid, polyacrylic acid, poly(styrene-alt-maleic acid), polyvinyl sulphate, polyvinylsulfonic acid, poly(2-acrylamido-2-methyl-1-propanesulfonic acid), poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-acrylonitrile), poly(4-styrenesulfonic acid), poly(4-styrenesulfonic-acid-co-maleic acid), hydrated 4-styrenesulfonic sodium salt,
and wherein when said 3D nanostructured porous membrane consists of at least two successive layers, a layer consisting of a positively charged polyelectrolyte alternating with a layer consisting of a negatively charged polyelectrolyte,
said 3D nanostructured porous membrane, in particular consisting of 1 to 150 successive polyelectrolyte layers,
and more specifically, said 3D nanostructured porous membrane consisting of 15 successive layers of poly(4-styrenesulphonate sodium) (PSS) and/or poly(allylamine hydrochloride) (PAH), alternating with one another.

### Claim 9 (depends on 8)

The microfluidic cell culture chip according to claim 8, which contains a solid lower module, comprising
a lower unit
which contains an upper face, a lower face and at least one side face, and comprising at least one tubular-shaped duct comprising an upper orifice of diameter d4 having an axis (t) passing through the centre of said upper orifice and perpendicular to said support, and a lower orifice of diameter d5,
a base,
said lower unit being integrated in said base, and forming a whole with said base, said lower unit and said base are composed, in particular of bioprinted plastic, polycarbonate, tissue culture plastic or SU8,
said value of the diameter d4 of said upper orifice of said duct being greater than or equal to the value of the diameter d5 of said lower orifice of said duct and said value of the diameter d4 being greater than or equal to the value of the lower diameter d2 of said at least one perforation,
said upper face of said lower unit comprising said upper orifice of the at least one duct, and said lower orifice leading to, itself or by intermediate means, the outside of said base.

### Claim 10 (depends on 9)

The microfluidic cell culture chip according to claim 9, wherein said lower module and said central module are assembled such that the upper orifice of the at least one duct, of said upper face of said lower unit, opens over at least one of said perforations of said support (1) of said central unit, and the lower orifice of the at least one duct, itself leads to the outside of the chip, or via an intermediary constituted by a reservoir, capable of recovering liquids and making it possible for a conveyance outside of the chip via an outlet duct leading to the outside of the base of said lower module,
said upper face of said lower unit and said lower face of said support of said central unit having an identical shape and an identical surface to one another,
and said lower unit and said central unit being assembled by attachment elements situated respectively on each of the bases of the lower module and of the central module, so as to assemble sealed said upper face of said lower unit (108) and said lower face of said support of said central unit.

### Claim 11 (depends on 10)

The microfluidic cell culture chip according to claim 10, wherein said upper orifice of said at least one duct leads to one single perforation of said support, and wherein said perforation of said support is in whole, facing said upper orifice of said at least one duct:
when the value of the lower diameter d2 of said perforation is equal to the value of the diameter d4 of said upper orifice of said duct, and the axis (w), passing through the centre of said lower section of the perforation and perpendicular to said support, and the axis (t), passing through the centre of said upper orifice of said duct and perpendicular to said support, are combined, or
when the value of the lower diameter d2 of said perforation is less than the value of the diameter d4 of said upper orifice of said duct, and the axis (w), passing through the centre of said lower section of the perforation and perpendicular to said support, and the axis (t), passing through the centre of said upper orifice of said duct and perpendicular to said support, are combined, or distinct from one another by a distance of a value, less than or equal to [(value of d4−value of d2)/2].

### Claim 12 (depends on 11)

The microfluidic cell culture chip according to claim 11, wherein said lower module comprises at least two ducts,
and wherein each of the upper orifices of the at least two ducts leads to a perforation
and the at least two lower orifices of the at least two ducts lead to the outside of the chip, respectively to at least two distinct sites,
or the at least two lower orifices of all of the at least two ducts lead to one same intermediary constituted by a reservoir, capable of recovering liquids and making it possible for a conveyance to the outside of the chip via an outlet duct leading to the outside of the base of said lower module.

### Claim 13 (depends on 8)

The microfluidic cell culture chip according to claim 8, which contains an upper module comprising:
a solid and hollow upper unit, which consists of a solid surface defining an open volume (chamber), and the edges of the solid surface being capable of being in contact with said central unit of said central module and delimiting the surface of the opening of said open volume;
and a base,
said upper unit being integrated in said base, and forming a whole with said base.
and wherein said upper unit and said base are, in particular composed of bioprinted plastic, polycarbonate, tissue culture plastic or SU8.

### Claim 14 (depends on 13)

The microfluidic cell culture chip according to claim 13, wherein said upper unit of said upper module and said central unit of said central module being assembled, such that said surface of the opening of said upper unit is positioned above said upper face of the 3D nanostructured porous membrane of said central unit,
to form a closed space, delimited by said solid surface of said upper unit and said upper face of the 3D nanostructured porous membrane,
said surface of the opening and said upper face having an identical shape and an identical surface to one another,
said upper unit of said upper module and said central unit of said central module being assembled by attachment elements situated on each of the bases of the upper module and of the central module, so as to assemble sealed said surface of the opening of said upper unit and said upper face of the 3D nanostructured porous membrane of said central unit,
and advantageously said at least one solid face of said upper unit has two orifices respectively leading to an inlet/outlet duct,
making it possible for said closed space to communicate with the outside of said microfluidic chip via said inlet/outlet ducts which lead to the outside of the base.

### Claim 15 (depends on 14)

The microfluidic cell culture chip according to claim 14, comprising an upper module, a central module and a lower module, and wherein said modules above are assembled such that
said surface of the opening of said upper unit of said upper module is positioned above said upper face of the 3D nanostructured porous membrane of said central unit of said central module,
and the upper orifice of the at least one duct, of said upper face of said lower unit of said lower module, opens on at least one of said perforations of said support of said central unit,
said surface of the opening of said upper unit and said upper face of the 3D nanostructured porous membrane of said central unit having an identical shape and an identical surface to one another,
said upper face of said lower unit and said lower face of said support of said central unit having an identical shape and an identical surface to one another,
and said upper module, said central module and said lower module being assembled by attachment elements situated on each of the respective bases
in particular wherein said inner face of said protuberance is in whole, facing said upper orifice of said duct:
when said inner face of said at least one protuberance is in whole, facing said at least one perforation of said support:
such that when the value of the upper diameter d1 of said at least one perforation is greater than or equal to the value of the diameter d3 of said at least one protuberance, and the axis (y), passing through the centre of said circular base and perpendicular to said support, and the axis (x), passing through the centre of the upper section of said perforation and perpendicular to said support, are combined; or
such that the value of the upper diameter d1 of said at least one perforation is greater than the value of the diameter d3 of said at least one protuberance, and the axis (y), passing through the centre of said circular base and perpendicular to said support, and the axis (x), passing through the centre of the upper section of said perforation and perpendicular to said support, are distinct from one another by a distance of a value, less than or equal to [(value of d1−value of d3)/2];
and when said perforation of said support is in whole, facing said upper orifice of said duct:
such that the axis (y), passing through the centre of said circular base of said protuberance and perpendicular to said support, and the axis (t), passing through the centre of said upper orifice of said duct and perpendicular to said support, are combined, or distinct from one another by a distance, less than or equal to [(value of d4−value of d3)/2] when the value of d4 is greater than the value of d2.

### Claim 16 (depends on 8)

A method for producing a microfluidic cell culture chip according to claim 8, wherein the production of said central unit of said central module comprises:
a step of extruding a resorbable polymer solution, through the at least one perforation of said support to form at least one 3D nanostructure on the side of said upper face of said support, said at least one nanostructure being, in particular in the shape of a dome, followed by
a step of polymerising said resorbable polymer solution to make rigid said at least one 3D nanostructure, said at least one 3D nanostructure forming a resorbable polymer mould on the side of said upper face of said support after polymerisation, followed by
a step of applying at least one continuous layer of at least one polyelectrolyte covering the upper face of said support and the surface of said resorbable polymer mould, to constitute the 3D nanostructure porous membrane, by
a step of dissolving said resorbable polymer mould to obtain said lower face of said 3D nanostructured porous membrane positioned secured to said upper face of said support and said inner face of the at least one protuberance positioned in whole, facing said at least one perforation,
said resorbable polymer being, in particular selected from among chitosan, agarose and alginate.

### Claim 17 (depends on 3)

A method for producing a microfluidic cell culture chip according to claim 3, wherein the production of the central module comprising the central unit, comprises:
(i) a step of assembling a support part, consisting of a side frame, an open upper face and a solid lower face comprising a cut in the format of the central unit, and a mould, in the shapes and dimensions of said support part (I, i), comprising at least one moulded 3D nanostructure on the upper face, said mould being, in particular, made of plastic,
(ii) a step of pouring a resorbable polymer solution on said upper face of said mould, followed by
(iii) a step of polymerising said resorbable polymer solution to make rigid said resorbable polymer solution and form a resorbable polymer matrix comprising at least one negative mould of said at least one 3D nanostructure, followed by
(iv) a step of removing said mould, to obtain said resorbable polymer matrix comprising at least one negative mould of said at least one 3D nanostructure formed in said cut of the solid lower face of said support part, followed by
(v) a step of assembling said support part, with a perforated part, comprising a support consisting of a non-resorbable membrane perforated by at least one perforation integrated in a base,
said perforated part being in the shapes and dimensions of said support part, and the number of perforations of said perforated part being identical to the number of moulded 3D nanostructures in said mould used in step (i),
such that said at least one perforation of said support, is aligned with said at least one negative mould of said at least one 3D nanostructure,
followed by
(vi) a step of applying at least one continuous layer of at least one polyelectrolyte on the continuous surface consisting of the lower face of said support and the lower face of said resorbable polymer matrix comprising at least one negative mould of said at least one 3D nanostructure at the said at least one perforation of said support,
to constitute a 3D nanostructured porous membrane comprising at least one protuberance, followed by
(vii) a step of dissolving said resorbable polymer matrix comprising at least one negative mould of said at least one 3D nanostructure,
(viii) a step of removing the support part to obtain said perforated part comprising on the lower face thereof, a 3D nanostructured porous membrane, and on the side of the upper face thereof, said at least one protuberance.