Membrane for reconstructed tissue comprising pores of several diameters

Abstract

The present invention relates to membranes comprising at least two types of porosity: a porosity of small size but of high density, allowing for the support and nutrition of culture cells in such a way as to obtain a reconstructed tissue model, anda porosity of large size but of low density, allowing for the circulation of some cell types and the passage of cytoplasmic extensions from one compartment to another.

Claims

1. A monolayer membrane for cell culture comprising at least two types of pores: (i) pores having an average diameter between 0.1 and 1 m, at a density of 110.sup.5 to 110.sup.8 pores per cm.sup.2 of membrane, and (ii) pores having an average diameter between 2 and 12 m, at a density of 0.510.sup.2 to 510.sup.3 pores per cm.sup.2 of membrane, with the two types of pores crossing through the thickness of the membrane from one side to the other.

2. The monolayer membrane according to claim 1, said membrane being a polycarbonate membrane.

3. A method for manufacturing a membrane according to claim 1, comprising providing a monolayer membrane for cell culture, comprising a first type of pore having an average diameter between 0.1 and 1 m, at a density of 110.sup.5 to 110.sup.8 pores per cm.sup.2 of membrane; and perforating said monolayer membrane in such a way as to produce a second type of pore that has an average diameter between 2 and 12 m, at a density of 0.510.sup.2 to 510.sup.3 pores per cm.sup.2 of membrane.

4. The method of manufacturing according to claim 3, wherein the membrane is perforated by laser perforation.

5. A cell culture device comprising at least one compartment of which at least one of the walls comprises or is constituted by a membrane according to claim 1.

6. The device according to claim 5, said device comprising two compartments and said wall comprising or being constituted by said monolayer membrane separating the two compartments.

7. A method for manufacturing a cell culture device according to claim 5, comprising providing a cell culture device for cell culture, which comprises at least one compartment of which at least one of the walls comprises or is constituted by a monolayer membrane which comprises a first type of pore having an average diameter between 0.1 and 1 m, at a density of 110.sup.5 to 110.sup.8 pores per cm.sup.2 of membrane; and perforating said monolayer membrane in such a way as to produce a second type of pore that has an average diameter between 2 and 12 m, at a density of 0.510.sup.2 to 510.sup.3 pores per cm.sup.2 of membrane.

8. A method for the preparation of a reconstructed biological tissue comprising at least two cell types, which comprises: a1) seeding a first cell type of the tissue to be reconstructed in a at least one compartment of the culture device according to claim 5 in such a way as to allow for adhesion of the first cells to the membrane of the culture device, a2) after a period required to allow for the adhesion of the first cells to the membrane of the culture device, and b) maintaining the first and cells of a second type in culture for a period and in conditions suitable for enabling the characteristic cellular organization of the tissue.

9. A reconstructed biological tissue comprising at least one group of cells (i) comprising at least one first cell type on a monolayer membrane according to claim 1.

10. A method for preparing a reconstructed biological tissue comprising at least one cell type, in a cell culture device according to claim 5, comprising the following steps: a) seeding a first cell type of the tissue to be reconstructed in said at least one compartment of the culture device, in such a way as to allow for its adhesion to the membrane of the culture device, and b) maintaining the cells in culture for a period and in conditions suitable for enabling the characteristic cellular organization of the tissue.

11. A method for manufacturing a membrane according to claim 2 comprising providing a monolayer polycarbonate membrane for cell culture, comprising a first type of pore having an average diameter between 0.1 and 1 m, at a density of 110.sup.5 to 110.sup.8 pores per cm.sup.2 of membrane, and perforating said monolayer membrane in such a way as to produce a second type of pore that has an average diameter between 2 and 12 m, at a density of 0.510.sup.2 to 510.sup.3 pores per cm.sup.2 of membrane.

12. A cell culture device comprising at least one compartment of which at least one of the walls comprises or is constituted by a membrane according to claim 2.

13. A method for manufacturing a cell culture device according to claim 6, comprising providing a cell culture device for cell culture, which comprises two compartments separated by a wall, wherein the wall comprises or is constituted by a monolayer membrane which comprises a first type of pore having an average diameter between 0.1 and 1 m, at a density of 110.sup.5 to 110.sup.8 pores per cm.sup.2 of membrane; and perforating said monolayer membrane in such a way as to produce a second type of pore that has an average diameter between 2 and 12 m, at a density of 0.510.sup.2 to 510.sup.3 pores per cm.sup.2 of membrane.

14. A method for the preparation of a reconstructed biological tissue comprising at least two cell types, which comprises: a1) seeding a first cell type of the tissue to be reconstructed in a compartment of the culture device according to claim 6 in such a way as to allow for adhesion of the first cells to the membrane of the culture device, a2) after a period required to allow for the adhesion of the first cells to the membrane of the culture device, seeding the second cell type in the other of said two compartments of the cell culture device according to claim 6, and b) maintaining the first and second cells in culture for a period and in conditions suitable for enabling the characteristic cellular organization of the tissue.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Optical microscope photograph of the membrane of polycarbonate of a Nunc insert with a high density of pores of 0.4 m in which has been added a low density of pores of 5 m.

(2) FIG. 2: Photograph of a histological section of reconstructed epidermis on a conventional membrane that only has a porosity of 0.4 m or on membranes in which pores of 5 m have been added.

EXAMPLES

Example 1: Manufacture of Membranes and Inserts According to the Invention

(3) The inventors have shown that it was possible using industrial inserts of the market used for cell culture and tissue reconstruction to manufacture a system that has two different diameters of pores or more.

(4) Nunc inserts (Cat No 140700) comprising a polycarbonate membrane of 0.5 cm.sup.2 and of 11 m in thickness, provided with pores of 0.4 m with a density <0.8510.sup.8 pores/cm.sup.2 were perforated by a laser perforation technique, in such a way as to produce pores of a diameter of 5 m with a density of 250, 500 and 1000 pores per cm.sup.2 (FIG. 1).

(5) These pores are more precisely produced by using a femtosecond type laser with a single Bessel beam pulse of about a hundred nanojoules per hole.

Example 2: Reconstruction of a Tissue on the Membrane According to the Invention

(6) Using the conventional techniques for reconstructing human epithelium models, the inventors reconstructed on the membrane of example 1 a reconstructed human epidermis (RHE) having morphological (histology) and functional (cell viability, barrier function) characteristics equivalent to those of a standard RHE skinethic model (FIG. 2).

(7) After seeding of the inserts by keratinocytes, the inserts were placed directly at the air-liquid interface and the reconstruction continued for 17 days without changing the medium.

(8) The inventors used two types of membranes: membranes in which the pores of 5 m in diameter are distributed homogeneously over the entire surface of the membrane, and membranes in which the pores of 5 m in diameter are distributed heterogeneously over the membrane.

(9) Several densities of pores of 5 m in diameter were moreover tested: 250 pores per cm.sup.2, 500 pores per cm.sup.2 and 1000 pores per cm.sup.2.

(10) The RHE models obtained on these membranes were compared to the RHE models prepared on a standard membrane that only had pores of 0.4 m in diameter.

(11) The RHE models obtained on the membranes according to the invention have the same morphological characteristics of an epidermis (basal layer, spinous layer, grainy layer and stratum corneum) as an RHE model obtained on a standard membrane. However, they have the additional advantage of allowing the passage of cells and/or of cytoplasmic extensions through the membrane, a passage which is impossible with standard membranes

(12) The presence of pores of 5 m in diameter in the polycarbonate membrane of this RHE model allows indeed for the passage, in this example, of cells from the lower compartment to the upper compartment in order to mimic the process of lymphocystic invasion such as those observed in certain inflammatory skin disorders.