FR2834466A1 - NEW MEANS FOR CELL HOSTING AND THEIR APPLICATIONS - Google Patents

Abstract

The invention relates to the use for housing substantially flat membrane cells, made of a biocompatible material, having a plurality of separate longitudinal channels. Application to the manufacture in particular of implants or bioartificial organs, or culture systems in vitro cells.

Description

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"New means for cell hosting and their applications"
The invention relates to new means for hosting cells and their applications, in particular for the development of implants or bioartificial organs or cell culture systems in vitro.

 It is known that the use of bioartificial pancreas has been proposed for the treatment of diabetes. Currently, this treatment involves the subcutaneous injection of insulin, usually several times a day to regulate blood sugar. This system does not maintain a constant blood sugar level during the day and can cause episodes of severe hypoglycemia in the diabetic patient. In order to better supplement the functions of the deficient pancreas, it has been proposed to implant islets of Langerhans immunoprotected by a semi-permeable membrane in the patient (Mikos 1994, Reach 1999, Colton 1995). The objective is to allow the islets to receive the signal, namely the concentration of glucose in the blood, and to respond to it by a synthesis and secretion of insulin making it possible to ensure the regulation of glycemia.

These islets must function as within a healthy pancreas.

However, in the case of an implantation of a bioartificial pancreas in the peritoneal cavity or subcutaneously, the exchanges between the immobilized islets and the blood are purely diffusive. In addition, the supply of oxygen, necessary for the survival and proper functioning of cells, is also done diffusively.

 The geometries of bioartificial pancreas to date all come up against a problem of change of scale, which results in the nécrcse of the islets of Langerhans. This necrosis results on the one hand from distances too high for the diffusion of oxygen, and on the other hand from a density of islets too large aiming to reduce the overall volume of the bioartificial organ. The use of microcapsules, in particular of alginate, has also been envisaged because the volume / surface ratio

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 of exchanges is optimized. However, during implantation in the peritoneal cavity, there is the problem of anchoring these elements, and of recovering them in the event of a problem. Macroencapsulation in semi-permeable hollow fibers overcomes this pitfall, and also offers increased mechanical strength. In this case, however, the change of scale for transposition to humans has so far failed. It seems difficult to propose a geometry sufficiently compact to accommodate the number of islands required for effective replacement. Mass transfers by diffusion appear to be limited, in particular with regard to oxygen, and impose neovascularization on the external surface of the immune barrier.

 Other teams have proposed using a flat geometry, easily extractable from the location, to minimize diffusive paths. These include the work of the companies Islet Sheet Medical (Antanavich 2000) and Baxter with the product Théracyte @ (Rafael 1997).

 The Théracyte system consists of a very fine diffusion chamber equipped with 2 superimposed membranes: an external membrane with pores favoring the formation of neovessels and an internal membrane with pores allowing immunoprotection of the islets. The diffusion distances are therefore very short due to the very small internal volume, it is however very difficult to place in this chamber islets of Langerhans whose average diameter is 200 m. The volume of an island being approximately 4.10-3 l, one can place approximately 10 000 islets in this room, under a maximum density. With an optimized density, of the order of 30%, it is possible to place approximately 3000 islets necessary for supplying the pancreas of a diabetic rat.

 Islet Sheet Medical offers to house the islets in a 3-layer alginate sandwich. The total thickness of the film is approximately 400 m, which again ensures a very short diffusion distance. The density of the islands in the gel is at

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 better than 35%. We therefore find the same type of dimension as those mentioned above.

 The work of the inventors has shown that by using new geometries of accommodation structure of islets of Langerhans, it was possible to overcome the limits mentioned above in relation to implants of artificial pancreas. In addition, these geometries have proved to be applicable to other types of implants or bioartificial organs.

 The object of the invention is therefore to use such structures to house cells, in particular defective cell types in given pathologies.

 It also aims to take advantage of the properties of these structures to produce implants or bioartificial systems or cell culture systems in vitro.

 The structures used according to the invention for housing cells are characterized in that they are substantially flat membranes, made of a biocompatible material, having a plurality of separate longitudinal channels, housing cells.

 These membranes will be designated below by the expression "polycrous membranes".

 Appropriate membranes correspond in particular to those which are the subject of patent FR 2 641 709 in the name of Lyonnaise des Eaux.

 The distribution and maintenance of cells within each channel are obtained by the presence of a biocompatible gel.

 As suitable gels, there may be mentioned alginates, collagens, or even agarose.

 After filling with gel, the channels are closed. A UV-crosslinkable adhesive such as that described in application FR 00 14 969 of 20 November 2000 in the name of Aquasource will be used with advantage. In its most general definition, this adhesive is based on a cross-linkable / polymerizable adhesive under ultraviolet radiation. An amount

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 precisely dosed adhesive is introduced into the ends of the channels for hardening.

 The different geometrical characteristics of the membrane are adjusted according to the specifications to, on the one hand, ensure the proper functioning of the cells housed and, on the other hand, allow easy recovery of the implanted device.

 The polycrous geometry (length, width and thickness of the membrane) can be adapted according to the needs and the intended application in humans or animals. The shape of the channels, originally circular in section, can be modified to ensure the accommodation of a sufficient number of islands or cells with optimal density. The density of the gel itself depends on the final solution sought. The filter layers of the membrane (internal and / or external) are adjustable to meet the needs in terms of transfer of glucose, insulin and oxygen material, but also to ensure the immunoprotection of the islets. Different polymer materials can be envisaged, depending on the mechanical properties and the biocompatibility sought.

 Depending on the desired channel shape, the geometry of the die used to produce the membrane will be adapted. If the channel is of circular section, its diameter is chosen to allow the accommodation of a given cell type. For example, in the case of the islets of Langerhans, this diameter can vary from 200 μm to approximately 1 mm. The channel can also have an oblong section, in order to place several islets in parallel on the same layer, while minimizing the diffusive paths for the substances of interest, for example, glucose, insulin, oxygen in the case of said islets.

 These structures are particularly suitable for hosting primary allogeneic or xenogenic cells, or from lineages, to be protected from the surrounding environment. Mention will be made in particular of the islets of Langerhans, but also hepatocytes, renal cells, of the thyroid, of the parathyroid or of the adrenal glands.

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 The implantation of these structures in humans or animals allows the long-term survival of the cells they contain.

 The invention therefore relates to the use of said membranes for the preparation of implants or bioartificial organs.

 As regards for example the case of islets of Langerhans, an adequate regulation of the glycemia is observed and one does not come up against the problems encountered by the other systems of the prior art.

 The invention also relates to the use of these polycrous membranes in extracorporeal applications. Such applications are of great interest in particular for producing a bioartificial liver, with perfusion of blood or plasma in the membrane containing the hepatocytes in culture, and re-administration of the treated blood or plasma to the patient.

 The invention also relates to the use of said polycrous membranes for the development of cell culture systems.

 Other characteristics and advantages of the invention are given in the description which follows with reference to FIGS. 1 to 3 which represent respectively: - FIG. 1 a plane polycrous membrane with channels of circular section, enclosing a row of cells, - FIG. 2, another polycrous membrane as used according to the invention, comprising channels of oblong section, and 2 rows of cells in each channel, and - FIG. 3, a polycrous membrane with an undulating external surface.

Use of a polycrous membrane
This membrane is advantageously manufactured according to the process described in the patent FR 2 641 709 cited above.

 The characteristics of the membrane are modulated according to needs, in particular in terms of: - Material: any type of biocompatible polymer: polysulfone, polyethersulfone, PEVA, PVA, modified cellulose,

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 - Homogeneity: the structure can be homogeneous or have a very porous support and a fine filtering layer which will define the cut-off threshold. The filter layer can be internal or external. In particular, it may be a very porous outer layer, with pore diameters greater than 0.8 μm, in order to promote neovascularization around the implant.

 - Thickness: the thickness of the membrane must be low enough to promote diffusive transfers, while ensuring good mechanical strength. This thickness, added to the diameter of the canal will define the total thickness of the implant or bioartificial organ.

 - Cut-off threshold: the nutrients as well as the species secreted by the cells immobilized inside the channels must pass freely through the membrane.

However, the immunoglobulins must be retained: consequently, the cut-off threshold can vary between 20 kDa and 150 kDa.

 - External shape: the external face of the membrane can be smooth or have characteristics allowing to increase its roughness. For example, the external surface can be wavy to favor the formation of new vessels which will reduce the diffusive paths between the blood and the immobilized cells (Fig. 3).

These structures have advantages over the devices mentioned above:
1. Original geometry by itself: although the geometry is plane, it is totally different from the previous devices mentioned above.

 2. Adjustment of the density of islands facilitated by filling in one dimension (axis of the channel). This parameter being essential for the survival of the islands in the long term, the filling channel by channel makes it possible to ensure a very good homogeneity on the whole of the planar device.

 3. Excellent mechanical strength of the assembly provided by the very structure of the membrane, hence the absence of risk of

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 decrease in internal volume, or delamination of the different gel layers, as observed with structures of the prior art.

 4. External surface favoring the formation of new vessels. Depending on the data in the literature, the size of the external pores of the membrane can be adapted to optimize neovascularization. It is also possible to increase the roughness of the material, and even, using a suitable die, to create a corrugated external surface.

 5. Possibility of using certain channels for nutritional or oxygen supply.

 Examples of structures of polycrous membranes are illustrated in FIGS. 1 to 3. FIG. 1 corresponds to a plane polycrous membrane 1 comprising channels 2 of circular section, each containing a row of cells 3 in a gel 4. FIG. 2 shows a membrane with channels of oblong section 5 containing two rows of cells 6 and 7 in each channel, which makes it possible to have the desired cell density, suitable for certain types of culture. FIG. 3 illustrates a structure with an undulating external surface 8, which makes it possible to promote neovascularization.

 Application to the accommodation of islets of Langerhans Replacement of a pancreas in rats.

 For approximately 3000 islets with a diameter of 200 μm, at a density of the order of 30%, an overall internal volume of 42 l is used. For example, with an internal channel diameter of 300 µm, a total channel length of 60 cm was used, i.e. for example 20 channels 3 cm long. With an interchannel distance of 300 μm, the overall dimensions of the structure are then: Length = 3 cm, width = 1.2 cm, the thickness of the implant being for example 500 μm, which corresponds to a total volume of 180 mm3.

 Certain channels can also be devoid of cells and be used for nutrient or oxygen supply.

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 The density of the cells can be modulated as a function of the volume of biocompatible gel introduced into the channels. This parameter is essential to ensure good oxygenation of the cells.

Bioartificial pancreas
The structure obtained as indicated above is implanted in the peritoneal cavity or subcutaneously. Due to its dimensions, it is easily removable.

BIBLIOGRAPHICAL REFERENCES
Mikos AG, Papadaki MG, Kouvroukoglou S, Ishaug SL, Thomson RC. Mini-review: islet transplantation to create a bioartificial pancreas. Biotechnol. Bioeng. 1994; Reach G. Perspectives in the treatment of insulin-dependent diabetes: reflections on five revolutions. Encycl Méd Chir 1999.

Colton CK.Implantable biohybrid artificial organs. Cell Transplant. 1995; 4 (4) 415-36
Antanavich RD, Dorian R. Retrievable bioartificial implants having dimensions allowing rapid diffusion of oxygen and rapid biological response to physiological change, processes for their manufacture, and methods for their use. US Patent 2000, US006165225A.

 Rafael E, Tibell A, Arner P. Microdialysis for in vivo evaluation of permeability of immunoisolation devices.

Transplant. Proc. 1997; 29: 2134-2135.

Claims (10)

1. Use for hosting substantially flat membrane cells, made of a biocompatible material, having a plurality of separate longitudinal channels.  2. Use according to claim 1, characterized in that the distribution and the maintenance of the cells within each channel are ensured by a biocompatible gel, such as an alginate, a collagen or agarose.  3. Use according to claim 1 or 2, characterized in that the channels are closed with an adhesive.  4. Use according to any one of the preceding claims, characterized in that one or more channels are devoid of cells and are used for nutrient intakes or oxygen intakes.  5. Use according to any one of the preceding claims for the accommodation of islets of Langerhans.  6. Use according to claim 5, characterized by the use of channels of circular section, having a diameter of 200 m to 1 mm, or oblong.  7. Use according to any one of the preceding claims for the preparation of implants or bioartificial organs.  8. Use according to claim 7, for the development of an artificial pancreas.  9. Use according to any one of claims 1 to 6 for the development of in vitro cell culture systems.  10. Cell hosting structures, characterized in that they comprise membranes containing cells, as used in any one of claims 1 to 6.