ES2799499T3 - Bioreactor composed of a watertight chamber and internal matrix for the generation of medical implants with cells - Google Patents

Abstract

Bioreactor for the generation of medical implants with cells characterized in that it comprises a unicompartmental (1) or multi-compartment (2) watertight external chamber that contains one or more matrices (3,4,5,6,7) inside, being manufactured simultaneously these inner matrices (3,4,5,6,7) and the outer chamber (1,2) of the bioreactor through a process of rapid prototyping; in which the external chamber (1,2) comprises a shape adapted to the implant shape to have cells (internal matrix), and because it allows the implants to be removed from the interior of the bioreactor by cutting the external chamber (1,2 ) in locations comprising grooves (9) just before implantation, enabling sterility of the implant.

Description

DESCRIPTION

Bioreactor composed of a watertight chamber and internal matrix for the generation of medical implants with cells

Object of the invention

The present invention refers to a bioreactor composed of a watertight chamber and one or more matrices contained in its interior, which are manufactured simultaneously, whose matrix can be two-dimensional or three-dimensional, with variable porosity and designed by simple or complex shapes. capable of withstanding the seeding and culturing of cells to, after removal from the watertight chamber, originate medical implants with cells for the replacement / regeneration of tissues or organs of animals or humans previously damaged by trauma or disease.

State of the art

In the replacement or regeneration of tissues or organs after physical damage caused by trauma or disease, auto-, allo- or xenografts are currently used in clinical practice. However, these medical grafts have limitations such as the low availability of healthy tissue, immunocompatibility problems or cross infection between different animal species. To overcome these restrictions in its use in current clinical practice, tissue engineering emerged as a promising alternative. This strategy includes the development of two- or three-dimensional porous matrices that support colonization and cell culture, the ex vivo cultivation of cells and the presence of growth factors that induce cell differentiation and / or growth. These factors can be used independently, in groups of two or three to allow tissue regeneration.

With the development of two- or three-dimensional porous matrices being a key stage in the success of a tissue engineering strategy, these matrices must comprise a set of physicochemical properties (eg, porosity, interconnectivity, roughness, surface area, mechanical properties, hydrophilicity ) that allow cell adhesion, migration, proliferation and differentiation. The maintenance of cells in culture within these two- or three-dimensional porous matrices can be achieved through the use of static or dynamic culture systems. It has been shown that cell culture in a suitable biochemical environment and in the presence of mechanical stimuli can promote the generation of medical implants with cells. Therefore, there is great interest in replicating the physiological environment of a target tissue in vitro , through the use of biomechanical systems of dynamic simulation, scientifically known as bioreactors. These bioreactors not only provide cultured cells within two- or three-dimensional porous matrices with equal concentrations of nutrients and oxygen, but also allow the removal of by-products resulting from their metabolic activity. In addition to these characteristics, bioreactors also allow a more uniform colonization of porous two- or three-dimensional matrices by cells. Therefore, these dynamic culture systems are a better method of quantitative control over cell culture parameters, and can provide an unlimited number of medical implants with cells.

WO2010 / 016023 discloses a bioreactor suitable for implant tissue culture, comprising a chamber adapted to receive a matrix supporting tissue culture. The chamber is provided with ports to achieve fluid flow. The bioreactor is provided with a flange to allow removal of the chamber wall by removing the adhered sheet to remove tissue.

JP2002335950 discloses a storage / transport container for a membrane tissue comprising a liquid-permeable support that can cut the flat-type membrane tissue comprising the biological material from both lateral sides and keep the membrane tissue in a state maintaining its original shape without deteriorating its biological characteristics, and an openable gas-tight and / or fluid-tight container body which can receive at least one holder together with a prescribed storage / transport liquid and maintain the membrane tissue held on the support in a wet state with the storage / transport liquid.

The present invention aims to develop medical implants with cells with improved biomechanical properties, through the development of colonization and cell culture methods on two- or three-dimensional porous matrices. Implicit is the development of a bioreactor that contains, by itself, one or more two- or three-dimensional porous matrices, allowing minimal handling of medical implants with cells to minimize the risk of contamination during ex vivo cell culture .

Description of the invention

The present invention refers to a bioreactor that can be used in the colonization and culture of cells inside support matrices contained therein, originating medical implants with cells for the replacement / regeneration of tissues or organs of animals or humans, such like bone, cartilage, skin, and muscle.

The bioreactor is mainly composed of one or more matrices, with variable porosity, three-dimensional or Two-dimensional, contained within a one-compartment or multi-compartment watertight chamber.

Both the culture chamber and the matrix contained within it are manufactured simultaneously through a rapid prototyping process, from one or multiple materials. These materials can be biodegradable or not, inert or not, polymeric or composite, transparent, translucent or opaque, such as polycaprolactone (PCL), polylactic acid (PLA) or polyglycolic acid (PGA).

During the manufacture of the bioreactor and its internal matrix an additional water soluble support material can be used, which is also deposited during the rapid prototyping process. This material only acts as a support for the deposition of the material or materials deposited during the manufacture of the chamber and its matrix, subsequently being removed by immersion in water or aqueous solution.

The architecture of the chamber and its internal matrix should preferably be designed to be self-supporting, not requiring the use of additional support material.

The chambers and their internal matrices can be sterilized by various methods depending on the materials used. They can be sterilized by immersion in ethanol, by exposure to ultraviolet radiation or exposure to ethylene oxide, in case none of the materials used shows any adverse reactivity, or even by sterilization in an autoclave, when the materials used are sufficiently stable when exposed. at high temperatures. In case the external camera is prototyped from one or more transparent or translucent materials, it becomes even possible to analyze its interior by optical means. In case the internal matrix is also prototyped from transparent or translucent materials, the interior of the internal matrix can also be analyzed by optical means.

In order to improve the performance of the internal matrix contained within the chamber, it is possible to coat its surface by filling or perfusing the interior of the chamber, through the internal matrix, with solutions or gases capable of generating these coatings.

The internal matrix can be designed using as initial reference simple shapes such as cubic, cylindrical, tubular or other shapes, or from complex shapes obtained from the analysis of tissues or organs by means of computed tomography or magnetic resonance techniques.

The design of the external chamber is carried out having as initial reference the shape of the internal matrix and by extruding its external surfaces.

At least two tubular structures are added to the design of the external chamber which are intended to connect the interior of the chamber with the exterior and allow the entry and exit of fluids and / or gases from / to the interior of the chamber. The location of these tubular structures should be determined taking into account the best possible efficiency in draining the fluids inserted into the chamber, as well as the improvement of the fluid flow dynamics inside the chamber.

The bioreactor can be integrated into a perfusion culture system that allows the culture medium to circulate through the interior of the external chamber and through the pores of the internal matrix of the bioreactor.

The culture medium is supplied to the chamber through the tubular structures for medium inlet / outlet, preferably located at the ends of the culture chamber, and is circulated to and from an aerated culture medium reservoir by means of a peristaltic pump.

When integrated into a perfusion culture system, the bioreactor is capable of continuous perfusion of the expansion or differentiation culture medium, improving the massive transport of cells into the matrix previously with cells. The continuous perfusion of the culture medium carried out by the culture system is capable of acting as a stimulus on the cells, being able to regulate or increase the proliferation and differentiation of cells. Perfusion of the medium through the cell arrays can be performed both unidirectionally and bidirectionally.

After culturing, the cell matrix can be removed from the interior of the bioreactor by cutting the outer chamber at specifically designed locations, using surgical tools.

Brief description of the drawings

Figure 1 shows a longitudinal section of the device containing a simple three-dimensional porous structure inside.

Figure 2 shows a longitudinal section of the device containing a two-dimensional porous structure in its inside.

Figure 3 shows a longitudinal section of the device containing a double three-dimensional porous structure inside.

Figure 4 shows a longitudinal section of the device that contains a tubular three-dimensional porous structure inside.

Figure 5 shows a longitudinal section of the device containing a complex tubular three-dimensional porous structure, manufactured using a three-dimensional model obtained from computed tomography or magnetic resonance analysis of a tissue or organ.

Figure 6 shows the device in isometric view.

Figure 7 shows a longitudinal section of the device.

Figure 8 shows a partial section of the device in isometric view.

Figure 9 shows an implant after removal from the outer enclosure in isometric view.

Figure 10 shows the device described in Figures 6 and 9 integrated into a dynamic perfusion cell culture system.

Detailed description of the invention

The following description refers to a preferred configuration of the invention using the figures of this document in order to allow a better understanding of the invention.

The bioreactor is essentially composed of a unicompartmental 1 or multi-compartment 2 watertight chambers that contain inside one or more matrices, possessing porosity that can be uniform or variable, homogeneous or heterogeneous, present throughout the matrix 3,5,6 , 7 or part of the matrix 4, three-dimensional 3,4,6,7 or two-dimensional 5, which have simple 3, 4, 5, 6 or complex shapes 7.

The simple shape external design of the matrix 3,4,5,6 can be achieved by using simple shapes as initial references such as cubic, cylindrical, tubular or other shapes, while the external shape design of the complex internal matrix 7 is achieved resorting to complex shapes obtained from analyzes performed on tissues or organs using technologies such as computed tomography or magnetic resonance imaging. The internal pores of the internal matrix 3,4,5,6,7 can be generated by manually or automatically adding pores inside the internal matrix design 3,4,5,6,7 or, also after by automatic addition, by defining specific properties for material deposition during the conversion of the final three-dimensional design into commands to control the equipment that will manufacture the internal matrix 3,4,5,6 by rapid prototyping.

The design of the external chamber 1,2 is carried out in turn taking as an initial reference the external shape of the internal matrix 3,4,5,6,7 and modifying, by extrusion of the external faces, a simplified replica of the same internal matrix 3,4,5,6,7, such that the designed internal walls of the device 1,2 are very close to the external walls of the internal matrix design 3,4,5,6,7, but also far enough apart so that they do not touch each other. This characteristic allows that, after the simultaneous prototyping of the internal matrix 3,4,5,6 and the external chamber 1,2, the means of seeding, culturing and cell differentiation subsequently used inside, be more efficient in the seeding, feeding, and stimulation of cells that will adhere to the inner and outer surfaces of the inner matrix 3,4,5,6,7 when they are circulated through them, within the outer chamber 1,2, since all the culture medium used will remain permanently in close contact with the cells adhered to the inner and outer surface of the inner matrix 3,4,5,6,7.

Given this characteristic, and despite being simultaneously prototyped, the internal matrix 3,4,5,6,7 and the external chamber 1,2 do not join at any point, facilitating the removal of the internal matrix 3,4 , 5,6,7 inside the external chamber 1,2 where the sowing and cell culture period took place.

Two tubular structures 8 are preferably added to the design of the external chamber 1,2 which connect the interior of the chamber 1,2 to the exterior and allow the entry and exit of fluids and gases from / to the interior of the chamber 1,2. The location of these tubular structures 8 must be determined taking into account the best possible efficiency in the drainage of the fluids introduced into the chamber, as well as the improvement of the fluid flow dynamics inside the chamber 1,2.

To the design of the external chamber 1,2, grooves 9 are also added, which will later be used to facilitate its precise cutting at predetermined locations, thereby facilitating removal of the matrix 3,4,5,6,7 from the interior of the external chamber 1,2.

The culture chamber 1,2 as well as the internal matrix 3,4,5,6,7 contained within it are manufactured simultaneously, through a process of rapid prototyping from one or multiple materials. These materials can be biodegradable or not, inert or not, polymeric or composite, transparent, translucent or opaque.

When a rapid prototyping process is used to manufacture the bioreactor, which is composed of a culture chamber 1,2 and an internal matrix 3,5,6,7, an additional water soluble support material can be used, which is also deposited during the prototyping process. The sole purpose of this material is to support the deposition of the material or materials from which the chamber and the matrix will be made, and can subsequently be removed by immersion in water or aqueous solution.

Preferably, the architecture of the chamber 1,2 and its internal matrix 3,5,6,7 should be designed to support themselves, eliminating the need for additional support material.

When the outer chamber is prototyped from one or more transparent or translucent materials, it also becomes possible to analyze the interior of the chamber by optical means. If the internal matrix 3,4,5,6,7 located inside the chamber is also subjected to prototyping from transparent or translucent materials, it also becomes possible to analyze the interior of that matrix 3,4,5,6, 7.

The chambers 1,2 and their internal matrices 3,4,5,6,7 can be sterilized by various methods depending on the materials used. They can be sterilized by immersion in ethanol, by exposure to ultraviolet radiation or ethylene oxide, in the absence of adverse reactivity, or even by sterilization in an autoclave, in case the materials used are sufficiently stable when exposed to high temperatures.

In order to improve the performance of the internal matrix 3,4,5,6,7 contained within the chamber 1,2, it is possible to carry out coatings on its surfaces by filling and / or perfusing the interior of the chamber, through of the pores of the matrix, with solutions or gases capable of generating these coatings. Additional coatings can also be made on the outer walls of chamber 1,2 for various purposes, such as increasing the watertightness of chamber 1,2.

The bioreactor can be integrated into the interior of a culture system illustrated in figure 10. This system is composed of the bioreactor in one of its possible configurations, comprising an external chamber 1,2 containing one of the various kinds of internal matrices. 3,4,5,6,7, connected to a culture medium tank 10 by tubes connected to their tubular structures 8. Apart from the two medium inlets / outlets, the tank 10 also has an additional connection for the inlet and Gas outlet that is purified by an air filter 11.

The culture medium is collected from the culture medium tank 10, it is pumped by a peristaltic pump 12 into the outer chamber 1,2, it is perfused through the pores of the inner matrix 3,4,5,6, 7 and finally it is pumped by the same peristaltic pump 12 back into the culture medium tank 10. This process and circuit is repeated for each individual culture cavity.

For the circulation of the culture medium, tubes made from formulations such as silicone should preferably be used since they are highly permeable to gases such as carbon dioxide and oxygen, increasing the exchange of gases between the circulating medium and the surrounding atmosphere. .

This culture system can be used not only for culturing, but also for seeding cells on the inner and outer surfaces of the matrix 3,4,5,6,7 for cell growth. Given its small internal volume, this device requires very low volumes of culture medium. For this reason, it is possible to perform dynamic seeding procedures using highly concentrated cell suspensions without using extremely large numbers of cells. In this way, the cells have a greater probability of adhering to the internal and external surfaces of the matrix 3,4,5,6,7 since they are highly concentrated and circulate more often through it, making the process seeding more efficient.

When integrated within the perfusion culture system described in Figure 10, this bioreactor is capable of enhancing cellular mass transport through the matrix with 3,4,5,6,7 cells. The continuous culture medium perfusion provided by the culture system can act as a stimulus on cells, resulting in the regulation or enhancement of cell proliferation and differentiation.

In addition to allowing seeding and cell culture by means of a perfusion-based method, this device also allows the use of other methodologies such as static or shaken seeding. When performing static seeding, a cell suspension is injected into the chamber filling all its internal empty spaces and, in particular, the pores of the internal matrix 3,4,5,6,7. After sealing the tubular structures 8 with caps, the cell suspension is kept within the chamber 1,2 and the pores of the matrix 3,4,5,6,7 for a period of time sufficient for the cells contained in the cell suspension adhere to the internal matrix 3,4,5,6,7. During this seeding, the device can be kept in static conditions or also under agitation in order to increase the possibility that the cells will come into contact with the surfaces of multiple matrices 3,4,5,6,7 and thus increase the possibility of adhesion, as well as allowing a more homogeneous distribution of adhered cells on the surface of the matrix 3,4,5,6,7.

After the seeding period, the cell suspension containing unattached cells is removed from the interior of the device and replaced by a new differentiation and / or expansion medium. Also, during the period of differentiation and / or expansion, the device containing differentiation and / or expansion medium within it can be kept under static conditions or alternatively subjected to agitation.

In order to maintain a sterile environment, with stable and adequate temperature and humidity, the bioreactor, integrated or not in a perfusion system, is placed inside a cell culture incubator.

Claims (11)

i. Bioreactor for the generation of medical implants with cells characterized in that it comprises a unicompartmental (1) or multi-compartment (2) watertight external chamber that contains one or more matrices (3,4,5,6,7) inside, being manufactured simultaneously these inner matrices (3,4,5,6,7) and the outer chamber (1,2) of the bioreactor through a process of rapid prototyping; in which the external chamber (1,2) comprises a shape adapted to the implant shape to have cells (internal matrix), and because it allows the implants to be removed from the interior of the bioreactor by cutting the external chamber (1,2 ) in locations comprising grooves (9) just before implantation, enabling sterility of the implant. Bioreactor according to the previous claim, in which the culture chamber (1,2) and the internal matrices (3,5,6,7) are supported by themselves, although allowing the use of an additional support material, They are also deposited during the prototyping process, which can later be removed by immersion in water or aqueous solution. 3. Bioreactor according to any of the preceding claims, wherein the design of the external shape of the simple matrix (3,4,5,6) is achieved using simple shapes as initial references such as cubic, cylindrical, tubular or other shapes, while that the design of the external shape of the internal complex matrix (7) is achieved by resorting to complex shapes obtained from tissue or organ analysis performed using technologies such as computed tomography or magnetic resonance imaging. 4. Bioreactor according to any of the preceding claims, in which the design of the external chamber (1,2) has as an initial reference the external shape of the internal matrix (3,4,5,6,7) in order to that the designed internal walls of the device (1,2) are sufficiently separated so that they do not touch the external walls of the internal matrix design (3,4,5,6,7) with a spacing similar to the pore diameter of the internal matrix, the diameter of which is preferably between 0.25 mm and 3 mm. Bioreactor according to any of the preceding claims, in which the internal walls of the external chamber (1,2) are sufficiently separated so that they do not touch the external walls of the internal matrix (3,4,5,6,7 ) forming a cavity, whose internal filling volume guarantees the immersion of the internal matrix, by means of which culture media and / or solutions or gases can be circulated. 6. Bioreactor according to any of the preceding claims, in which it allows coatings to be made on its surfaces by filling and / or perfusing the interior of the chamber, through the pores of the internal matrix, with solutions or gases that are capable of to generate those coatings. Bioreactor according to any of the preceding claims, in which the outer chamber has at least two tubular structures (8) that connect the interior of the outer chamber (1,2). Bioreactor according to any of the preceding claims, in which it is manufactured from one or more transparent or translucent materials. Bioreactor according to any one of the preceding claims, in which the cells / tissues incorporated inside the internal matrices (3,4,5,6,7) grown inside the bioreactor can be subjected to static or dynamic conditions by means of the integration of the bioreactor in a perfusion and / or shaking system. 10. Bioreactor according to any of the preceding claims, wherein the internal matrices (3,4,5,6,7) are perfused uni- or bidirectionally. 11. Culture system for the generation of medical implants with cells, wherein it comprises a bioreactor according to any of claims 1-10 for the generation of medical implants with cells, an external chamber (1,2) containing one of the various kinds of internal matrices (3,4,5,6,7), connected to a culture medium tank (10) by means of tubes connected to their tubular structures (8) and an additional connection for the inlet and outlet of gases which are purified by an air filter (11).