EP2788469A1 - Single-step method and device for the generation of stratified tubular tissue substitutes - Google Patents

Abstract

Device and method for manufacturing stratified tubular tissue. The device comprises a lower rectangular part (3) over which a porous membrane (4) is centrally placed, a malleable part with a closed (5) or open (6) configuration, provide with tubular structures (9) and defining at least two contiguous cavities in its lower surface, placed over the lower rectangular part (3) and over specific zones of the porous membrane (4), and a third part (7) with screws (8), which compresses the malleable part (5 or 6) and particularly against the porous membrane (4).

Description

DESCRIPTION

SINGLE—STEP METHOD AND DEVICE FOR THE GENERATION OF

STRATIFIED TUBULAR TISSUE SUBSTITUTES

OBJECT OF THE INVENTION

The present invention is related to the manufacturing of stratified and tubular tissue substitutes for the replacement/regeneration of composite animal or human tissues, which may have been previously damaged by means of disease or trauma. The present invention describes a device which enables the simultaneous culture of multiple and different cellular types into porous membranes, as well as the method to manufacture stratified tubular tissue substitutes from those porous membranes cultured with multiple different cellular types.

BACKGROUND

Live tissues are assemblies of cells arranged in a specific organized fashion. In some cases, cells are all structurally and functionally alike, forming simple tissues (e.g. cartilage, epithelial and adipose tissues). However, most tissues in the human body contain a mixture of cells with distinctive functions, which may be termed compound tissues (e.g. bone, skin, nervous and vascular tissues. Cells that form tissues can be divided into parenchymal

- 1- substitute sheet cells, which maintain tissues, and support cells, which provide the structural scaffolding of a tissue. Support cells comprise a set of highly developed cell types with complex metabolic functions and produce an extracellular matrix (ECM) , which largely defines the physical characteristics of a tissue. The support cells together with ECM are organized in an elaborate and hierarchical order to achieve multi-scale functions and to mutually regulate the cellular activity by soluble bioactive molecules, cell-cell direct contact, or cell-ECM interaction. This elaborate structure also provides individual cells with different microenvironments, where cells experience specific cues and show corresponding responses towards tissue function.

Tissue Engineering has been recognized, for some time, as a promising alternative to the use of autografts or allografts for tissue reconstruction and regeneration. This approach utilizes cells, biomaterial scaffolds and signaling molecules for the repair of diseased or damaged tissues. Despite the great progress in this field, development of clinically relevant size tissues with complex architecture remains a great challenge. This is mostly due to limitations of nutrient and oxygen delivery to cells and limited availability of scaffolds, which can mimic the complex live tissue architecture. Biomaterial scaffolds are designed to support cell and tissue growth, aiming at a macroscopic level to be compatible with the

-2- substitute sheet mechanical loading of the surrounding organs and tissues but without the need to recreate the complexity and nanoscale detail observed in tissues and organs at the extracellular level. In a Tissue Engineering and Regenerative Medicine approach, the development of a synthetic ECM is a critical issue, since we need to learn from nature how to engineer biomaterials that will help in recapitulating the early events of morphogenesis that lead to the formation of the hierarchical organization of the ECM and drive the cells to build fully functional adult tissues. To maintain the proper cell phenotype in 3D tissue engineering one needs to achieve biomimetic design to replicate the ECM, seeding/infiltration of cells into a biomaterial scaffold and culturing the seeded scaffold withadequate nutrient supply.

The in vivo microenvironment for each cell type varies from tissue to tissue and from site to site, and this variation provides and conveys specific cues to cells for specific functions. Besides the recognized complex hierarchical organization, cells in tissues constantly experience mechanical stimuli. Therefore, culturing cells in the appropriate biochemical environment and in the presence of mechanical stimuli will provide a more realistic extracellular microenvironment and, consequently, will improve cell-cell and cell-matrix interactions. Various developed bioreactor systems introduce convective flow of the medium to perfuse in vitro-grown 3D tissue

-3- substitute sheet constructs, e.g. spinner flasks, rotary vessels or perfusion flow systems. The use of these bioreactor systems can allow a homogeneous cell seeding of the biomaterial scaffold, a good nutrient distribution through the scaffold, and an efficient removal of metabolites at the cellular and sub-cellular levels. Consequently, there is a huge scientific interest in obtaining the appropriate mechanical environments to improve the quality and functionality of the in vitro-generated hybrid constructs.

DESCRIPTION OF THE INVENTION

The present invention describes a device, which enables the simultaneous culture of multiple and different cellular types into porous membranes as well as the method to fabricate stratified tubular tissue substitutes from those porous membranes cultured with multiple different cellular types.

The device herein described allows to separately seeding and culture multiple and different cell types into separate areas of one same porous membrane.

The device allows delimitating multiple watertight areas on the surface of porous membranes by compression of those same membranes over specific zones. This compression is carried out by placing the membrane in between a lower rectangular plate and a malleable part, which defines

-4- substitute sheet cavities on its lower surface. This malleable part is in turn compressed against the porous membrane and the lower rectangular plate by means of a third part which is screwed to the lower rectangular plate.

The malleable part, which is part of this device, can be closed, making the cavities that it delimitates closed, or open, making the cavities that it delimitates open to contact with the exterior by means of upper apertures. In order to enable the injection/removal of cellular suspensions and/or culture medium from and to the interior of the closed culture cavities, tubular structures are used, which pierce the walls of the malleable part and connect each of the cavities to the exterior.

This device, in particular in its closed configuration, can be integrated into a dynamic cell culture system able to automatically seed the porous membrane's separate areas, which are delimitated by the device' s culture cavities as well as to renew the culture medium inside those cavities.

Due to the transparency of most of the materials utilized in its construction, this device allows as well for the observation of the interior of the internal chambers through the walls of its parts.

-5- substitute sheet Due to the characteristics of the materials utilized in its construction, the device can as well be easily sterilized by chemical or thermal methodologies.

The method herein described enables the construction of stratified tubular tissue substitutes by resorting to porous membranes possessing specific zones of their surfaces seeded with multiple and different cell types.

The method herein described consists of rolling a porous membrane, which is seeded with multiple and different cell types, around a porous rolling structure. This rolling is started in the extremity of the porous membrane containing the cell type that should be situated in the more internal layer of the stratified tubular tissue substitute and finalized in the extremity containing the cell type which should be located in the tissue substitute's most external layers.

The method herein described is finalized after a culture period, which allows for the adhesion of cells to the surfaces of adjacent membranes. By removing the tubular and porous rolling structure a stratified tubular tissue substitute is generated.

-6- substitute sheet BRIEF DECRIPTION OF THE DRAWINGS

Figure 1 shows in exploded isometric view the device in closed configuration.

Figure 2 shows in isometric view the assembled device in closed configuration.

Figure 3 shows in isometric view a partial section of the assembled device in closed configuration.

Figure 4 shows a longitudinal section of the device in closed configuration.

Figure 5 shows a transversal section of the device in closed configuration.

Figure 6 shows in exploded isometric view the device in open configuration.

Figure 7 shows in isometric view the assembled device in open configuration.

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

Figure 9 shows longitudinal section of the device in open configuration.

-7- substitute sheet Figure 10 shows a transversal section of the device in open configuration.

Figure 11 shows the device in closed configuration integrated into a complete dynamic cell culture system.

Figure 12 shows the porous membrane and rolling structure before rolling the porous membrane containing three different cellular populations over its surface in order to generate a stratified tubular structure .

Figure 13 shows the porous membrane partially rolled around the rolling structure in order to generate a stratified tubular structure.

Figure 14 shows the porous membrane totally rolled around the rolling structure.

Figure 15 shows a transversal section of the porous membrane rolled around the rolling structure and showing its inner stratified structure possessing different cellular populations located into different layers .

Figure 16 shows the stratified tubular tissue substitute after removal of the rolling structure.

-8- substitute sheet DETAILED DESCRIPTION OF THE INVENTION

This description refers to a preferential configuration of the invention resorting to the figures in this document in order to allow for a better understanding of the invention.

The device, which may possess a closed 1 or open 2 configuration, comprises a lower rectangular part 3, over which a porous membrane 4 is centrally placed. A malleable part, which can possess a closed 5 or open 6 configuration, defining three contiguous cavities on its lower surface, is placed over the lower rectangular part 3 and over specific zones of the porous membrane 4. By using a third part 7 and screws 8 the malleable part 5 or 6 is compressed against the porous membrane 4. Given that the malleable part 5 or 6 defines three cavities on its lower surface, only the extremities and zones situated in between cavities are compressed. In this way, the surfaces submitted to compression become watertight surfaces consequently generating three watertight cavities.

Tubular structures 9 are added to the closed type malleable part 5) , which pierce its lateral walls and are used for injection, removal and circulation of fluids such as cell suspensions and culture media from/to the interior of the cavities previously delimitated by the device. In

-9- substitute sheet this configuration, each device cavity should be connected to the exterior by at least two of these tubular structures 9, in a way to allow the entry of fluids and gases through one tubular structure and the simultaneous exit of excess fluids and gases through the other tubular structure. If desired, by simultaneously controlling the entry and exit of fluids and gases, it is possible to exert positive or negative pressures to the interior of each cavity.

In case of open type malleable part 6, cell suspensions and culture media are simply placed, removed or circulated into the cavities through their upper apertures.

The lower rectangular part 3 is preferably manufactured from polycarbonate or glass. The preferential utilization of these materials in the manufacturing of this part is related with their chemical, mechanical and optical properties since they are biologically inert, extremely resistant to solvents, possess good dimensional stability and good resistance to high temperatures. The resistance to solvents and high temperatures confers great versatility in terms of the sterilization process to be used since it allows sterilization both through exposure to solvents and to high temperatures (autoclaving) . Additionally, these materials confer an advantage by being transparent, allowing for the content of each cavity to be visualized through the inferior and upper part of the device.

-10 substitute sheet The malleable parts 5 and 6 are manufactured through a molding process, preferably from silicone. Like polycarbonate and glass, silicone is biologically inert, resistant to solvents and to high temperatures. Therefore, these parts can also be sterilized both by exposure to solvents and to high temperatures (autoclaving) . Due to the silicone transparency, these parts allow the content of each cavity to be visualized through its lateral walls, as well as, in the case of the closed malleable part 5, through its upper wall. Additionally, silicone is permeable to gases, enabling the exchange of gases between the culture cavities and the exterior. This feature is particularly important in the case of devices with closed configuration 1.

As for the compression part 7 and screws 8, as they do not come into direct contact with the interior of the culture cavities or the porous membrane 4, they do not need to be transparent or inert and can be manufactured from a greater variety of materials, as long as they are dimensionally stable and resistant to solvents and high temperatures .

The porous membrane 4 should preferably be manufactured from a material or combination of biocompatible and biodegradable materials, which can be processed by various methods, such as electrospinning . The size of the membrane pores should also be preferably less than the diameter of cells to be cultured onto the membrane

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substitute sheet surface in order to enable an efficient retention of each specific cell type into specific layers of the tubular structure to be fabricated.

Although the devices herein described possess three cavities, these can vary both in number and in size, according to the intended application.

The devices herein described can simultaneously contain three different cell cultures (one in each cavity) , which can vary in various ways, such as cellular type and density or culture medium used.

In its closed configuration 1, the device can be integrated into a culture system illustrated in figure 11. This system comprises the device 1 connected to a culture medium reservoir 10 by tubes connected to its tubular structures 9. In addition to the connections for entry/exit of medium, the reservoir 10 also possesses a further connection for the entry and exit of gases which are purified by an air filter 11.

Briefly, the culture medium is collected from the culture medium reservoir 10, pumped by a peristaltic pump 12 to the culture cavity inside the device and finally pumped by the same pump 12 again to the culture medium reservoir 10. This process and apparatus is repeated for each one of the individual culture cavities.

-12 substitute sheet For the circulation of culture medium, tubing made from formulations, such as silicone, should preferably used be since they are highly permeable to gases such as carbon dioxide and oxygen, increasing the gas exchange between circulating medium and surrounding atmosphere.

In order to keep a sterile environment, with stable and adequate temperature and humidity, the system is placed inside a cell culture incubator.

The culture system can be used not only for culture but as well as for the seeding of cells onto membranes for cellular growth. Given its small dimensions, 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 amounts of cells. In this way, cells have a greater chance to adhere to the porous membrane's surfaces 4 since they are highly concentrated and are circulated more often through the membrane's surfaces, making the seeding process more efficient.

After sterilization and assembly of the device in its closed 1 or open 2 configuration, containing in its interior an equally sterilized porous membrane by properly compressing its extremities and inter-cavity areas, the device is ready for the start of the cellular seeding over the porous membrane 4 surface.

-13 substitute sheet Cellular seeding can be performed by different ways depending on the device's configuration. When an open configuration device 2 is used, a cell suspension can be simply transferred to the interior of the cavities, through their upper apertures, over the porous membrane' s 4 surface. Since there are three contiguous independent cavities, it is possible to transfer suspensions composed of different cell types or combinations of cell types to each one of the cavities. The cell suspension should be of sufficient volume to cover the porous membrane' s surface delimitated by each cavity. After having cell suspensions transferred to the cavities, a lid should be placed over the device in order to avoid evaporation.

When using a closed configuration device 1, the cell suspension is injected into the cavities through one of the tubular structures 9, which connect the cavities to the exterior. The injection can be performed using a syringe attached to the external part of the tubular structure. In this case, the second tubular structure of each cavity should be kept open so that the air, and probably medium excess, are expelled from the chamber and so avoiding excessive pressure. After this procedure all tubular structures should be closed with lids.

When using a closed configuration 1 it is also possible to perform dynamic cellular seeding by means of perfusion using the culture system described in figure 11.

-14 substitute sheet After connecting the system tubing to each cavity tubular structures 9, a cell suspension, previously transferred to the culture medium reservoir 10, is pumped and circulated through the interior of each one of the device cavities.

The required time for performing each one of the seeding methods is variable, depending on various factors such as the type of cells use and operator preferences .

After the cell seeding period, an additional culture medium volume is added to the interior of the cavities or, in case a dynamic seeding/culture system is used, to its culture medium reservoir 10. From this point on, the cell culture period is started. This period can be meant for expansion and/or differentiation, according to the type of supplements included into the culture medium, and can be kept for variable periods of time. During the culture period, culture medium should be regularly renewed, totally or partially, according to the intrinsic necessities of each cell type in culture and to the operator' s preferences. This renewal is performed using the same procedures and apparatus as in the seeding step, after total or partial removal of the culture medium contained into the culture cavities and/or dynamic culture fluidic circuit .

-15 substitute sheet At the end of the cell culture period, culture medium is totally removed from the culture cavities and/or dynamic culture fluidic circuit and the device disassembled.

As a result, a porous membrane seeded 13 with cells in three separate areas of its surface is obtained. Each separate area contains one cell type or combination of cell types different from the ones found in the other seeded areas .

This seeded and cultured porous membrane 13 possessing three different cell types into separate areas of its surface is then rolled around a porous cylindrical or tubular rolling structure 14 in order to generate a stratified tubular structure 15 around that same rolling structure 15. The rolling should be initiated from the porous membrane extremity which is closer to the internal cellular colony 16, that is, the cellular colony which should be located in the more internal layers of the generated stratified tubular structure 15. After that, the intermediate cellular colony 17 is rolled, which shall be located in the intermediate layers of the stratified tubular structure 15, and finally the external cellular colony 18 which shall be located in the more external layers of the stratified tubular structure 18.

The rolling tubular structure 14 should preferentially be porous in order to actively or passively allow a more

-16 substitute sheet efficient nutrition of the internal 16 and intermediate 17 cellular colonies while rolled around the rolling structure 14.

After the rolling process, the stratified tubular structure 15 should preferably be kept for a certain period of time rolled around the rolling structure 14 and immersed in culture medium in order to allow the cells contained into the various layers to adhere to the surfaces of membranes in adjacent layers. Furthermore, some kind of biocompatible adhesive, such as, for example fibrin-base sealants, can be applied to the membrane superficial extremities in order to reinforce the formed stratified tubular structure 15 stability.

Finally, after a sufficient culture period over which a consistent cellular matrix can be generated into the stratified tubular structure 15, the tubular rolling structure 14 is removed from the interior of the stratified tubular structure 15. In this way, a ready-to-use stratified tubular tissue substitute 19 is obtained.

The porous rolling structure 14 should preferably be manufactured from politetrafluorethylene (PTFE) . The choice of PTFE for fabricating this structure is justified by its reduced friction coefficient, which facilitates the process of removing the structure from inside the stratified tubular structure 15 and so preventing from damage to the

-17 substitute sheet latter structure. Besides, PTFE is characterized by its excellent dimensional stability, constant mechanical properties, inertness and biocompatxbxlity. Finally, it shows also great resistance to solvents and to high temperatures being easily sterilizable by use of solvents or by autoclaving.

-18 substitute sheet

Claims

1. Device for the separate seeding and culture of multiple and distinctive cell types in separate areas of the same membrane, characterized in that it comprises a lower) rectangular part (3) over which is centrally placed a porous membrane (4) ; a malleable part, which can possess a closed (5) or an open (6) configuration, defining at least two watertight contiguous cavities on its lower surface, placed over the inferior rectangular part (3) and over specific zones of the porous membrane (4) ; and a third part (7) that, by the use of screws (8), compresses the malleable part (5 or 6) against the porous membrane (4) .

2. Device, according to claim 1, characterized in that it allows a closed (5) or an open (6) configuration.

3. Device, according to the previous claims, characterized in that at least two tubular structures (9) are added to the closed type malleable part (5) ) , which penetrate its lateral walls and are used for injection, removal and circulation of fluids such as cell suspensions and culture media from/to the interior of the cavities previously delimitated by the device.

4. Device, according to the previous claims, characterized in that the malleable part with a closed (5) or open (6) configuration, which defines at least two

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substitute sheet contiguous cavities on its lower surface, causes that only the zones of the porous membrane (4) placed under its extremities and zones between cavities are compressed upon compression against the porous membrane (4) and the lower rectangular part (3), making the surfaces subject to watertight surfaces and generating two watertight cavities.

5. Device, according to the previous claims, characterized in that it allows the use of any cell type, from animal or human origin, cell lines or primary cells, in procedures of seeding, adhesion and/or culture.

6. Device, according to the previous claims, characterized in that the porous membrane (4) is preferentially manufactured from a biocompatible material and in that the size of the membrane pores is preferably smaller than the diameter of cells to be cultured onto the membrane surface.

7. Device, according to the previous claims, characterized in that the lower rectangular part (3) , the malleable parts (5) and (6) , the compression part (7) and the screws (8) are manufactured from materials resistant to solvents, possessing good dimensional stability and good resistance to high temperatures, preferentially polycarbonate, glass and silicone; the lower rectangular part (3) being manufactured from a biologically inert material preferably polycarbonate or glass; and the

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substitute sheet malleable parts (5) and ( 6) being manufactured from a biologically inert material and through a molding process preferably from silicone.

8. Device, according to the previous claims, characterized in that it is manufactured from transparent materials, preferentially polycarbonate, glass and silicone, able to be sterilized both by chemical and thermal methods .

9. Dynamic culture system using the device for the separate seeding and culture of multiple and distinctive cell types in separate areas of the same porous membrane, characterized in that it is composed by the closed configuration of the device (1) connected to a culture medium reservoir (10) by tubes connected to its tubular structures (9); the reservoir (10) possesses one connection for the entry/exit of medium and an additional connection for the entry and exit of gases, which are purified by an air filter (11); the culture medium is collected from the culture medium reservoir (10), pumped by a peristaltic pump (12) to the culture cavity inside the device and finally pumped by the same pump (12) back into the culture medium reservoir (10) ; this process and apparatus being repeated for each one of the individual culture cavities contained into the device in its closed configuration (1) .

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10. Dynamic culture system, according to claim 9, characterized in that the circulation of the cell seeding suspensions or the expansion or differentiation medium is continuous or discontinuous and performed unidirectionally or bxdirectionally.

11. Dynamic culture system, according to the claims 9 and 10, characterized in that it uses tubings made from materials such as silicon, which are permeable to gases, such as carbon dioxide and oxygen, in order to increase the gas exchange between circulating medium and surrounding atmosphere.

12. Method for the generation of stratified tubular tissue substitutes, characterized in that it comprises a first phase of seeding cells on specific areas of the porous membrane (4) located inside and delimitated by internal cavities of a device with a closed (5) or open (6) configuration; followed by a second phase of expansion and/or differentiation of cells cultured at the surface of the porous membrane (4) ; followed by a third phase of rolling of the seeded and cultured porous membrane (13) around a rolling structure (14); and finally followed by a fourth phase of post-culture and removal of the tubular rolling structure (14) from the interior of the stratified tubular structure (15), generating a stratified tubular tissue substitute (19) .

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substitute sheet

13. Method for the generation of stratified tubular tissue substitutes according to claim 12, characterized by a rolling phase where, after disassembling of the device with a closed (5) or open (6) configuration used in the culture phase, the seeded and cultured porous membrane (13) generated in the interior of that device is rolled around a porous cylindrical or tubular rolling structure (14) initiated from the porous membrane' s extremity which is closer to the internal cellular colony (16), that is, the cell type which should be located in the more internal layers of the generated stratified tubular structure (15) , and finalized in the external cell type (18) , which shall be located in the more external layers of the stratified tubular structure (18) , in which some kind of biocompatible adhesive can be applied to reinforce the stability of the formed stratified tubular structure.

14. Method for the generation of stratified tubular tissue substitutes according to claims 12 and 13, characterized by a post-culture phase, where the stratified tubular structure (15) is immersed in culture medium to allow the cells contained into the various layers to adhere to the surfaces of membranes in adjacent layers; then, the tubular rolling structure (14) is removed from the interior of the stratified tubular structure (15), generating a stratified tubular tissue substitute (19) .

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substitute sheet

15. Method for the generation of stratified tubular tissue substitutes according to claims 12 to 14, characterized in that a porous rolling structure (14) is manufactured from a material with reduced friction coefficient, good dimensional stability, constant mechanical properties, inertness, biocompatibility, and with great resistance to solvents and to high temperatures, preferentially politetrafluorethylene (PTFE) .

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substitute sheet