Patent Application
20050049581
This invention refers to a hybrid circulatory system with which the transportation of cells and/or substances within a biological organism can be mimicked, in particularly the human body. The natural example is blood with plasma, transferring substances and blood cells, e.g. from hematopoietic-, immune-, and stem cell systems. Such circulatory systems are essential in the development of methods in cell biology, medical therapy, regenerative medicine, tissue engineering, and stem cell applications. Such systems can provide cells for extracorporeal organ-systems, e.g. bio-artificial liver support. Likewise, cells can be prepared and produced, especially progenitor cells for cell transplantation in cell-based therapy. These systems are generally of interest for the production of certain types of cells or metabolic products like mediators, effectors, antibodies, proteins, vaccines and such; whereby organ typical cells can be cultivated, differentiated, and propagated, while communication between cells of different location plays a role, e.g. hybrid bone marrow.
Devices for metabolic exchange, e.g. bioreactors, cell perfusion devices, and general modules, especially for liver support systems, are already known as alternative method for animal experiments, the production of biological cell products, or in the area of organ support.
A particularly effective module is described in the EP 059 034 A2 (Gerlach, J. C.)/U.S. Pat. No. 08/117,429: 1993. The described module for the culture and utilization of metabolic performance and/or maintenance of microorganisms consists of a casing with at least three independent membrane systems arranged inside. Of these membrane systems at least two independent membrane systems are developed as hollow fiber membranes. These hollow fiber membranes form a tightly packed 3D interwoven spatial network. The microorganisms are immobilized in the cell compartment of the network and/or to the hollow fiber membrane surfaces.
A first independent hollow fiber membrane system serves for the media inflow. A second independent hollow fiber membrane system serves for the gas supply of the microorganisms with oxygen, and the removal of CO2. The outflow of the media is guaranteed through a third independent membrane system.
Each individual, independent hollow fiber membrane system consists of a multitude of individual hollow fiber membranes, whereby the hollow fibers of a system communicate through at least one inflow head, respectively one inflow and outflow head. Thereby, the simultaneous media supply through the inflow head to the hollow fibers of each independent system is guaranteed. Furthermore, the individual hollow fibers are interwoven with each other.
These independent hollow fiber membrane systems create a multi-compartment system in a spatial, tightly packed, interwoven network inside the module in such a way that almost anywhere in the network the organisms have almost identical conditions for substrate supply. Therewith, the conditions in physiological organs with arteries and veins, e.g. the liver, with the arrangement of hepatocytes in lobules are largely simulated. Through the independent arrangement of different membrane systems the module presents the advantage of a decentralized transport of nutrients, products for synthesis, gases, to/from a multitude of microorganisms independent of their position inside the module, the same way as it is in the cell environment of natural organs. The outflow of media is ensured through the third independent membrane system. This membrane system can be a hollow fiber membrane, an exchangeable flat membrane, or an exchangeable capillary membrane. It is crucial that the third membrane system is independent form the other two hollow fiber membrane systems.
One design suggests that the tightly packed network in the inside is constructed from independent hollow fiber membrane systems. In this case all independent membrane systems are hollow fiber membrane systems that are arranged in the inside. Here, one independent hollow fiber membrane system serves the inflow of media, a second hollow fiber membrane system serves the outflow of media, and a third system serves for the supply of other substances, e.g. oxygen. The tightly packed network consists of these three independent systems. Alternative to mass exchange from one to the other hollow fiber membrane system is their use for counter-directional flow operation.
The tightly packed network can be constructed in various ways as long as it is guaranteed that the microorganisms inside receive an identical substrate supply. The tightly packed network can consist of, for instance, tightly packed layers each with alternating layers of independent systems. The second layer, also consisting of individual hollow fiber membranes, is arranged on the same plane, however opposite the first layer, rotated by, for example, 90 degrees.
These layers alternate and create a dense package. The third independent hollow fiber membrane system that, once again, consists of individual layers of hollow fiber membranes, infuses the first two layers vertically form top to bottom and thereby “interweaves” the first two independent layers.
A further design plans for three independent hollow fiber membrane systems with alternating, overlaying layers that are all arranged in one plane but each rotated about 60 degrees.
This tightly packed network is arranged inside the module. Because each independent system communicates with at least one inflow, respectively one inflow and outflow, even distribution of inflowing media as well as steady oxygenation is ensured. Through the third independent system for the outflow of media, the media can continuously and consistently be eliminated from anywhere in the module.
In a further design, in addition to the three hollow fiber membrane systems, an additional independent membrane system is used inside the module for media outflow. For that purpose and exchangeable flat membrane or an exchangeable capillary membrane can be mounted on the outer casing.
A further design plans that the tightly packed network is generated from two independent hollow fiber membrane systems, whereby one serves for the inflow of media and the other for oxygenation. A third independent membrane system, which is an exchangeable flat-or capillary membrane and mounted on the outer casing serves for the outflow of media.
The tightly packed network in the inside, which is generated from the two hollow fiber membrane systems, is designed analogous afore described systems.
The use of hydrophilic or hydrophobic polypropylene, polyamid, polysuphon, cellulose, or silicon-rubber is preferred for hollow fiber membranes. The selection of hollow fiber membranes depends on the molecules planned for substance exchange. However, all state of the art hollow fiber membranes, known as substance exchange devices (or mass exchange devices), can be used.
By using three independent hollow fiber membrane systems, which form a tightly packed network, a capillary system of fluid impermeable capillaries, e.g. stainless steel or glass can be used, which can serve to control the temperature inside the module. This system also facilitates the even cooling of the module, its inside and the infuse microorganisms, below −20 degrees Celsius. In a further design all other hollow fiber membrane systems can also be used for temperature control, respectively cool down below the freezing point.
In a further design the outer casing is made from a poured cast, whereby it is ensured that an access way form outside into the volume of the capillaries is possible.
In another design the module exhibits various additional access ways. One access way serves as inflow device for microorganisms into the module. Additional access ways serve for instance for pressure-, pH-, and temperature measurements inside the modules.
This bioreactor already exhibits excellent results in regards to substrate supply and substrate removal. A further module that has been submitted simultaneously on the same day, by the same inventors, along with this registration is known as “Module for the culture and utilization of metabolic performance and/or for the maintenance of microorganisms”(German patent application #103 26 744.1 of 13 Jun. 2003, J. Gerlach). This module consists of a body that is arranged inside a water-/germ tight container, whereby the body is designed with open pores that can communicate with each other. Simultaneously this body exhibits at least one channel like hollow pathway system whose individual hollow pathways infuse the body and intersect and/or overlay each other. Because the body inside the container is made of porous material, whose pores can communicate with each other, the connection between the pores via their connections to the independent, channel like hollow pathway systems is guaranteed. Inside the module, microorganisms, particularly cells, inside the pores of this porous body, are immobilized without completely filling it up. Through the independent, channel like, hollow pathway systems, arranged inside the body, a consistent supply and waste disposal of the microorganisms inside the open pores, especially the cells, can occur from anywhere in the body with a low substrate gradient. Because the arrangement of the pathways, mass exchange is comparable to the module described above. The hollow fiber membranes and the hollow channel-like pathways with their walls to the open porous body serve comparable functions. This module replicates the organ supply similar to the natural organ. With this module, because of the open pores of the hollow pathway system, a bioreactor is available that facilitates an optimal substrate supply and removal of a relatively large amount of microorganisms over longer periods of time.
A channel like hollow pathway system is advantageously developed in such a way that it consists of collateral channels arranged in one plane. It is advantageous if the channel like hollow pathway system consists of several such planes that overlay each other in a predetermined distance. The distance between the individual channels of a hollow pathway system in a plane and between the individual planes is preferably in the range from 0.5-5 mm. The diameter of the individual channels is preferably 0.1-3 mm. The body of the module can exhibit at least two such hollow pathway systems that intersect and/or overlay each other.
This facilitates a substrate exchange across both hollow pathway systems, respectively between both hollow pathway systems, via counter current flow and therefore with relatively high mass exchange capacity and low substrate gradients.
An advantageous design is arranged with intersecting hollow pathway systems. Therefore one hollow pathway system, preferably consisting of several overlaying planes, infuses the body form on direction, and the second hollow pathway system infuses the body in the other direction at, for example, a 90 degree angle. Because the planes are arranged in afore mentioned distance the supply and removal of substrate from the microorganisms, inside the pores of the open porous body, is guaranteed almost anywhere in the body. This module naturally includes all additional designs in regards to the geometrical arrangement of the hollow pathway systems to each other, provided that an almost identical substrate supply and removal process from anywhere is the is secured. The two hollow pathway systems can intersect inside the body at a predetermined angle. They can also be arranged parallel on top of each other whereby the counter current principle is optimally utilized.
If the module exhibits a third independent hollow pathway system, it is preferably constructed from parallel-arranged hollow pathways in another plane. These hollow pathway systems also infuse the body, for instance, vertically form top to bottom, interweaving the first two independent hollow pathway systems with each other and integrating other decentralized functions like oxygenation or CO2 removal.
The module with the third hollow pathway system includes all geometrical arrangements, provided that an almost identical substrate supply and removal process for the microorganisms, meaning the cells, is secured from anywhere in the body. Analogous a fourth or additional hollow pathway system can be integrated, whereby additional functions like cell drainage, cell injection, cell extraction, and movement/pressure/flow application for cell removal are made possible.
In this module the first independent hollow pathway system can serve for media inflow. The second independent hollow pathway system serves for the supply of the microorganisms, for instance with oxygen, respectively for the removal of CO2. This can also occur by threading gas perfuseable oxygenation hollow fibers taken from blood oxygenator production into the hollow pathway system. The media outflow is then secured via the third independent hollow pathway system. Alternatively the first and third hollow pathway system can be operated in counter current flow, whereby a cell perfusion is achieved through pressure gradients between the two systems. Afore mentioned channel like hollow pathway systems infuse the porous body of the described module. The dimension of the pores of the porous body of the module is selected in such a way that the pores exceed the size of a cultivated cell. The pores of the porous body therefore exhibit a diameter of preferably 10-1000 micrometer. Importantly, these pores communicate with each other via pore wall openings to facilitate an optimal in- and outflow of media across a multitude pores, whereby the pores are connected through openings of preferably 5-500 micrometer in size. This arrangement guarantees that the inflowing media can reach every part of the porous body via the independent hollow pathway systems, and like wise the outflowing media can be disposed of, via the pores and their connections to the channels of the hollow pathway system, from every part of the porous body. Therewith a media perfusion, flushing of cells, migration of cells as well as substrate exchange is possible through the pores. Therefore the porous body can also be referred to as an open porous foam-/sponge like structure. This bioreactor describes a device that facilitates the organ like reorganization of biological cells, especially in co-culture of parenchymal and non-parenchymal cells of an orgen.
The porous body that is arranged inside the casing can exhibit any geometrical shape. It is important that the porous body has a volume that is able to hold enough cells, respectively microorganisms, for various different applications. Therefore, the porous body exhibits a volume of preferably 0.5 ml-10 liters.
The geometrical shape is not determined. Preferred is a block form because it permits easy infusion of one hollow pathway systems from one side to the other and another hollow pathway system from an additional side. Preferred are cuboids or other rectangular hollow block forms.
Only modules exceeding three hollow pathway systems require a more complex outer form.
The porous body in block form can be generated from one piece, or the porous body is constructed form networks of several overlaying, disc/slide like, individual layers that are retained by the container.
In regards to afore mentioned second alternative, the disc/slide like arrangement, it is advantageous if at least one plane of the disc/slide like individual layers are infused with channel like ridges. These channel like ridges are arranged on the surfaces and shaped in such a way that they, in connection with the very next individual layer, form a channel like hollow pathway system. Therefore, the ridges are for instance shaped like a semi-channel so that, via interconnection with the next following individual layer, a complete channel is formed. The advantage of this arrangement is that it is technically very easy to equip the individual discs/slides with ridges. Preferably, the individual discs/slides can also be constructed in such a way that, viewed from the front wall, they exhibit the second channel like hollow pathway system in form of infused channels.
Consequently, the construction of these individual layers and their connections create a porous body with two independent hollow pathway systems. One hollow pathway system is created by the ridges in the individual layers, whereas the second hollow pathway system is created by the channel like hollow pathways already infused into the individual disc/slides. Drilling ridges into the remaining plane of the discs/slides can form a third hollow pathway system.
The porous body, as afore described, is arranged inside a casing. The configuration of a watertight-/germ tight container and open porous body is arranged in such a way that the channel-like hollow pathways of a system meet in at least one inflow and outflow heads. These inflow and outflow devices are configured in such a way that they pass through the container ensuring the supply and waste removal of the hollow body, arranged inside the container, from the outside. For this purpose two different designs are possible. One is that the inflow and outflow devices are part of the container itself and the arrangement of the body inside the container creates the connections. The other is to connect the inflow and outflow devices with the porous body, in which case the arrangement is enclosed by the sterile and water tight container.
The container can be in form of a solid casing or a foil. A container is the preferred application whereby the use of an injection-molding casing is advantageous. All known, state of the art materials, for example from polycarbonate, are possible for the injection-molding casing. It is advantageous if the container and the connections are constructed from bio-absorbable/bio-degradable material to potentiate the use of the module as medical implant.
Any known state of the art material can be used for the porous body that exhibits afore defined dimensions in regards to the pores and their connections, which leads to an open porous foam-/sponge like structure. As afore mentioned in connection with the container a biodegradable material can be used here as well.
Preferably, the material consists of sintered ceramic powder, especially the use of hydroxyapatite. Hydroxyapatite belongs to the group of calcium phosphates, which include ceramic materials with varying parts of calcium and phosphate. Hydroxyapatite is a compound that occurs in nature but can also be manufactured synthetically. The clinical use of hydroxyapatite as bone replacement material is an already know state of the art application. The motivation for the clinical use of hydroxyapatite is to apply a compound with a similar chemical composition as the mineral part of bone marrow. Hydroxyapatite exists in 60-70% as a natural component in the mineral part of the bone marrow. Hydroxyapatite powder can be generated via precipitation method from a watery solution, for instance by adding ammonium phosphate in a calcium nitrate solution and basic pH. A sintering process at 1000 to 2000 degrees Celsius will result in compounding the powder particles (Wintermantel et al.: Biokompatibler Werkstoff und Bauweise: Implantate fur Medizin und Umwelt, Berlin Springer 1998: 256-257). Wintermantel describes the manufacturing of a porous solid body from hydroxyapatite, for example open porous foam like structures, where hydroxyapatite powder is mixed with organic additives and then cauterized under high temperatures.
A further module that has also been described and simultaneously submitted with the description at hand, by the same inventors, titled “Bioreactor for cell self-assembly in form of an organ copy; procedures for the production and the application of cell culture, differentiation, maintenance, proliferation and/or use of cells.” (German patent application #103 26 746.8 of 13 Jun. 2003, J. Gerlach). In this case the bioreactor consists of a container that holds a open porous body whose pores also communicate with each other. In addition, the body contains at least two independent, branching out hollow pathway systems that cross and/or overlay each other and infuse the body. These hollow pathway systems depict natural organ copies, e.g. arteries and veins. Cells also settle inside the open pores of the body and are immobilized.
Therewith a bioreactor in form of an organ copy is made available. The hollow structures of the bioreactor allow for the maintenance of a larger cell mass with high density, whereby the fluid exchange to and from the cells via blood plasma or media occurs decentralized and avoiding large substrate gradients. The hollow structures include copies of arteries, veins, as well as other organ typical vessels for example liver portal veins of the liver, liver biliary tract canaliculi, and the Hering Channels with the liver stem cells.
Essential with this bioreactor is that its immunological inactive porous body exhibits open pores that communicate with each other. The pores exhibit a size that is larger then the size of the cells of the respective organ. Therefore the pores have a diameter of preferably 10-1000 micrometer and they are connected through pore wall openings. These openings, preferably formed channel- like, are preferably 5-500 micrometer in size. Through this arrangement the communication between the pores via the pore wall openings and with the hollow structures of the organ copy is secured. Via the pores a media perfusion, inflow of cells, cell migration as well as substrate exchange is made possible. Afore described structure of the porous body can also be referred to as an open porous foam-/sponge like structure. This bioreactor describes a device that facilitates organ typical reorganization of biological cells.
Importantly, the bioreactor is constructed from an immunological inactive, perfuseable open porous foam-/sponge like structure, whereby cells are settled inside the hollow spaces, and the pores of the foam-/sponge like structure. Via the pores media perfusion, inflowing of cells, cell migration as well as substrate exchange is made possible. Therewith, afore mentioned bioreactor is significantly improved with respect to known, state of the art, bioreactors in regards to mimicking substrate exchange structures, performances, and characteristics/attributes of natural organs.
This bioreactor describes a device that facilitates organ typical reorganization of biological cells. It is characteristic for this bioreactor that the specific hollow structures for the cell maintenance are arranged the same way as they occur in the natural organ.
All known state of the art materials, that produce open porous foam-/sponge like structures according to the invention, are well suited. Suitable are for instance ceramics, e.g. hydroxyapatite. Hydroxyapatite exists in form of a powder and, with additives and pore forming materials, can be frothed to foam-/sponge like structures and then sintered.
This bioreactor is preferably located in a sterile and water tight container. Suitable are foiled or accordingly dimensioned containers. In this case connections are provided, which are in connection with at least one hollow structure of the organ cast to guarantee the appropriate supply and waste removal in the bioreactor. In reference to the design of the connections, naturally several in-and outflow devices of the organ, inside the container, can be combined to one in-and outflow device.
In addition, it is advantageous with this bioreactor that the container and the connections can be generated from bio-absorbable, respectively biodegradable material which potentiates the use of the bioreactor as implant.
Afore described three registrations are, in their entirety, included in the registration at hand in regards to their disclosure content, design of the module/bioreactor, since such bioreactors can also be applied as bioreactor in the invention at hand.
Other bioreactors are already known from WO 00/75275 (Mac Donald, USA) and EP 1 185 612 (Mac Donald, USA).
Above described modules are generally suited for cell culture, proliferation and differentiation of cells, whereby the cells are encased in the respective containers of the modules and supplied through hollow pathway systems. Therewith, besides cell production, also the synthetic performances of the enclosed cells can be utilized, because the cell products can be led away from the reactor. However, the disadvantage of these bioreactors is, that they are not able to facilitate complex systems of cells in a circulation requiring the communication of several organs, or the migration between several organs. An example is the preservation of early stem cells in the bone marrow, maturation, or differentiation of immune cells at several further locations in the body. Hereunto the biological interactions in the organism with several independent organs within the blood/plasma circulation are much too complex. Particularly in the biological systems of the human body, the differentiating cells run through spatially varying stations that have to be passed through in a chronologically defined rhythm. During this process rest- and activity phases occur in different locations in the organisms in regards to cell differentiation. In addition, growth- and differentiation factors synthesized by various organ systems interact with each other via the circulatory system.
The invention at hand creates a hybrid circulatory system that implements such an interactive organ circuit structure.
This task is solved via the hybrid circulatory system according to claim 1 as well the application according to claim 37. Advantageous, advanced developments of the hybrid circulatory system are described in depending claims.
As per the invention, bioreactors are interconnected in a circulatory system, whereby a revolving media circuit ensures substrate exchange between at least two bioreactors. The substrate exchange can include mediators, soluble receptors, effectors, antibodies, and metabolic products like differentiation factors, growth factors, hormones, and such.
The substrate exchange can be controlled via the molecular cut off of the membranes used. This exchange can also include cell transfer. The cell exchange can also be controlled via the pore size of the membranes used
This invention permits cells to circulate between individual bioreactors. Thereby, for example, bone marrow cells can pass through the individual developmental stages as they occur in the human body. This means, differentiating bone marrow stem cells will first proliferate in a bioreactor providing a cell environment similar to bone marrow, from which they will be transported to a bioreactor with an environment corresponding to that of spleen tissue, or followed by a bioreactor that resembles the thymus and/or the liver. Then, the differentiating bone marrow stem cells are transported (or can actively migrate) into a bioreactor resembling the lymph nodes. It is also possible to, intermittently, set up small bioreactors with a cell specific environment resembling lymph nodes through which the cells have to pass.
The cell specific environment is generated in such a way that the differentiating cells are cultivated in co-culture with supporting cells of the respective organ like stroma cells, endothelial cells, and/or connective tissue cells. This can occur either inside the same compartment, via a semi permeable membrane (or a hollow fiber membrane structure) separate from the differentiating cells. In later case, the two compartments exchange mediators and effectors relevant for the differentiating cells that are generated by the cell specific environment.
Similarly, bioreactors with lymph node-like cell structures (or other organ typical bioreactors) can be connected with, for example, the circulatory system via a semi permeable membrane to restrict uptake into the circulatory system to certain mediators or effecters instead of cells.
The bioreactors cannot only be arranged in a row, but in copying the natural system, it is also possible to parallel arrange individual bioreactors into the circulatory system.
Alternatively it is possible to only circulate metabolic products of individual bioreactors in the circulatory system rather then circulating cells from one bioreactor to another. In this case it is possible to cultivate a particular cell in a stationary bioreactor, which will be supplied with mediators and effectors, necessary for their growth and proliferation, through other bioreactors that are connected to the circulatory system via a semi permeable membrane.
Thus it is also possible, for instance, to proliferate a stem cell culture and therewith produce stem cells in an indirect exchange with animal feeder cells. Furthermore, a human- to human stem cell/feeder cell structure can therefore be enabled. These techniques may be called compartmentalized co-culture.
Otherwise it is possible to generate certain mediators, effecters and such, and subsequently isolate them from the circulatory system. This is particularly advantageous when the respective mediators and effecters are not yet known, however under the given conditions can be generated as they occur in the biological body.
Thus it is possible to create a complete cycle of the maturation of, for instance, blood cells, the differentiation of immune cells, or the maintenance of proliferating stem cells inside a bioreactor. Should the circulatory system be set up to generate antigens, it is possible to produce immune cells that respond to antigens, which facilitates the production of vaccines.
Likewise it is possible to produce viruses, viral components or products that are necessary for the development of vaccines, which, in this context, are considered metabolic products of the cultivated cells.
Based on the complex interactions of organ systems in a human organism, the hybrid circulatory system permits the preservation of the early stem cells and their selective proliferation while conserving the early stem cell pool.
The invention permits the simulation of specific biological processes, for instance the growth of stem cells, stem cell differentiation by mediators produced in distant organ systems, cellular migration across lymphatic structures (spleen, lymph nodes), physiological migratory paths of the cells with ease and activity across several tissue stations, migration across tissue of different germ layers, as well as concluding proliferation and differentiation to immune cells or maturation to blood cells.
The circular media transfer of the circulatory systems can serve for the transfer of cells or the transfer of cellular signals, respectively chemical mediators or signals between the bioreactors and tissue structures. An analogous transfer can also occur within one reactor that contains two different compartments, for example one compartment for the culture of cell lines and another compartment for the co-culture for an organ specific environment, simulating the in vivo macro environment of individual cell lines. Additionally, a selective contact of individual cells in a bioreactor, with defined molecules of determined size, can be achieved via the technical, in any pore size set, exclusion barrier of individual molecules into the bioreactor.
Following, a few examples of hybrid circulatory systems are explained:
In
These reactors describe the essential elements o a circulatory system in which bone marrow cells can be cultivated, proliferated, and differentiated.
The interaction of the bone marrow cells inside the bioreactor 3 with the cells/mediators of other bioreactors also facilitates the preservation of the early bone marrow stem cells ensuring the long-term conservation/preservation of the entire system.
Alternatively, this circulatory system from
In the invention and the circulatory system, it is ideal that each reactor contains, proliferates, and/or differentiates the necessary organ specific cells.
Overall, the circulatory system is able to imitate not only the circulatory system of the body but also the entire system of the blood circuit and organs.
As fourth component of each unit 20a through 20c, unit 24a, 24b, and 24c is added with which the temperature of all system components is controlled via warm air.
A refrigerator maintaining a temperature of 4 degrees Celsius is made available for all units 20a through 20c in which for instance the fresh media supply is stored. A medium circulation is arranged between the individual units 20a through 20c so that the bioreactors 21a through 21c, contained in units 20a through 20c, can exchange substrates or cells.
The three-way valves in the circulatory system can be positioned either to infuse the individual cell systems directly via the cell compartment, or via semi permeable membrane.
| Number | Date | Country | Kind |
|---|---|---|---|
| 103 26 749.2 | Jun 2003 | DE | national |
