1. Field of the Invention
The invention concerns a process for producing a three-dimensional bioartificial tissue having viable cells in or on a matrix, and by which cells and matrix can be cultivated into a tissue or a precursor of a tissue, a vascularized tissue of biological materials, obtained by this process, and an experimental reactor for scientific purposes and for producing clinically usable tissues and organs.
2. Background Description
Bioartificial tissues are understood to be tissues produced in vitro from natural biological materials, which are, therefore, not natural tissues, but tissues which as nearly as possible simulate natural tissues.
The process according to the invention and the matching reactor are particularly suitable for producing a bioartificial heart muscle tissue.
Morbidity and mortality in the Western population are largely related to loss of heart function and heart tissue (cardiac failure). This is a problem of great socioeconomic relevance. Bypass surgery or heart transplants are increasingly necessary as ultimate therapeutic measures. Transplants, though, involve problems such as lack of suitable donor organs or the stress on transplant patients by lifelong treatment to suppress rejection reactions.
The problems of transplantation affect not only hearts, but also other organs which are no longer functional and which must be replaced. Because of these problems, major attempts have been made in medical research in the areas of xe2x80x9ctissue retentionxe2x80x9d and xe2x80x9ctissue and organ replacementxe2x80x9d. Research endeavors on xe2x80x9ctissue and organ replacementxe2x80x9d comprise use and better adaptation of xenogeneic organs as well as the culture of three-dimensional tissues from natural starting materials so as to be able to replace at least parts of organs.
In the past, early cellular forms (e. g., embryonal stem cells or infant heart cells) have been injected into recipient myocardium (individually and as clumps of cells).
Prototypes of rat heart muscles in collagen, enriched with nutrients and growth factors have been made to produce, or, initially, to simulate, heart muscle from simple biological basic materials. Contractions have already been demonstrated with them. However, these experiments have not dealt with functional three-dimensional tissue which would be suitable for transplantation and which could replace damaged organ tissue (R. L. Carrier et al., xe2x80x9cCardiac Tissue Engineering: Cell Seeding, Cultivation Parameters and Tissue Construct Characterizationxe2x80x9d, Biotechn. Bioengin. 64 (5), 580-90, 1999).
The artificial pieces of heart tissue so obtained were only a few millimeters thick and never survived longer than a few weeks. After implantation, they were unable to integrate themselves into the recipient tissue so as significantly to improve the strength of beating (B. S. Kim et al., xe2x80x9cOptimizing Seeding and Culture Methods to Engineer Smooth Muscle Tissue on Biodegradable Polymer Matricesxe2x80x9d, Biotechn. Bioengin. 57 (1), 1998; R. K. Li et al., xe2x80x9cIn vivo Survival and Function of Transplanted Rat Cardiomyocytesxe2x80x9d, Circ. Res. 1996; 78: 283-288). Thus the objective of the invention is to provide a process for producing an improved three-dimensional bioartificial tissue. In particular, vascularization of the tissue is attempted, so that it can be supplied and thus maintained in a viable state.
To achieve that objective, the generic concept of the process is designed so that at least one vessel is inserted in the tissue at the beginning of its production. The vessel is supplied from the outside, so that vascular propagation occurs in the course of tissue cultivation and a three-dimensional, vascularized bioartificial tissue is obtained.
Because the tissue is permeated by a vessel even during its cultivation, it can develop three-dimensionally as in nature. The main vessel branches, leading to a vascularized tissue. The vascularized tissue produced can, on transplantation, be connected to the vascular system, so that it remains nourished and viable. There is a more natural modeling of the tissue conditions even during the tissue culture itself. Culturing over longer periods is possible because the supply to alls cells, even those in the interior of the cell assembly, can be better assured. A longer culture can be advantageous, for instance, if a better degree of cell differentiation is to be attained. Culture in the experimental reactor, which is described in more detail below, also allows provision of a physiological environment for organogenesis (e. g., atmospheric pressure or vacuum, defined gas concentrations (partial pressures), biochemical environment, etc.).
A bioartificial tissue in the sense of the invention is understood to be a tissue not taken, as such, from an organism, as by surgery, but rather simulated artificially using biological materials. That is generally done by assembling certain cells into a cell assembly, applying them to a stabilizing substrate, or putting them in a matrix. The substrate or matrix can itself be of synthetic or, alternatively, biological origin. Polymers, especially biodegradable polymers, may be considered as synthetic matrices. They can also be in the form of layers or networks. Collagen in particular is also considered as a matrix material. A segment of tissue, removed from a human or animal, generally acellularized by chemical and/or mechanical methods, can also be used as the source of the collagen matrix.
The bioartificial tissue should contain viable cells so that they can form a three-dimensional biological tissue which has the longest possible stability and viability.
In one simple embodiment, collagen and viable cells can be mixed, and further cultivated (i. e., supplied with a nutrient solution renewed continuously or at intervals). Various simulated tissues have already been recommended and used. The detailed structure of this tissue depends on the nature of the tissue or organ.
The principal problem with tissues comprising biodegradable matrices and cells, especially artificially simulated heart tissue, is their limited dimensions and survivability, because for a long time it has been impossible to nourish them by blood or nutrient medium in a natural manner (see N. Bursac et al., xe2x80x9cCardiac Muscle Tissue Engineering: Toward an in vitro and in vivo model for electrophysiological studiesxe2x80x9d, Am. J. Physiol. 277 (2), HH433-H444, August 1999; R. K. Li et al., xe2x80x9cSurvival and Function of Bioengineered Cardiac Graftsxe2x80x9d, Circulation 1999, 100 [Suppl. II]; II-63 to II-69).
The invention is, then, based on the recognition that the ability of a tissue to survive and develop its function is critically dependent on its vascularization (the occurrence of vessels within the tissue). Therefore the process of the invention provides that at least one vessel, which is supplied from the outside, is inserted into the artificial biological tissue at the beginning of its production. By supplying the vessel with a suitable nutrient solution or blood, vessels propagate naturally in the course of the cultivation.
The vessel can be inserted into the matrix, such as an acellularized collagen matrix, before it is inoculated with the cells desired for the artificial biological tissue; or, if the production of the artificial tissue involved only mixing of cells and matrix material, the vessel can be inserted into that mixture at the beginning of the culture.
A vessel of natural origin from a human or animal can be used as the vessel, for instance, rat aortas, especially for scientific experiments. But it can also be an artificial vessel, particularly one of a biologically compatible polymer. It is also possible to use the entire vascular structure of an organ to be supplied.
Preferably, a native vessel with a large lumen, as nearly as possible specific for the species, is used. The extended vessel lumen can have lateral openings or branches. The openings are produced in the simplest case by cutting off side branches from the main branch of a native vessel. On culture of the artificial tissue, vascular development proceeds into the tissue.
Alternatively, an inherently porous vessel can be used. The porosity simplifies the beginning of vascular branching, and could be produced by chemical and/or mechanical treatment of the vessel. The mechanical treatment can also comprise repeated pressure stressing of the vessel, for instance, forcing a pulsing flow through the vessel. If an artificial vessel is used, tubular fiber networks appear particularly suitable. The porosity of the synthetic material can be adjusted as desired by technological means. For instance, a tissue-like synthetic material with freely selectable porosity can be used. The inherently porous vessel can be prepared at points with a material or means providing a stimulus for angiogenesis, such as the protein VEGF (vascular endothelial growth factor).
It is further important for the process that the vessel be supplied, from the inside or outside, with blood or a culture medium. That can be done by perfusing the vessel in the usual manner. That is, it can be connected to a circulatory system of blood or culture medium. Substances promoting angiogenesis, such as VEGF or other known growth factors (TGF, PDGF, VGF) can be added to the culture medium. Such substances, and, in particular, growth factors, can also added directly to the tissue matrix or generally to the tissue mixture.
The perfusion can preferably be done under pressure. In the simplest case, it is provided that the perfusion supply line be of greater diameter than the perfusion drain line, so that a pressure develops within the vessel. Such a pressure is also maintained in very organ in nature, so that perfusion under pressure contributes to simulation of natural conditions. Because of the pressure, liquid is forced into the tissue, resulting in a mechanical stimulus that promotes vascular development. Perfusion under pressure can also be accomplished in other ways with suitable pumps and throttling elements.
It is considered particularly advantageous for the perfusion to be pulsatile, as this most nearly approaches a natural supply of the tissue, pulsing with the heart beat.
The technological realization of pulsatile perfusion, under more or less pressure, is basically known at the state of the art, and therefore need not be described in further detail here.
With the supply and drain lines needed for the perfusion, the vessel can also be positioned and fixed within the tissue. That applies particularly for tissue cultures of gel-like mixtures.
In one preferred embodiment, cardiomyocytes are used as cells within the tissue to be artificially vascularized, so as to give an artificial heart tissue. The principle of vascular development is also applicable to other bioartificial tissues, though, particularly to skin tissue. In the latter case, keratinocytes are used preferably as cells.
Kidney tissue, lung tissue, and other tissues can be obtained in corresponding ways. Complete bioartificial organs (heart, kidney, lung) can be obtained by specifying the geometric form of the culture and inserting a vascular tree for an entire organ.
By means of the invention, it is possible for the first time to obtain a vascularized three-dimensional tissue distinguished by the fact that at least one vessel passes through it, from which other vessels branch off into the tissue. The vascularized transplants can be integrated better into the receiving bodies, as they can be supplied continuously from the beginning. When the tissue is used as a transplant, the vessels of the artificial biological tissue can be connected with the vascular system of the receiver""s own body, so as to assure immediate supply of the newly inserted tissue. For artificial heart tissue, there are the particular advantages that the tissue segments can, for the first time, exceed a thickness of a few millimeters and that the survivability of the vascularized, three-dimensional tissue is distinctly superior to that of a non-vascularized tissue.
The invention also covers an experimental reactor for controlled production of an artificial biological tissue, particularly by the process of the invention for producing a vascularized artificial tissue. This experimental reactor is characterized by at least one tissue culture chamber, at least one inlet and outlet for this, or each, chamber, and by two opposite plane, parallel and optically transparent boundary walls of this or of each tissue culture chamber. The experimental reactor is particularly intended for making biological and physiological research on bioartificial tissues easier. For that purpose, the reactor is designed so that the contents of the culture chamber can be examined and observed by optical procedures. The reactor can be exposed directly to optical methods of examination during the culture process by means of the two or more [see Note 1] opposite plane, parallel and optically transparent boundary walls. The contents of the reactor culture chamber(s) can be observed directly with a microscope; and still or motion pictures can be made of the contents.
With multiple separate tissue culture chambers, serial investigations can be carried out with variation of certain parameters, so that research on bioartificial tissues can also be distinctly systematized and made easier. There are many possibilities in the suggested experimental reactor for influencing the culture process and applying various growth-promoting measures. The blood and nutrient supply of the bioartificial tissue can be observed well and optimized by varying different parameters.
In this way the experimental reactor is simply designed and economical to produce. The experimental reactor is preferably provided with at lest one closable inlet and outlet per chamber. The preferred closure means comprise cocks, valves, hose clamps, and/or diaphragms. The inlets and outlets can at the same time serve to delimit and fix the vessels to be positioned within the tissue culture in the process according to the invention. However, separate means can also exist for fixing the vessels, such as means for fixing an extracellular matrix, such as an acellularized collagen substrate.
One of the plane, optically transparent boundary walls of the chamber can preferably be made as a lid. It is advantageous to provide at least one additional closable inlet, or an addition point for each chamber to influence the medium or the substrate. These can, for instance, be supports with threaded connections, with diaphragms inserted in the screw holes [see Note 2]. Certain substances which can affect the development of the artificial tissue in the culture can be added through these addition sites with a syringe (see above, e. g., VEGF).
The reactor inlets or outlets are preferably connected to a micropump and a tubing system through which blood or nutrient medium is provided. The drain line or lines is/are preferably connected to a waste tank.