Regenerative Medicine: The Challenge of Qualified Biobanking and Controlled Self-Healing
All phases of tissue growth have a particular meaning for the body’s own self-healing powers. Researchers in the Fraunhofer Group for Life Sciences have managed to influence all these phases – from the stem cell and its differentiation to a specific cell structure, to tissue and through to the implant – by systematic stimulation and control mechanisms. For each stage of this process, the Group can utilize research and development results on novel therapeutic approaches, its own methods, and its specialist know-how. Particularly good results have been obtained with adult glandular stem cells. After targeted differentiation, these are currently being tested in cardiac infarction and skin replacement models. Complex cell-therapeutic concepts have proven beneficial, for example, to control immunological processes and to treat tissue damage after stroke.
To support healing processes or as a replacement in case of extensive tissue loss, the Fraunhofer Group for Life Sciences can grow biological tissue, in particular cartilage and skin, in its own GMP laboratories where all necessary regulatory expertise is available. Up to now, this has been a manual process for the most part, but the development of novel, fully automated tissue engineering plants is already well advanced.
Biomarkers play a key role in the life sciences. They can be successfully identified and validated by comparing as many samples and case cohorts as possible. A prototype of networked biobanking is the CRIP bio-database (Central Research Infrastructure for molecular Pathology), which is used to store data obtained by standardized sampling and sample asservation procedures. Actual samples are stored and preserved for future use in EUROCRYO, the European research database maintained by the Fraunhofer-Gesellschaft. The Group thus has at its disposal a biobank which is unrivaled worldwide regarding its system and content.
The cell is the basis
Regenerative medicine aims to copy the healing and repair processes which continually take place in the human body. These natural healing and defense mechanisms may perform differently in every individual body. If a body has reached the limits of its own natural capabilities for dealing with a particular lesion, further healing processes can be stimulated or mimicked with the means provided by regenerative medicine. Biological replacement cells or tissues are used and the body’s own repair processes are stimulated so as to restore tissue or organ functions.
The core expertise of the Fraunhofer Group for Life Sciences includes work with stem cells, and in particular adult stem cells. The extensive experience of the Group with these systems forms the basis for the development of novel handling systems and devices for the transfer, differentiation, and dedifferentiation as well as for the cloning of single cells.
The Fraunhofer Group for Life Sciences has worldwide unique expertise in the technology and processes of cryoconservation of cells and small tissue segments. Cells may thus be stored without loss of quality and subsequently used for research, biotechnological applications, or therapeutic purposes. In the European research cryobank of the Fraunhofer-Gesellschaft, EUROCRYO, valuable and unique collections of cells (live samples) are stored in a way designed to meet the requirements of modern biotechnology. Retrospective investigation of samples, therefore, provides relevant information even after decades. Core elements of the underlying concept are miniaturization and highly parallel sample storage. Thanks to novel storage substrates, the microsystem-based cryotechnology developed for this purpose allows the number of possible cryosamples to be increased by a factor of 100 during the first miniaturization step. To rule out any risk of sample confusion, an electronic storage unit with a capacity of up to 1 GB and which is readable and writable even at temperatures around -180 °C, is kept together with the samples. The methods developed are characterized by high vitality rates and are thus particularly well suited to stem cell research and preservation. The services offered by the Group based on this expertise include the design, construction monitoring, and setting up of cryobanks and cryo-specific equipment for medical, biology, and drug research applications right up to industrial scaling.
New strategies for targeted stem cell differentiation
Recent findings in stem cell research have provided evidence that a very efficient method for making stem cells differentiate into a particular cell type is the coculturing of stem cells with other cells or tissue biopsies. The hypothesis is that important external signals controlling the identity of a cell are given locally by the target tissue; which means, for example, that an adult stem cell penetrating into an injured heart will receive the decisive signals to differentiate into a heart cell from the heart tissue itself.
In this research area, the Fraunhofer Group for Life Sciences benefits from the results and experience gained during many years of stem cell research. Glandular stem cells are cocultured in direct or indirect contact with tissue biopsies – depending on the type of cell and tissue. In the case of direct coculture, tissue biopsies are added to adherently growing cells in the culture containers. For indirect coculture, tissue biopsies and stem cells are placed in separate coculturing containers to enable spatial separation. This system allows an exchange of messenger substances and signals, however, at the same time it prevents the biopsy cells from getting through to the stem cells. This method has up until now been successfully used to induce an accumulation of nerve cells via brain biopsies and an accumulation of cardiomyocytes via heart biopsies. First results of these investigations are already being implemented in the development of strategies for heart tissue regeneration. To this end, stem cells are cultured on biodegradable meshes and induced to differentiate into cells similar to cardiomyocytes. These meshes are currently being tested in a model of myocardial infarction.
Reprogramming of cells
Not only stem cells, but all therapeutically relevant cell populations in general bear the hope of enabling successful treatment of diseases which so far have been considered untreatable. This holds true in particular for diseases involving a loss of cells and thus of functionality. The possibilities for therapeutic use, however, go far beyond the actual replacement of cells. Benefits have also been demonstrated for the prevention or mitigation of degenerative processes.
A major challenge for biomedicine in this context is to make adult stem cells available in sufficient quality and quantity for therapeutic purposes. The Fraunhofer scientists have developed a method for producing patient-specific, individualized stem cell lines from somatic cells without viral integration or genetic modification (induced pluripotent stem cells – iPS). These iPS cells display the typical characteristics of embryonic stem cells without, however, engendering ethical controversy.
A special focus in this research work is the generation of iPS without the use of viruses. Different types of reprogramming, including those with specially developed media compositions, can be explored, for example fusion, nuclear transfer, and stem cell extracts.
Immunodiagnostics and cellular diagnostics
For the demonstration of cells and cell functions in tissues and the prediction of therapeutic responsiveness, the Fraunhofer Group for Life Sciences offers a variety of analytical and diagnostic techniques: cell-imaging diagnostics and follow-up, cell function analysis, innovative tissue typing, and pharmacogenomic investigations on inflammatory processes. Using the cells and tissue cultures developed in the Group, the Fraunhofer scientists can investigate the functionality of diagnostic and therapeutic concepts in complex systems close to the real-life situation.
Immunomodulation and cell therapy
The scientists of the Fraunhofer Group for Life Sciences develop strategies for immunological tolerance induction, tolerance monitoring, and controlling the functions of the immunological system. These enable successful immunological management of tissue rejection reactions. Through cell isolation and preparation, cell apheresis, and cell proliferation in vitro, the Group further develops therapeutic concepts. To this end, the scientists of the Group use cell suspensions, mainly with progenitor and stem cells. A focus of research in this area is on new cell-therapeutic approaches to treat tissue damage resulting from stroke. With its possibilities for low-temperature conservation and a standardized and patented isolation method the Fraunhofer Group for Life Sciences has at its disposal a variety of stem cell cultures which are available nowhere else in the world. These are cultures of different tissues from widely differing animal sources right through to human stem cell cultures, with excellent growth rates, good differentiation possibilities, and suitable for long-term culturing.
Manufacture of cellular therapeutic agents
The clinical testing of medications for novel therapies requires top-quality clinical investigational drug products, which must be manufactured in compliance with GMP regulations. The Fraunhofer Group for Life Sciences not only has clean-room facilities for the production of biopharmaceuticals, but also clean-room facilities specially designed for the manufacture of cellular and tissue preparations and complying with the most recent technical and regulatory standards. A special feature of these facilities is the subdivision into separately accessible clean-room suites, meaning that individual cellular products for each client can be continuously provided. A consistently high quality level is ensured by an excellently equipped quality laboratory, highly qualified staff, and a comprehensive quality management system.
The treatment of oncological diseases with cellular therapeutic agents is one of the most recent treatment strategies, which is currently being tested worldwide in clinical studies. The Fraunhofer Group for Life Sciences has the capability to produce and optimize both cytokine-induced killer cells and dendritic cells specifically for each patient. All steps from process development through to acquiring the manufacturing license are accomplished by the same organization.
An advanced stage has been reached in the development and practical implementation of a GMP manufacturing process for an autologous cellular therapy for ischemic stroke.
Risk analysis for regenerative strategies
The development of novel regenerative strategies requires the biocompatibility of the products to be tested as comprehensively as possible in murine and human systems. This assures speedy development of the products up to their preclinical and clinical application. In the Fraunhofer Group for Life Sciences, risk profiles of products are being generated by using in vitro and in vivo analyses. They take into account, for example, immunological tolerability, cytotoxic, proliferative, and stimulating effects on primary, stem, or tumor cells, as well as thrombogenic, hemolytic, or genotoxic activity. A large variety of specialized mouse models are employed for complementing in vivo experiments.
In addition, the Group collaborates with a competent partner who has an accredited diagnostic laboratory in order to carry out clinical studies.
The bio-database CRIP – “Central Research Infrastructure for molecular Pathology”
“Bio-bases” are collections of biological materials and corresponding data.
An infrastructure across different biobases and institutions – also referred to as “bio-database” – is gaining more and more importance in biomedical research and development for tasks such as the identification and validation of biomarkers. The “Central Research Infrastructure for molecular Pathology” CRIP is such a bio-database, which has been operated inside the Fraunhofer Group for Life Sciences since 2007 and is being further developed as a prototype for other research areas. The rights of the partner bio-bases regarding the respective data and samples stored in the bio-database continue to be safeguarded, as are the personal rights of the sample donors and the data privacy regulations. Studies on multifactorial diseases and individualized treatments require ever increasing numbers of samples and case cohorts, which a single clinic would not be able to collect or only over a very long time span. This is why medical bio-bases in particular have to also be networked to each other by means of bio-databases. Prerequisites for this include standardized sampling and sample asservation procedures as well as a data network which fulfills the relevant ethical and legal standards.
Human tissue samples are of particular relevance for research as they enable investigation of both locally restricted diseases (such as cancer or inflammation) and organ-specific manifestations of systemic conditions. In contrast to other body substances such as blood or serum, tissue samples are not reproducible. The consolidation of tissue banks in bio-databases – for example in the “Central Research Infrastructure for molecular Pathology” (CRIP) – therefore aims to overcome a bottleneck in research.
The term tissue engineering refers to the reproduction of natural tissue from primary cells under laboratory conditions. This tissue can be used to support healing processes, to regenerate tissue that has become dysfunctional, and to replace destroyed tissue such as burnt skin. The procedure to manufacture a ready-for-use tissue engineering product involves two main phases. The first phase consists of establishing and verifying a procedure that leads to the desired product; the second phase consists of adapting this procedure to the medical regulatory requirements. This process is subject to a high degree of formalization and authorized documentation. The Fraunhofer Group for Life Sciences as a competent research partner in medicine and medical technology develops tissue engineering processes and products, including appropriate carrier structures; GMP laboratories for the manufacturing of autologous transplants are also available.
Another focus is the development of three-dimensional test systems with organ-specific properties; these are suitable for in vitro testing of substances in medical engineering, cosmetics, and the pharmaceutical and chemical industries.
Manufacturing technology for tissue engineering
Tissue engineering has been a focus of research for several years already, and many biotechnological laboratories are producing tissues such as cartilage or skin. So far, however, the cultivation of transplants such as skin grafts is a costly process, because most steps are performed manually. The provision of tissue structures at reasonable price and above all quickly continues to be one of the major challenges of tissue engineering. Only by complete revision of the production technology, processes, and methods via interdisciplinary collaboration of researchers from the life sciences and the engineering sciences will it be possible to unleash the enormous potential of tissue engineering in an integrated approach.
This challenge has been taken on by the scientists in the Fraunhofer Group for Life Sciences: together with colleagues from the Fraunhofer institutes for manufacturing engineering and automation technology they are working on a fully automated tissue production process. The machine-based process has been subdivided into individual modules, so that the requirements for the manufacture of different tissues can be met. Further development work is required before the final machine for “tissue engineering on demand” will be ready for the market. But it is already clear today that this will open up a variety of new possibilities in the medical area.
Automated tissue engineering is also an interesting option for chemical, cosmetics, pharmaceutical, and medical companies that require tolerability testing of their products.
New stem cell sources for tissue engineering
The treatment of acute and chronic skin lesions has long been a domain of secondary health care with sophisticated plastic-reconstructive interventions. Every year, thousands of burned individuals and hundreds of thousands of patients with chronic wounds of different origins require their wounds to be closed in order to prevent loss of fluid and electrolytes, infections, metabolic dysfunction, immunosuppression, pain, and amputation. The development and use of artificial skin substitutes that are capable of achieving the most relevant properties of human skin allow wounds to be closed immediately and support regeneration.
Biological skin substitutes are three-dimensional systems consisting, for example, of collagen fibers and elastin, enabling immediate wound closure and serving as a fiber scaffold for permanent regeneration of functional autologous dermis. A further optimization of artificial skin substitution methods might be able to be achieved by combination with stem cells differentiating into skin structures or releasing growth factors that would accelerate and/or improve the regeneration process. One focus of the Fraunhofer Group for Life Sciences in the area of cell differentiation is the characterization and utilization of adult stem cells from exocrine glandular tissue. Multi-potent stem cell populations with similar properties can be isolated from pancreas tissue, salivary glands, and skin. Studies in this area have already demonstrated that a skin substitute colonized with pancreatic stem cells leads to accelerated wound healing and improved vascularization of total skin defects in a murine animal model.
Biomaterials are opening up new possibilities
The interaction between the material and the relevant biological system is of pivotal importance for the success of tissue engineering. By combining expertise in materials science and experience in cell biology, the Fraunhofer Group for Life Sciences develops bioactive, biocompatible, or bioinert materials for use in medicine and medical engineering – one example of the possibilities of interdisciplinary cooperation within the Group. Using plasma technology, for example, implant materials can be prepared in such a way that proteins will not adsorb non-specifically; on the other hand, a coating with growth factors may be applied so as to promote the adhesion of mammalian cells. The biocompatibility of surfaces is of great importance also for artificial extracellular carrier structures. Only good biocompatibility will guarantee the proper functioning of the adjacent biological systems.