Cultivating Human Skin Cells for Transplantation
The Development of a Technology of Cultivating Human Skin Cells for Transplantation at Treating Skin Defects
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
State Research Center of Virology and Biotechnology VECTOR, Russia, Novosibirsk reg., Koltsovo
- Delivery Systems International, Russia, St Petersburg
- Tufts University / International Institute for Prosthetic Rehabilitation of Landmine Survivors, USA, MA, Stoughton\nMassachusetts General Hospital / Center for Engineering in Medicine, USA, MA, Charlestown\nState University of New York University at Buffalo / School of Medicine and Biomolecular Sciences, USA, NY, Buffalo
Project summaryThermal injuries remain one of the urgent problems of surgery as their number increases worldwide, especially in industrially developed countries. For instance, in Russia patients with burns make up 20% of all traumatological patients; 26% of them need hospitalization, and lethality rate is 4.7- 11%. In the USA approximately 2 million people get burned every year; 130 thousand of them are hospitalized, and 10-12 thousand die. About 5 thousand patients need reconstructive operations.
Trophic ulcers occur in the population at the rate of 1-1.5%. Self-healing of an ulcer with a diameter >6-9 cm is practically impossible. High demands for donor material are also observed in plastic surgery. For example, donor skin annual demands at a 1 department of plastic surgery are 5.000 cm2.
Restoration of skin is one of the most important problems at treating patients with large and deep burns, trophic ulcers, large traumatic skin injuries and wounds healed by secondary tension. Autodermoplastics with whole or perforated skin pieces covering the surfaces of granulate wounds is the most efficient method. However, the problem of deficiency of skin for transplantation is very common. Known methods of covering burn surfaces with allotransplants (including corpse ones), xenotransplants, amniotic membrane and synthetic materials cannot solve these problems and do not exclude the use of autotransplants (Sarkisov D.S. et al., 1995). In addition, for many patients with large burns, autodermoplastics is an additional trauma, which can be regarded as a risky operation. Besides, these patients do not have enough skin for autotransplants, and multistage plastic operations prolong the treatment periods, which exhausts the organism and causes complications. That’s why studies aimed at the development of new, less traumatic methods of reconstruction of injured skin are very urgent.
Cultivation of the patient’s skin cells in vitro followed by transplantation to the wound is the most promising method. However, successful growth of keratinocyte layers in culture and their transplantation - is a rather complex and many-factor process that depends on the patient age and state, the conditions of dissociation and cultivation of cells, the used support or substrate and many other factors. This technology is successfully used in critical situations, however, it requires optimization before it can be generally used in practice of medical institutions. Therefore, the selection of conditions for production, cultivation and transplantation of human skin stem cells grown in vitro and suitable for reconstruction of skin at healing burns is an urgent task of scientific and practical value.
At SRC VB “Vector”, Institute of Cytology RAS, the Pavlov State Medical University (St.-Petersburg) and JSC “DELSI”, there are favorable conditions for cooperative development of new methods of reconstruction of burned or otherwise injured skin. At Research Institute of Cell Cultures of SRC VB “Vector” and the Institute of Cytology RAS, a basis has been created for research work on production, cultivation and certification of cells of different human and animal organs and tissues; there is a group of high-skilled specialists and necessary equipment and premises. Studies on developing the methods for production and cultivation of cells of epidermal and mesodermal layers of human skin are in progress; conditions for enzymatic dissociation of tissues have been selected (Yurchenko et al., 1997.). Samples (strains) of human skin cells – keratinocytes and fibroblasts have been obtained from different donors: patients with burns, healthy donors and embryonic material. The first auto- and allotransplantations of human skin cells have been performed according to vitally important indications (Kolosov, Efremov et al., 1996, Kolokoltsova et. al., 1997). Allotransplantation of human fibroblasts gave the best results (Kolokoltsova et. al., 1997).
The effects of fibroblasts, collagen and laminine on the wound healing process have been studied in laboratory animals (Blinova et al., 1997). It has been shown that matrigel significantly accelerates healing of crushed wounds and promotes keratinocyte growth in deep wounds (Gorelik et al., 2000). The role of feeder cells in attachment and flattening of keratinocytes from newborn animals has been revealed (Gorelik et al., 1995). The effect of the elements of extracellular matrix on migration activity has been shown using keratinocytes of newborn rats (Gorelik et al., 1997; Gorelik et al., 1998). It has been shown that different isoforms of laminine produce different effects on the process of adhesion of basal and differentiated humans keratinocytes (Gorelik et al., 2001).
JSC “DELSI” has a great experience of work with biopolymeric materials. For instance, within the scope of projects of the International Science and Technology Center (ISTC), this company succeeded in the development of microcapsulated and microgranulated peroral forms of live measles vaccine. Besides, systems of peroral delivery of pharmaceutical products (piracetam, aspirin and erythomycine) and different biologically active food additives have been developed. Different capsulation matrices have been developed; these matrices allow producing pH-dependent membranes that release active components actively due to pH change. Matrices on the basis of chitosan and its derivatives and gelatin resorbed in the organism have been developed. The microcapsulation and microgranulation techniques were implemented in a form of two patented devices: a vibrator and a sprayer. The designed devices allow the production of microcapsules and microgranules either in the mode of drops with membranes forming when they get into calcium chloride solution or by spraying into liquid nitrogen with the formation of frozen granules. The diameter of capsules (granules) may vary from 50 microns to 3 mm. The laboratory has equipment for production and analysis of materials on the basis of natural polysaccharides; performing chemical modification; preparing solutions; and producing film and fibrous materials from these solutions.
The staff of Institute of High molecular Compounds RAS has knowledge, experience, methodology and know-how of investigation of fiber-forming ability of shrimp and crab chitin. A method of structural and chemical modification of chitin has been developed to overcome the fragility of fibers and to produce filaments and films on the basis of chitin with high antimicrobial activity. The produced filaments are to be used as resolving surgical suture; they are passing the stage of preclinical trials and can be the basis of felt and woven materials used as supports for cell cultivation.
The department of experimental medicine of the Pavlov State Medical University (St.-Petersburg) has been trying experimental modeling of different diseases for 30 years. Transplantation of cell cultures of different organs (endocrine organs, spleen, cardiomyocytes and skin) has been the main trend of the department activities over the past 15 years. High-skilled specialists work at then department, and necessary premises and equipment are available. An experimental model of skin defects (burns, trophic ulcers and wounds) has been developed for experiments on investigating the functions of different transplant types used to remove these defects. The University includes the laboratories of clinical and biochemical diagnostics, the immunological, typing, molecular biology and other laboratories that are necessary for complex evaluation of transplants in vivo.
Great professional experience of the specialists of the above institutions, the availability of the necessary material and technical basis, management of up-to-date methods of substitution therapy as well as original ideas of the Project participants about the ways of perfecting the technology of skin restoration using cell products and matrix materials are the basis for the development of perfect technologies for treating burns and other skin wounds of different etiologies.
Project’s Goal :
The goal of the proposed Project is to develop a technology for isolation, cultivation and transplantation of human skin cells suitable for restoration of skin at various defects.
The Project activities will be carried out in three stages (three years). New perfect technologies of production, cultivation and application of cell products for restoration of human skin at healing burns and other wounds will be obtained as a result of the carried out research work.
The developed methods will allow developing new modifications of substitution therapy depending on the character of skin injuries and the origin of skin defects, and solving the problem of donor material deficiency, which is especially urgent for patients with large areas of skin defects, infected patients and children.
The study of the possibility of using human skin stem cells will open a new promising trend. The use of diploid human fibroblasts that have been certified and deposited for storage in ampoules in sufficient number will provide for the delivery of required cell material in patients with serious burns and in critical situations. Preliminary control of the quality of cell products will exclude the risk of infection with different viruses in the process of allotransplantation. The maintenance of optimal conditions at transplantation in practical medicine will reduce the lethality rate among patients with burns, decrease the sizes of donor skin pieces, shorten the hospitalization periods and give better therapeutic and cosmetic effects.
Carrying out experimental studies will allow us to extend the scope of application and to develop a whole spectrum of modifications of cells products adapted for treatment of different skin injuries. The carried out studies will provide new data on the mechanisms of skin regeneration, which could become the basis for the development of optimal conditions in other fields of substitution cell therapy.
The developed technology for obtaining cell products can be used not only in medicine to cover wound surfaces or in cosmetology to remove skin defects, but also as skin models in vitro to test medical and cosmetic preparations and to analyze the effects of ultraviolet rays, radiation and other physical and chemical factors on human skin.
The Project implementation will open new opportunities for practical medicine in Russia and abroad and create the basis for the development of new trends of biomedical research. Undoubtedly, the technology for production of cell products containing certified human cells would have commercial value. Production of cell products and complete development of a technology for their application can be organized not only at SRC VB “Vector” and the Institute of Cytology RAS, but also in any other foreign company having an experience of work with cell cultures and gene-engineered preparations.
The Project implementation will promote the application of experience and expertise of research scientists, who were formerly engaged in the area of biological defense, to developing a new technology of great value both for fundamental science and the needs of practical health service. In addition, the involvement of SRC VB Vector and “DELSI” specialists will allow the social problem of the staff retraining for the public health needs to be solved, which is consistent with ISTC goals and objectives.