Search for methodological approaches of organotypic cultivation for following cryopreservation

Abstract

Introduction. A number of modern pharmaceuticals and transplantation methods require the use of small organs and tissues fragments with the preserved interactions of cells. These can be organotypic cultures, cell-engineering structures, 3D prints. Cryopreservation of such objects greatly expands the possibilities of their application. When working with multicellular structures, it is necessary to overcome specific problems, such as the phenomena of central necrosis, infiltration with a cryoprotectant, and features of crystal formation. The aim of the research was to find methods of organotypic cultivation and evaluation of mouse organ slices for following cryopreservation. Materials and methods. The organs of BALB/c mice were used. Using a vibratome, sections (slices) were made from animal organs embedded in agarose blocks. The possibility of using various concentrations of agarose in different organs was determined: liver, brain, spleen, heart, kidney, testicles, ovaries, lungs, and uterus. Light microscopy and screening methods for evaluating objects were used. Results and discussion. A necessary step in the manufacture of slices is pouring in agarose from 3 to 5% at a temperature of not more than 45°C, which allows for making slices thickness from 0.02 to 0.5 mm. The quality and thickness of the samples depend on the structure of the tissue, the number and density of connective tissue fibers, the size of the structural and functional elements. It is possible to obtain thin slices of nervous tissue – from 0.05 mm. Slices of the liver and kidneys should be made with a thickness of about 0.3 mm, which corresponds to the size of their structural and functional units (lobules and glomeruli, respectively). Activities with the lungs, spleen, and heart are complicated by the density of connective tissue and the lack of tight fixation in agarose. The production of thin slices of the testicles is not possible due to the slight connection between the tubules, which are separated from each other during the cutting stage. For screening evaluation of slices, trypan blue staining, the use of light microscopy, and tetrazolium and resazurin tests are possible. Conclusions. The quality of the slices for organotypic cultivation and cryopreservation depends on the density of the connective tissue, the size and the relationship between the structural elements. It is possible to obtain high-quality slices of nervous tissue, liver, and kidneys.