Vol 17, No 4 (2022)

The 120th anniversary of professor N.A. Shevchenko, the prominent Soviet scientist which made an worthy impact to the development of evolutionary and experimental histology will held in 2023.

Analyzed his activities in different years at the departments of histology of the S.M. Kirov Military Medical Academy and I.P. Pavlov Leningrad Medical Institute. Professor N.A. Shevchenko conducted a large-scale study of the endothelium of large blood vessels, substantiated the concept of its heteromorphy, established the patterns of reparative regeneration, and studied inflammatory growths of the endothelium. They confirmed the position of the outstanding histologist academician N.G. Khlopin about the endothelium as an angiodermal type epithelium.

The authors shares personal memories of the teacher.

Inflammation is a non-specific process that underlies the pathogenesis of many diseases, including COVID-19. The study of the mechanisms and molecular pathways of this phenomenon is carried out using various models, including in vitro investigations on cell cultures. The most accessible model for reproduction is the lipopolysaccharide-induced one.

This review analyzes the scientific papers in which the model was represented in vitro. It was found that inflammation caused by lipopolysaccharide is realized by activating classical signaling pathways (nuclear factor "kappa-bi", mitogen-activated protein kinase, etc.), changes of expression of circular and non-coding RNAs, etc. Evaluation of the working model of inflammation was carried out by many parameters, the most important of which was the concentration of pro-inflammatory cytokines (IL-6, IL-8, IL-1β, TNF-α). Inflammation through stimulation with lipopolysaccharide was reproduced in cultures of different cells, however, it was most easily reproduced in the culture of monocytes and macrophages, which is explained by their conversion to the M1 phenotype. The co-cultivation of different cells made it possible to study the mechanisms of the pathological process in the model of inflammation more fully.

The review summarizes innovative advances in the field of organoids as a tool for accurate cancer modeling. The conditions for reproducing the microenvironment in vitro based on organoid technology are generalized, various methods for cultivating the tumoroids are considered, and an analysis of their properties is carried out on the example of colorectal cancer.

The final part of the review summarizes the literature data on the use of tumoroids in predicting the therapeutic response of a tumor to chemotherapeutic drugs, studying the mechanisms associated with resistance, and optimizing strategies and potential treatments for patients with malignant tumors. Currently, the tumoroid model is widely used in personalized medicine, basic research, screening of antitumor drugs, and to create libraries of tumors with various mutational profiles.

Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of diseases caused by mutations in at least ten genes. ARCI is characterized by varying degrees of hyperkeratosis and the presence of scales on the surface of the patients’ skin since birth. Despite the variety of mutations and phenotypic manifestations of ARCI, from 32 to 68% of cases are due to a mutation in the transglutaminase-1 gene.

Currently, ARCI therapy is aimed at reducing the symptoms of the disease. To alleviate clinical symptoms, symptomatic therapy with moisturizers, keratolytics, retinoids and other cosmetic substances that improve the condition of the patients' skin is used.

There is a great need for the development of therapy aimed at the root cause of the development of the ARCI. Graft transplantation is usually used to correct eyelid defects in ARCI. Gene and cell therapy are developing as promising methods for the treatment of ARCI, with the help of which it is possible to correct the functional activity of mutant genes, in particular, transglutaminase-1. The review discusses current research on gene and cell therapy approaches and their future prospects in the treatment of patients with various forms of ichthyosis.

BACKGROUND: Spheroids are self-assembled clusters of cells mimicking a tissue-like architecture. Since the structure of complex three-dimensional cellular models is not stable, the formation of core spheroids and further maintenance are crucial stages within the cultivation process. There are a lot of options described for the establishment of 3D cell models. A wide range of reagents is presented from simple hydrogels to complex natural and synthetic composites. However, cultivation of 3D models is still a technically challenging task requiring adaptations of protocols for particular purposes.

AIM: To compare methods of the formation of gomogeneous (3D) and heterogeneous (3D-2) spheroids from tumor and/or stromal cells of breast cancer using hydrogels such as agarose, gelatin and Matrigel™, as well as using ultra-low-adherent plates.

MATERIALS AND METHODS: Breast cancer cell lines MCF7, MDA-MB-231 SK-BR-3 and stromal fibroblasts BrC4f, BrC120f, BN120f were used as a models for 3D and 3D-2 cultures. Spheroids were obtained on a substrate of simple hydrogels or when cultured on a low-adhesive plastic. The processes of formation and growth of spheroids, as well as "crushed preparations" were visualized using a Nikon Eclipse Ti-S series fluorescent inverted microscope (Nikon, Japan).

RESULTS: We demonstrated, that gelatin-based hydrogel is not suitable as a substrate for obtaining 3D and 3D-2 spheroids for any of the cell lines used in the work. The use of only one type of hydrogels does not allow to obtain the entire repertoire of tumor, stromal, and heterogeneous 3D models. Agarose exhibited high output for stromal spheroids and Matrigel™ for tumor cells, and the use of ultra-low-adherent Nunclon™ Sphera™ plates was preferable for 3D-2 models combining both cell types. We also revealed that the application of cooled cultivation plastic and solutions is a technological advantage for handling spheroids growing in low-adherent plates.

CONCLUSION: The proposed approaches for the formation of both homogeneous (3D) and heterogeneous (3D-2) spheroids from tumor and (or) stromal breast cancer cells are a generalized guide to the most effective production of spheroids of various cellular composition.

BACKGROUND: Metabolic profiling of cells is an essential aspect of studies of intracellular metabolism. One of the parameters which can be used for this purpose is a rate of oxygen consumption during degradation or transformation of substrates by cells.

AIM: This study was aimed at the development of a method for assessment of cell physiology and biochemistry based on the metabolic profiling via registration of the cell respiratory activity, and use of this method for evaluation of inhibition and recovery of liver parenchyma cells affected by different compounds.

MATERIALS AND METODS: The method used a Clark-type oxygen electrode with the cells located at its working area, in order to measure dissolved oxygen concentration in the immediate surrounding of living intact cells thereby ensuring their metabolic profiling and physiological status assessment. The experiments were performed using Wistar rat liver parenchyma fragments. Three experimental series were performed: first (series A) was aimed at metabolic profiling of the liver parenchyma; the second (series B) determined the stability of parenchyma’s response to sodium citrate over time; and the third (series C) ensured real-time recording and assessment of the effect of hepatotoxicants on the liver tissue. Solutions of glucose, fructose, sucrose, sodium citrate, sodium pyruvate, L-glutamine, ethanol and hydroquinone were used as the substrates for liver tissue metabolic profiling, and solutions of methanol and clarithromycin as the toxic agents.

RESULTS: During the experiments, a metabolic profiling of tissue and real-time monitoring of toxicity effect on liver parenchyma cells have been demonstrated.

CONCLUSION: The developed method for assessing the physiological and biochemical characteristics of cells can be used to track the metabolic activity, inhibition and restoration of liver parenchyma cells when exposed to substrates and toxic compounds.

BACKGROUND: Currently, to effective bone regeneration, biocompatible matrices containing plasmid constructs with osteoinductor genes are developed.

AIM: To study in vitro the properties of gene-activated matrices based on collagen and platelet-rich plasma impregnated with polyplexes with the bone morphogenetic protein 2 gene.

METHODS: The methods of fluorescence microscopy, flow cytofluorimetry, ELISA, real-time PCR and MTT test were used in the study.

RESULTS: Using the MTT test and fluorescence microscopy to detect live and dead cells, it was shown that the obtained matrices have high cytocompatibility. Impregnation of plasmid constructs in hydrogel matrices based on collagen and fibrin ensures their prolonged release within 25 days. Using flow cytometry and fluorescence microscopy it was shown that polyplexes released from matrices are able to effectively transfect multipotent mesenchymal stromal cells derived from rat adipose tissue. 3 weeks after incubation of cells with matrices impregnated with polyplexes with BMP2 gene, it was shown a 25-fold increase in BMP-2 protein production by enzyme-linked immunosorbent assay. BMP-2 secreted by transfected cells induced osteogenic differentiation of multipotent mesenchymal stromal cells, as evidenced by increased expression of the osteopontin gene and extracellular matrix mineralization.

CONCLUSION: The developed matrices in the future can be used in gene therapy for damaged bone.