Vol 18, No 1 (2023)

Over the past decades, cell viability tests have been an essential research tool in cell biology, tissue engineering, and regenerative medicine. Assessment of cell viability is mandatory in the production and quality control of cell products for biomedical applications.

Methods of viability assessment can be broadly classified according to the underlying mechanism and how the results obtained are evaluated. This article presents variants of the most commonly used tests and protocols for assessing cell viability. Their advantages and disadvantages are presented, which should be considered when planning experiments, e.g., when developing cell preparations for regenerative medicine.

The authors point out the main factors influencing the choice of viability assessment method: efficiency, speed, safety, reproducibility, sample integrity, compatibility with biomaterial, and cell line type. Finally, the authors discuss separately cell viability tests that can be applied not only to 2D cell structures but also to 3D cell structures, which have recently become widespread due to more accurate modeling of biological processes.

INTRODUCTION: Plasma liquid biopsy with tumor cell-free DNA detection is one of the most promising technologies in pancreatic ductal adenocarcinoma diagnosis. Due to low levels of this biomarker in plasma its detectability turns out to be lower than expected. However, some patients with this disease present with biliary obstruction, which enables collection of bile for tumor cell-free DNA analysis.

THE AIM: To comparison of diagnostic potential of cell-free tumor DNA detection-based liquid biopsy of plasma and bile in pancreatic ductal adenocarcinoma patients.

MATERIAL AND METHODS: The pilot study included 15 primary untreated pancreatic ductal adenocarcinoma patients with biliary obstruction. Cell-free DNA was isolated from 5 mL of plasma and 5 mL of bile. Tumor cell-free DNA detection was performed using digital droplet PCR with KRAS gene mutations analysis: G12A, G12C, G12D, G12R, G12S, G12V, G13D и Q61H (183A>C), Q61H (183A>T), Q61K, Q61L, Q61R. False-positive droplets threshold was established during the analysis of plasma cell-free DNA samples from 15 healthy volunteers.

RESULTS: Tumor cell-free DNA was detected in 13 out of 15 bile samples, whereas among paired plasma samples only 9 were positive. All plasma positive samples had a paired positive bile sample. Tumor cell-free DNA levels were statistically significantly higher in bile than in plasma: 538.0 (4.1–1960.0) vs. 4.4 (0–27.6) copies/mL (p=0.005).

CONCLUSION: Bile liquid biopsy in pancreatic ductal adenocarcinoma patients with biliary obstruction is a promising alternative to plasma analysis, due to higher concentrations of tumor cell-free DNA.

BACKGROUND: The problem of male infertility particularly affects men with HIV infections, as semen volume and sperm motility are reduced by highly active antiretroviral therapy.

AIM: To analyze the metabolic parameters of spermatozoa, spermograms and spermatozoa morphology in HIV-infected men.

MATERIALS AND METODS: The study included 47 patients (aged 25–46 years) who were under observation at the clinic of Professor M.A. Florova (Samara). 2 groups were formed: main (n=22) — HIV-infected patients who want to have children (with an undetectable viral load (40–50 copies/ml) and taking antiretroviral therapy); control (n=25) — clinically healthy men with one child. The material was taken and the ejaculate was examined according to standardized methods proposed by WHO experts.

RESULTS: In HIV-infected men, spermatozoa motility was low (the number of progressively motile spermatozoa was 28.50±3.72%), the spermatozoa concentration was two times lower compared to the control group of men, the morphological characteristics of spermatozoa were significantly worse than in the control group: most often pathology of the head (21.75±1.10%) and neck of the spermatozoon (22.30±1.18%) was detected, which negatively affects the fertilizing ability.

In HIV-infected men, changes in metabolic metabolism were noted: the activity of creatine phosphokinase in both sperm plasma (661.95±1.08 U/l) and blood plasma (76.90±1.09 U/l) was lower, than in the control group (844.25±0.13 and 79.50±1.37 U/l, respectively), the glucose concentration (8.07±1.14 mmol/l) was 2 times higher than in the control group (3.06±1.09 mmol/l), the concentration of calcium 6.53±0.01 and sodium (119.20±1.23 mol/l) slightly exceeded those in the group of healthy patients (5.55±0.08 and 116.85±0.01 mol/l, respectively). Electron microscopic analysis revealed fragmentation of sperm DNA: the highest percentage of sperm with fragmented DNA was found in men with HIV infection (more than 23%).

CONCLUSION: It was revealed that in vivo fertilization in HIV-infected men is impossible in most cases. The study forms the basis for a future comprehensive assessment of the state of reproductive function in HIV-infected men to assess their fertility and the need for assisted reproductive technologies.

BACKGROUND: Limbal stem cell deficiency is a complicated pathology of ocular surface, which is not always helped by conservative methods of treatment and surgery is limited by available sources of tissue. Consequently, searching for new effective methods of its treatment is now gaining popularity. The most promising approach is transplantation of tissue-engineered constructs consisting of cultured limbal stem cells (LSCs) and variety of biopolymer carriers.

AIM: This study was performed to obtain and characterize a tissue-engineered constructs consisting of cultured LSCs and collagen membrane.

MATERIALS AND METODS: The study was performed at the Krasnov Research Institute at the Krasnov Research Institute of Eye Diseases and Koltzov Institute of Developmental Biology in cooperation with IMTEK Ltd. with a series of experiments. Two Chinchilla rabbits with an average weight of 3.5 kg and age of 6 months have been involved in this trial. LSCs were isolated and cultured in vitro from the healthy eye of rabbits using a method modified by the authors. The abtained cells were then cultured for 14 days and transplanted to the collagen membrane, which was then examined using immunohistochemical analysis.

RESULTS: The cells isolated from the biopsy were a mixture of fibroblast-type cells and cells with characteristics of LSC. They maintained high survivability, proliferativity, phenotype and stemness on the collagen carrier according to immunofluorescent study.

CONCLUSIONS: Thus, the abstained tissue-engineered constructs could be used for further transplantation to the affected eye with limbal stem cell deficiency under experimental conditions.