Optimisation of a Cytogenetic Sample Preparation Procedure for <i>In Vivo</i> Metaphase Analysis of Mammalian Bone Marrow Cells

Abstract

INTRODUCTION. Metaphase analysis is used to assess the mutagenicity of medicines, and its accuracy depends directly on the quality of cytogenetic preparations. Existing procedures for cytogenetic sample preparation require optimisation because of their time and labour intensiveness and potential for artefactual chromosome loss that leads to errors in the interpretation of analytical results.AIM. This study aimed to optimise a cytogenetic sample preparation procedure for mammalian bone marrow cells for in vivo metaphase analysis.MATERIALS AND METHODS. The study was performed in randomly bred male mice weighing 18–20 g. The authors prepared cytogenetic samples of bone marrow cells according to the procedures set forth in the Guidelines for Conducting Preclinical Studies of Drugs (A.N. Mironov (ed.), 2012), and GOST 34659-2020, Methods for Testing the Impact of Chemical Products on the Human Body. Assessment of Chromosomal Aberrations in Bone Marrow Cells of Mammals, as described and with modifications. Sample preparation involved using Hanks’ solution, sodium citrate and potassium chloride solutions, acetic acid, methyl alcohol, medium 199, HEPES, and Giemsa stain.RESULTS. The authors conducted an experimental comparison of the characteristics of cytogenetic preparations of mammalian bone marrow cells for in vivo metaphase analysis prepared using several methods. The samples prepared in accordance with the Guidelines for Conducting Preclinical Studies of Drugs exhibited the highest percentage of analysable metaphase plates, in comparison with those obtained using the other sample preparation procedures. The authors optimised the sample prepara tion conditions, including the composition of the hypotonic solution (0.56% KCl), the time of hypotonic treatment (20 min), the time of fixation (12 h), and the composition of the culture medium (liquid medium 199 with Hanks’ salts and glutamine). The authors introduced a step of preliminary chromosome fixation with 6% acetic acid, which generally contributed to an increase in the proportion of analysable metaphase plates by reducing the scattering and sticking of chromosomes. CONCLUSIONS. The authors presented a modified sample preparation procedure for cytogenetic preparations that, compared with the previous procedures, offers an increase in the percentage of analysable metaphase plates to 56%, a 12% rise in the percentage of full sets of chromosomes (n=40), and a 2–10-hour reduction in the preparation time.