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

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.

1. Durnev AD, Zhanataev AK. Relevant aspects of drug genetic toxicology. Bulletin of the Scientific Centre for Expert Evaluation of Medicinal Products. Regulatory Research and Medicine Evaluation. 2022;12(1):90–109 (In Russ.). https://doi.org/10.30895/1991-2919-2022-12-1-90-109

2. Deng W, Tsao SW, Lucas JN. A new method for improving metaphase chromosome spreading. Cytometry A. 2003;51(1):46–51. https://doi.org/10.1002/cyto.a.10004

3. Moralli D, Yusuf M, Mandegar MA. An improved technique for chromosomal analysis of human ES and iPS cells. Stem Cell Rev Rep. 2011;7(2):471–7. https://doi.org/10.1007/s12015-010-9224-4

4. Liehr T, Weise A, Person L. How to obtain high-quality metaphase spreads for molecular cytogenetics. Curr Protoc. 2022;2(2):e392. https://doi.org/10.1002/cpz1.392

5. Novgorodova IP. Comparative analysis of hypotonic solutions for cytogenetic studies of animals. Agrarian Science. 2021;(6):24–6 (In Russ.). https://doi.org/10.32634/0869-8155-2021-350-6-24-26

6. Ovsepyan VA, Yugov YuI. Method for obtaining preparations of metaphase chromosomes out of bone marrow cells in hemoblastose-suffering patients. Patent of the Russian Federation No. 2200318; 2000 (In Russ.). EDN: MMDIRO

7. Budantsev AYu, Demyanov AYu. Deformations during histological tissue processing. I. Methods for morphometric analysis of deformations. Cytology. 2017;59(5):362–8 (In Russ.). EDN: YQGDZP

8. Konenkov VI. Korolev MA. Churin AA. Studying the possible mutagenic properties of new medicine on the basis of complex lithium citrate, aluminum oxide and polymethylsiloxane. Siberian Scientific Medical Journal. 2019;39(5):62–7 (In Russ.). https://doi.org/10.15372/SSMJ20190507

9. Dolgova EV, Nicolin VP, Popova NA. Pathological changes in mice treated cyclophosphamide and exogenous DNA. Vavilov Journal of Genetics and Breeding. 2013;17(1):129–46 (In Russ.). EDN: RUGRAJ

10. Blumenthal AB, Dieden JD, Kapp LN, Sedat JW. Rapid isolation of metaphase chromosomes containing high molecular weight DNA. J Cell Biol. 1979;81(1):255–9. https://doi.org/10.1083/jcd.81.1.255

11. Fukami M, Shima H, Suzuki E, Ogata T, Matsubara K, Kamimaki T. Catastrophic cellular events leading to complex chromosomal rearrangements in the germline. Clin Genet. 2017;91(5):653–60. https://doi.org/10.1111/cge.12928

12. Mardin BR, Drainas AP, Waszak SM, Weischenfeldt J, Isokane M, Stütz AM, et al. A cell-based model system links chromothripsis with hyperploidy. Mol Syst Biol. 2015;11(9):828–40. https://doi.org/10.15252/msb.20156505

13. Pacchierotti F, Stocchi V. Analysis of chromosome aberrations in somatic and germ cells of the mouse. Methods Mol Biol. 2013;1044:147–63. https://doi.org/10.1007/978-1-62703-529-3_7