Optimization of cell culture and the spread of metaphase chromosomes for cytogenetic studies using Taguchi design in the deer

Abstract

Metaphase production is an important aspect of cytogenetic studies. To achieve acceptable metaphase spreads for cytogenetic study, cells, depending on source animals, require different operating conditions. It is therefore necessary to optimize the process to produce a protocol, which achieves the best result. This study aimed to establish the growth and arrest requirements of cultured Peripheral Blood Mononuclear Cells (PBMCs) from three breeds of deer (Axis axis, Rusa timorensis and Rusa unicolor) using Taguchi design. Twenty-four animals (8 in each study) were used in the study. Culture medium (CM) (RPMI1640+ or RPMI1640-, with or without 16.6 mM glucose and 4 mM glutamine), fetal bovine serum (FBS, 10% or 20%), agitation frequency (AF, once or twice), time of colcemid introduction (col, early or late) and the duration of cells in KCL (DKCL) (20 or 30 minutes) were the factors investigated. They were set at two levels in an L8 (2˄5) orthogonal array. Rusa timorensis had RPMI 1640+, 20% FBS, AF twice per day, early col and DKCL of 20 minutes as the optimal factor levels. Rusa unicolor on the other hand had RPMI 1640+, 20% FBS, AF once per day, late col and DKCL of 30 minutes as the optimal factor levels. Axis axis had RPMI 1640+, 20% FBS, AF once per day, early col and DKCL 30 minutes as the optimal factor levels. This work demonstrated the utility of the Taguchi design in optimizing the production of metaphase spreads from cultured PBMC in the deer.