Culture requirements and cytochemical characteristics of colony forming units-granulocyte/monocyte in cynomolgus monkeys

Abstract

For the preclinical safety assessment of recombinant human haematopoietic growth factors, the in vitro bone marrow progenitor cell assays could provide valuable information that can be used to select appropriate biologically responsive animal species and to predict the potential toxicity and dosage selection for in-life toxicology studies. The cynomolgus monkey is a commonly used non-human primate model for the assessment of efficacy and toxicity of such compounds; however, the information regarding their bone marrow progenitor cell assays is scarce. This study evaluated the culture requirements and the morphological and cytochemical characteristics of colony forming units-granulocyte/monocyte (CFU-GM) for this species. The optimal CFU-GM growth was achieved in methylcellulose cultures of minimum essential medium-alpha (MEM-alpha) that contained 5% leucocyte-conditioned medium (LCM) and 30% heat inactivated horse serum (HIHS), after 14 days of incubation in a humidified atmosphere. A triple cytochemical staining method using naphthol-ASD chloroacetate esterase (CAE), alphanaphthol acetate esterase (ANAE) and luxol fast blue (LFB) was used to characterise the monocyte/macrophage, neutrophil and eosinophil colonies, respectively. This cytochemical method showed the presence of approximately 15% pure neutrophil, 61% pure monocyte/macrophage, 4% pure eosinophil, 15% mixed granulocyte/monocyte (positive for at least two stains) and 3.5% unstained colonies in 14-day-old cultures. The monocyte, neutrophil and mixed colonies were smaller in diameter, compact and contained a larger number of cells than the eosinophil colonies. Since eosinophil colonies exhibit lower cell density, they may be easily missed in total CFU counts performed at low magnification. These in vitro assays could be used to predict the potency of and lineage-specific response to haematopoietic growth factors in cynomolgus monkeys.