A Practical Approach, Second Edition=Ronald D. Ho.pdf

IN VITRO METHODS FOR THE STUDY OF MECHANISMS OF DEVELOPMENTAL TOXICOLOGY 673Trowell MethodOrgan CultureLimb budLimbBoneHeartPalateVisceral yolk sacEyeReproductive organsTooth germRoller CultureFigure 16.8Organ culture of numerous structures can be initiated from embryos obtained during a broadrange of developmental stages, depending on experimental needs. Organs can be grown inculture on static grids or filters using the Trowell method (Exp. Cell Res., 16, 118, 1959), asdepicted in the upper part of this illustration, or in roller cultures, as shown in the bottom portion.Growth and differentiation are generally superior in roller cultures.bioactivation systems extend the utility of these experimental models. They allow for the study oforgan-organ interactions and direct xenobiotic effects on interrelated cells and tissues. Endpointsfor evaluation of organ cultures range from gross morphogenetic changes to cellular effects,biochemical alterations, metabolic transitions, and physiological responses to the molecular basesof differentiation, as well as the genetic events that regulate or are regulated by xenobiotics.1. Limb Bud and Related Perinatal Bone CulturesA number of toxicants produce persistent malformations involving the developing limbs. Thissuggests that these organs may be particularly sensitive to chemicals during development and may,therefore, be good systems in which to study mechanisms of developmental toxicology. 199,200Undifferentiated limb buds from the chick were first successfully grown in culture by use of aplasma clot environment. 201 Later, chick, rat, and mouse limb buds were cultured by use of theTrowell method, 198 where limbs were grown attached to Millipore ® filters in a humidified environment.Plasma clot techniques are difficult to work with in studies involving the addition of chemicalagents or radioisotopes because the clot material interferes with chemical distribution and quantitation.Initial studies by Aydelotte and Kochhar 202 established the limb bud as an important earlytool for the mechanistic study of chemical induced limb malformations. Limb buds removed from40 to 45 somite rodent embryos provided the greatest degree of growth and differentiation andcould be cultured successfully for up to 6 d, when they reached optimal differentiation (Figure16.9). Organ cultures of this type produce the distinct patterns of chondrogenesis and establishmentof the skeletal anlage seen with normal limb growth in vivo, but they require a longer period tocomplete differentiation and undergo developmental growth. The expected sequence of developmentand the programmed production of unique products of differentiation, however, show that thecultured limb buds bear close morphological and biochemical resemblance to the intact limb invivo. Subsequent modifications to the Trowell method by Neubert and Barrach 203 utilized a submergedroller bottle culture system. Their modification effectively improved the cultured limbs’morphological appearance, decreased the time needed for optimal growth and differentiation, andprovided greater accessibility of organs and tissues to growth factors, nutrients, oxygen, and testchemicals. This approach effectively overcomes many of the morphological infidelities associatedwith growing organs attached to a solid filter substrate and produces differentiated structures in© 2006 by Taylor & Francis Group, LLC