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

IN VITRO METHODS FOR THE STUDY OF MECHANISMS OF DEVELOPMENTAL TOXICOLOGY 681Micromass Cell CultureMidbrainABGD12Limb budsCell dissoclationABFigure 16.11Schematic diagram showing the procedure for midbrain or limb bud micromass cultures. Limbbuds or midbrains are dissected from GD 13 rat embryos or GD 12 rabbit embryos anddissociated to a suspension of single cells in medium containing trypsin and EDTA. (A) Highdensitycell droplets (micromasses) are cultured in small petri dishes or 24-well culture platesin a humidified incubator at 37°C with a 5% CO 2 atmosphere for 1 to 1.5 h. (B) After cells haveattached, cultures are flooded with medium, to which can be added test chemicals, inhibitors,modulators, bioactivation systems, and growth factors. (Adapted from Kistler, A. and Howard,W.B., Methods Enzymol., 190, 427, 1990. With permission.)A more recent advancement in the culture of mammalian embryonic cells for the study ofmechanisms has been the use of micromass cultures. This system, as with many other in vitroapproaches, has been touted as a possible rapid screen for teratogenic agents 257–259 but may findgreater utility in mechanistic studies. This method involves the removal of midbrains (CNS) andlimb buds (LB) of 34 to 36 somite rat or mouse embryos by microdissection, followed by successivewashing in calcium- and magnesium-free phosphate buffered saline solution and trypsin to weakencell adhesions (Figure 16.11). The cells are resuspended in an appropriate culture medium, dissociatedinto individual cells by repeated titration through a Pasteur pipette, and filtered on nylonmesh to remove clumps and provided a suitable starting cell suspension. Cell suspensions are platedon plastic petri dishes at concentrations of 1 × 10 5 to 4 × 10 5 cells per 20 µl in culture mediumdrops. Ninety minutes after initial plating, additional culture medium containing the test compound(and possibly an S-9 microsomal bioactivating system and additional modulators) is added. However,the S-9 mix can be toxic and therefore must be limited to a specific period of culture. 257 Atthe end of a 5-d culture period, the medium is removed, and the cells are fixed and stained formorphometric analysis.While midbrain cultures generally provide an excellent means to gather qualitative data, limbbud micromass cultures are typically stained with Alcian Blue to visualize regions of activechondrogenesis. Stained chondrogenic foci can be eluted and then measured spectrophotometricallyto provide quantitative data. In regions of initial plating of the LB micromass, massed mesenchymalcells generally differentiate in place, but other cell types will proliferate at the edge of the massand spread outward (Figure 16.12). This finding provides information about two distinct populationsin the same culture — a proliferating population and a differentiating population — which mayprove useful for various toxicological assays. When stained with mercury orange to assess smallbiothiol content (i.e., GSH and cysteine), cells in the differentiation zone are seen to contain muchless GSH than cells in the proliferative zone (Figure 16.13). This may predispose differentiatingcells to greater damage during oxidative stress. 260 Other biochemical analyses can be performedon these two populations and may be extrapolated to in vivo exposures.Numerous agents have now been tested with the micromass approach, but mostly duringscreening for teratogens, the intended use of the assay. 257–259 Validation of these procedures, especiallythe limb bud micromass technique, was attempted by Flint and Orton. 257 Eighteen differentknown teratogens were injected into time-mated rats. Sixteen hours later, the embryos treated inutero were removed and their limb bud cells were plated as micromass cultures. Of the 18 teratogens© 2006 by Taylor & Francis Group, LLC