Marysia Placzek and Kim Dale 1 Introduction

The characterization of molecular and antigenic markers that identify specific vertebrate cells has increased dramatically in recent years. As a result, patterns of cell differentiation and development can be observed in vivo, and subsequently, the tissue interactions and differentiation factors that may operate to establish these patterns can be examined in vitro. Three-dimensional collagen gels provide a culture environment in which in vitro assays can be established, and used to assess the biological activity of one tissue or protein in patterning cells within a second potentially responsive tissue. Initially developed as a means to culture embryonic neuronal tissue and examine the effect of trophic factors (1), such gels have been used more recently to identify tissues and molecules responsible for inductive (2-17), chemotropic (18-23), and chemorepulsive (24,25) interactions. The advantages of a three-dimensional culture system are especially marked when the amount of material that is available to assay is limiting, and so, to date, they have been especially useful in the development of functional bioassays for explanted embryonic tissue. When used in conjunction with in vivo assays, such as described in Chapters 17-19, results obtained from such three-dimensional in vitro assays are especially compelling and can be used to extend and evaluate rapidly an observation made initially in an in vivo bioassay. The ability to assay specific tissues in isolation has many advantages. Large numbers of experiments can be set up in a single day, and the effects of tissues and proteins can be examined in the absence of other tissue that would normally be adjacent and potentially interfering in vivo. Furthermore, the isolation and culture of appropriate pieces of tissue enable one to understand the mechanism underlying an observed effect; thus, for

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

instance, one can distinguish trophic from tropic interactions and inductive vs migratory effects in a manner that is impossible in an in vivo assay.

The three-dimensional culture system has a number of advantages compared to a two-dimensional culture system. The collagen gel supports well the integrity of explanted tissue, which develops in culture in a manner appropriate to an equivalent piece developing in vivo: the explanted tissue does not flatten or spread, as occurs when cultured on a two-dimensional substrate. Explanted tissue of very small size often develops appropriately, when similar-sized explanted tissue cultured on a two-dimensional substrate fails to survive. The ability to manipulate the orientation of tissue explants within the three-dimensional gel means that very small quantities of tissue can be used to assay the effect of a protein or one tissue on a second. Consequently, sufficient tissue can be easily gathered to perform experiments and controls that are statistically relevant. Furthermore, the ability to manipulate the orientation of two tissues relative to one another within a three-dimensional gel provides a means to assess whether the action of a tissue is mediated by a factor that is readily diffusible, or by a membrane-associated factor. Finally, subsequent to culture, the development of the explanted tissue can readily be examined by either immunohistochemical or in situ hybridization techniques.

Although other types of material, such as fibrin clots or matrigel, can be used in place of collagen, the collagen matrix is less costly and is easy to manipulate. The collagen, which is prepared and maintained under conditions of low pH and low temperature, begins to gel as the temperature and the pH are increased. These two paramenters can be altered to establish optimal conditions in which the collagen sets sufficiently slowly to allow tissues to be manipulated within it. Matrices such as agar or agarose cannot be used as substitutes for collagen—the high density of such gels appears to suffocate the explanted tissue, whereas collagen fibrils are sufficiently spaced to allow the diffusion of nutrients from the culture fluid through the collagen gel to reach the cultured tissue.

The protocol presented here describes how to prepare collagen that is suitable for culturing embryonic tissues, and how to process it to make a three-dimensional gel in which tissue explants can be manipulated and embedded. The techniques used to isolate and embed embryonic tissue are described, as are the ways in which growth and differentiation factors can be presented to tissue explants within these gels. In addition, a summary is provided of the manner in which such cultures can be manipulated to process by immunohistochemical or in situ hybridization techniques in order to examine the pattern of cell differentiation within tissue explants.

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