Neuronal Differentiation in Reaggregate Cell Cultures1

Nicholas W. Seeds,     Department of Biochemistry/Biophysics/Genetics, University of Colorado Medical School, Denver, Colorado

Publisher Summary

This chapter discusses various aspects of neuronal differentiation and cell interaction in brain-cell reaggregates. Aggregation of certain cell types is a normal event in embryogenesis. Although freshly dissociated cells spontaneously reassociate, their aggregation is enhanced by gently rotating flasks of cell suspensions to increase the collision frequency between cells. The chapter describes an experiment in which cerebellare cells from several different neurological mutant mice, which display abnormal cerebellar histogenesis, and cells from their normal littermates were compared for aggregatability. Aggregation is a rapid process, and initially, these reaggregates are a random distribution of loosely packed cells. The cells are often interconnected by filopodia but are lacking membrane specializations or cell junctions. It can be hoped that reaggregate and surface cultures of dissociated brain cells will allow neurobiologists to ask better questions and obtain even better answers concerning the functional relationships that make up the nervous system.

I  Introduction

II  Aggregation

III  Morphological Development

IV  Biochemical Development

A  Neural Enzymes

B  Hormonal Influences

C  Pharmacological Aspects

V  Cell–Cell Interaction

VI  Conclusion

References

I Introduction

Neural cell culture has come a long way since Ross Harrison (1907) first put living nerve cells into culture. Although one might question what major discoveries have been made, cell culture has had a dramatic impact on many facets of neurobiological research, including development, genetics, biochemistry, pharmacology, electrophysiology, immunology, and toxicology. Three basic types of cultures have been used in these studies. Expiant cultures retain intercellular relationships, and a variety of developmental events can be followed in the tissue, including neurite outgrowth and the specificity of connections formed between specific tissues or brain regions. Monolayer cultures of dissociated cells have lost some of the intercellular associations characteristic of neural tissue, but they permit the analysis of events at the single-cell level which has been very advantageous to electrophysiological and immunological analysis, conditioned media and growth factor studies, as well as the popular clonal cell lines of neuronal and glial origin. Unfortunately, many brain cells do not like to settle down on these glass or plastic surfaces, and the cells must be coaxed onto these substrates by coating them with collagen or polylysine. Many cells, especially neurons, prefer to aggregate with one another rather than attach to such substrates. Therefore, another cell culture approach has been to allow these cells to reaggregate and reestablish cell interactions characteristic of the tissue source. Reaggregate cultures have shown a variety of developmental phenomena characteristic of brain maturation and suggest that certain aspects of neural development may require a three-dimensional organization of specific cell types. If so, why bother dissociating the tissue in the first place? If the tissue had not been dissociated we would not have known that a three-dimensional organization requirement existed; and more importantly, with dissociated cells we can reconstruct the tissue in specific ways using various permutations and combinations of these different cell types! This presentation will focus on various aspects of neuronal differentiation and cell interaction in brain cell reaggregates.

II Aggregation

Aggregation of certain cell types is a normal event in embryogenesis. In the developing nervous system individual neural crest cells migrate away from the neural plate, only to reaggregate at distant sites to form autonomic, cranial, and spinal ganglia. The formation of aggregate cell cultures takes advantage of these cellular affinities that most cells in developing tissues show for like cell types or cells from the same tissue. The reaggregation of dissociated cells from amphibian blastula was first observed by Roux (1894). More recently reaggregation of cells from embryonic and neonatal avian and mammalian tissues has been extensively studied (see Moscona, 1973, for a review). Cells from different tissues form reaggregates which differ in size, shape, and internal structure. Although brain cells and other neural tissues readily aggregate, cells from various brain regions also produce reaggregates that can differ markedly in their relative size and shape [i.e, cerebral cortex cells form much larger aggregates than cerebellar cells (Garber and Moscona, 1972a)].

A variety of cellular, extracellular, and environmental factors influence aggregation in vitro (Table I). The age or degree of differentiation expressed by the tissue influences aggregate size; in general, the more differentiated the tissue, the smaller the reaggregates (Garber and Moscona, 1972a). Either enzymatic or mechanical procedures have been used to dissociate tissue into individual cells. A combination of enzymatic dissociation of neural tissue with trypsin and mechanical sieving of the loosened tissue (Seeds, 1971; Marks and Seeds, 1978b) gives a greater yield of viable cells which form larger aggregates than obtained with either dissociation method alone. Trypsinization may provide a more adhesive surface by uncovering recognition sites on the cell surface that function during morphogenesis and are masked when differentiation is complete (Moscona, 1973). However, some lots of trypsin contain toxic activities that can be deleterious. The numerous membrane fragments and dead cells found in the mechanically dispersed tissue preparations inhibit cellular aggregation, yielding smaller aggregates. The importance of aggregate size to neuronal differentiation will be discussed in Section V. The culture media can influence both aggregation and subsequent developmental events (Table II). Both basal Eagle’s medium and Dulbecco’s modification of Eagle’s medium have been shown to favor aggregation and biochemical maturation of neural cells in aggregate culture (Moscona, 1973; Seeds, 1971; Honegger et al., 1979). Fetal mouse brain cells failed to aggregate when placed in CRML 1066 or McCoy’s 5a medium. Although fetal calf serum is routinely used, Honegger et al. (1979) have shown the differentiation of brain aggregates in a serum-free media of defined protein supplements.

Table I

Factors Influencing Cell Aggregation

Tissue of origin

State of differentiation of tissue

Tissue dissociation methods

Culture medium

Rotation speed

Temperature

Ca2+

Cell-specific aggregation factors

Table II

Influence of Culture Media on Aggregation and Differentiationa

aAll media contained 10% fetal calf serum, while Eagle’s and Waymouth’s media contained an additional 0.4% glucose. Seventeen-day fetal mouse brain cells were cultured as reaggregates in the above media for 15 days prior to biochemical analysis of choline acetyltransferase and acetylcholinesterase activities.

Although freshly dissociated cells spontaneously reassociate, their aggregation is enhanced by gently rotating flasks of cell suspensions to increase the collision frequency between cells. The rotation speed, flask shape, and the volume of medium are also variables affecting aggregate size in the “rotation mediated” in vitro aggregation technique (Moscona, 1973). Aggregation is rapid, occurring within minutes to hours at 37°C; however, aggregation is temperature dependent and does not occur at temperatures below 15°C (Moscona, 1961), suggesting that some metabolic events or membrane fluidity changes are necessary for cell–cell adhesion. Bivalent cations have been implicated in cell aggregation for some time (Steinberg, 1958;...