Characterization of long‐term mouse brain aggregating cultures: Evidence for maintenance of neural precursor cells

Abstract

An extensive characterization of fetal mouse brain cell aggregates has been performed using immunohistochemical and stereological methods. Single cell suspensions from mechanically dissociated cortex and hippocampus were cultured in serum-free, B27-supplemented medium under constant gyratory agitation for up to 56 days. Three-dimensional aggregates started to form immediately after seeding and reached a final average size of 500 microm in diameter. Among the cell types identified, neurons were the most abundant cells in the aggregates, followed by astrocytes, microglia, and oligodendrocytes. Western blotting for synaptophysin and immunostaining for neurotransmitter-related molecules indicated the presence of well-defined phenotypic characteristics of the neurons in this culture system, suggesting functionality. Proliferating cells, many with neural precursor cell properties, were seen throughout the culture period and could be isolated from the aggregates even after 2 months in culture. Neural precursor cells were isolated from the aggregates after more than 1 month in culture; these cells were successfully differentiated into neurons, astrocytes, and oligodendrocytes. The aggregate culture system may provide a versatile tool for molecular dissection of processes identified in mouse models, including transgenic animals and manipulation of neural precursor cells.

An in vitro model for de‐ and remyelination using lysophosphatidyl choline in rodent whole brain spheroid cultures

“…GFAP positive astrocytes were primarily present at the rim of the spheroids. These features of the model were in agreement with previous studies (Berglund, et al, 2004;Honegger and Richelson, 1976;Trapp, et al, 1979). LPC has long been known to induce demyelination in vivo (Blakemore, et al, 1977;Hall, 1972;Jeffery and Blakemore, 1995;Waxman, et al, 1979;Woodruff and Franklin, 1999).…”

“…We tested whether the demyelination protocol set-up in rat could also be applied in a mouse spheroid model (Berglund et al, 2004). To induce similar demyelination in mice addition of three times 0.15 mM LPC was needed when compared with three times 0.12 mM used in rats.…”

In vitro modeling of central nervous system myelination and remyelination

“…As with other culture techniques, a key refinement was the development of serum-free chemically defined medium (Honegger et al, 1979), which allowed aggregate cultures to be used to further characterise myelination in vitro (Trapp et al, 1982), and identify factors that influence myelination (Almazan et al, 1985;Tosic et al, 1992)}. A further development was the preparation of aggregate cultures from mouse (Berglund et al, 2004), which allowed this technique to be used in studies using tissue from transgenic mouse lines .…”

A simple method for large‐scale generation of dopamine neurons from human embryonic stem cells

“…In an attempt to adapt this protocol to hESC-derived neural tissue, we dissociated the expanded neural progenitors with Accutase and suspended them as single cells in conditioned NS medium supplemented with B27 containing 10 ng/ml bFGF. We plated 7 million cells per 75-cm 2 flask on a gyrating shaker (ES-W, Kuhner AB, Switzerland) operating at 60 rpm inside an incubator at 378C and 5% CO 2 , as described previously (Berglund et al, 2004). On the third day of suspension culture, to the medium we added GDNF, BDNF, dbcAMP, and AA at the same concentrations mentioned above but no bFGF to induce maturation.…”

“…The sphere culturing technique has certain advantages. For example, spheres recapitulate some of the three-dimensional structure of the nervous system (Berglund et al, 2004), and neurons in spheres are more readily grafted without loss of viability . After detaching the neural progenitors, we cultured them on the shaker.…”