Characterization of Porcine Ventral Mesencephalic Precursor Cells following Long-Term Propagation in 3D Culture

Abstract

The potential use of predifferentiated neural precursor cells for treatment of a neurological disorder like Parkinson's disease combines stem cell research with previous experimental and clinical transplantation of developing dopaminergic neurons. One current obstacle is, however, the lack of ability to generate dopaminergic neurons after long-term in vitro propagation of the cells. The domestic pig is considered a useful nonprimate large animal model in neuroscience, because of a better resemblance of the larger gyrencephalic pig brain to the human brain than the commonly used brains of smaller rodents. In the present study, porcine embryonic (28–30 days), ventral mesencephalic precursor cells were isolated and propagated as free-floating neural tissue spheres in medium containing epidermal growth factor and fibroblast growth factor 2. For passaging, the tissue spheres were cut into quarters, avoiding mechanical or enzymatic dissociation in order to minimize cellular trauma and preserve intercellular contacts. Spheres were propagated for up to 237 days with analysis of cellular content and differentiation at various time points. Our study provides the first demonstration that porcine ventral mesencephalic precursor cells can be long-term propagated as neural tissue spheres, thereby providing an experimental 3D in vitro model for studies of neural precursor cells, their niche, and differentiation capacity.

Enzyme-Free Dissociation of Neurospheres by a Microfluidic Chip-Based Method

“…After coating, transfer the PR coated wafer onto the hot plate at 65 C for 25-30 min, then transfer the wafer to the other hot plate at 95 C for 70-80 min for the soft-baking process, followed by turning the hot plate off to allow the wafer to cool down to room temperature. 4. Place the wafer on the chuck stage of the mask aligner, align the chrome photomask to the PR coated wafer and expose the wafer to UV light (365 nm) at a dose of 600 mJ/cm 2 to photolithographically pattern the PR coated silicon wafer.…”

“…Neurosphere dissociations are commonly performed by using enzymatic methods which can generate high yields of single dissociated cells with reproducible results. However, to avoid protease and collagenolytic activities, which degrade cell membrane proteins [3], mechanical methods including trituration [4], manual cutting with scalpel [5], micro-scissors [6], tissue chopper [7], and microfabricated filter are used [8]. We have developed a microfluidic chip-based mechanical method to dissociate neurospheres [9].…”

The Olfactory Bulb in Newborn Piglet Is a Reservoir of Neural Stem and Progenitor Cells

“…Three previous studies have documented the isolation, culture, and characterization of neural precursor cells from porcine fetal brain [ 51 - 53 ]. One study isolated, from cerebrum of 60-day pig fetuses, and grew neurosphere-forming cells in the presence of FGF2 and EGF and demonstrated, by RT-PCR, expression of NPC and glial markers as well as Sox2 [ 51 ].…”

“…Another study isolated, from forebrain SVZ of 65-80 day pig fetuses, and grew neurosphere-forming cells in the presence of FGF2 and EGF over long-term during which neurospheres spontaneously differentiated into the 3 primary neural cell lineages [ 52 ]. A more recent study used fetal day 28-30 pigs to isolate and propagate in culture ventral mesencephalic NPCs [ 53 ]. Thus, our study builds on the value of the swine brain in NSC/NPC research and is the first to demonstrate the isolation, propagation, and differentiation of large numbers of multipotent NSCs from the postnatal piglet forebrain-SVZ and OB-SVZ.…”

Single-Cell Enzyme-Free Dissociation of Neurospheres Using a Microfluidic Chip

“…However, without the use of enzymes, neurosphere dissociation by pipetting alone can result in the death of more than 50% of the processed cells . In previous studies, other mechanical methods have been used to circumvent the limitations associated with enzymatic dissociation, including manual cutting of the spheres into smaller fragments using scalpels, microscissors, tissue choppers, , and more recently, a microfabricated “biogrid” device . While the scalpels, microscissors, and tissue choppers are simple and straightforward to use, the biogrid device has the advantage of being able to process large quantities of cells in closed systems.…”