Neuron Growth in Polydimethylsiloxane Microfluidic Devices
Tanner, Arla (2018)
2018
Biotekniikka
Teknis-luonnontieteellinen tiedekunta - Faculty of Natural Sciences
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Hyväksymispäivämäärä
2018-12-05
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tty-201811222724
https://urn.fi/URN:NBN:fi:tty-201811222724
Tiivistelmä
Central nervous system (CNS) is the control center of the body and it is composed of millions of inter connected neuronal cells or neurons. Neurons are polar and while maturating send processes called axons and dendrites. These processes or neurites connect via synapses and thus form neuronal networks that transmit signals throughout the body. CNS has a very limited potential to regenerate. To understand CNS regeneration, also neurite damage and the limitations in neurite regeneration must be studied. To help the study of axons and their regeneration in vitro, the growth of neuron somas can be restricted, and neurites compartmentalized from the culture. Neurite compartmentalization simplifies culture analysis and improves reproducibility of results.
In this study, neurons were cultured in microfluidic polydimethylsiloxane (PDMS) devices. PDMS devices had two compartments soma or plating compartment and neurite compartment separated by 15 restriction tunnels. PDMS devices were oxygen plasma treated, reversibly bonded to glass wafers, and coated with cell type specific procedures prior cell plating. Restriction tunnels were 10 µm in width, either 1 or 3,5 µm in height and either 100, 250 or 500 µm in length. Neurons cultured in PDMS devices in this study were four types of neurons differentiated from human pluripotent stem cells (hPSCs) and rat cortex neurons. The aim of the study was to find restriction tunnel dimensions that are most successful in axon compartmentalization and to study cell viability and neurite growth in PDMS devices. Cell cultures were analyzed with phase contrast microscopy, to study cell morphology and neurite network forming, immunocytochemistry, to confirm neural nature of cells, LIVE/DEAD assay, to study cell viability, and DAPI staining, to detect neuron somas from inside the tunnel structures.
It was found that neurons from both human and rodent origin could be cultured in these PDMS devices for up to four weeks. It seems that 1 µm high restriction tunnels were more successful in soma growth restriction of all neuron types tested than 3,5 µm high restriction tunnels. Although, neurites grew faster and in bigger bundles through tunnels that were 3,5 µm high than through tunnels that were 1 µm high. No remarkable differences in soma growth restriction or neurite growth were detected between the different restriction tunnel lengths. Variation in results between cell types suggest that restriction tunnel dimensions need to be optimized for each cell type and application. Results from this study can be used to further develop microfluidic devices that are used to culture neurons and compartmentalize neurites. Future studies could include integration of activity measuring microelectrode arrays (MEAs) to PDMS devices or co-culturing of neurons with for example glial cells.
In this study, neurons were cultured in microfluidic polydimethylsiloxane (PDMS) devices. PDMS devices had two compartments soma or plating compartment and neurite compartment separated by 15 restriction tunnels. PDMS devices were oxygen plasma treated, reversibly bonded to glass wafers, and coated with cell type specific procedures prior cell plating. Restriction tunnels were 10 µm in width, either 1 or 3,5 µm in height and either 100, 250 or 500 µm in length. Neurons cultured in PDMS devices in this study were four types of neurons differentiated from human pluripotent stem cells (hPSCs) and rat cortex neurons. The aim of the study was to find restriction tunnel dimensions that are most successful in axon compartmentalization and to study cell viability and neurite growth in PDMS devices. Cell cultures were analyzed with phase contrast microscopy, to study cell morphology and neurite network forming, immunocytochemistry, to confirm neural nature of cells, LIVE/DEAD assay, to study cell viability, and DAPI staining, to detect neuron somas from inside the tunnel structures.
It was found that neurons from both human and rodent origin could be cultured in these PDMS devices for up to four weeks. It seems that 1 µm high restriction tunnels were more successful in soma growth restriction of all neuron types tested than 3,5 µm high restriction tunnels. Although, neurites grew faster and in bigger bundles through tunnels that were 3,5 µm high than through tunnels that were 1 µm high. No remarkable differences in soma growth restriction or neurite growth were detected between the different restriction tunnel lengths. Variation in results between cell types suggest that restriction tunnel dimensions need to be optimized for each cell type and application. Results from this study can be used to further develop microfluidic devices that are used to culture neurons and compartmentalize neurites. Future studies could include integration of activity measuring microelectrode arrays (MEAs) to PDMS devices or co-culturing of neurons with for example glial cells.