Integration of synthetic astrocytes into neuroblastoma spheroids via droplet based microfluidics

Csatári, Júlia

4. term

Nanotechnology, Master

2019

2019-06-10

70

Tre-dimensionelle cellekulturer, såkaldte spheroider, bruges i stigende grad, fordi de efterligner væv bedre end flade cellelag. Med mikrofluidiske enheder kan spheroider samles i et præcist og ensartet mikromiljø, hvor celler ligger tættere og derfor interagerer mere. I dette arbejde præsenterer og vurderer vi en mikrofluidisk, gel-stilladsfri metode til at fremstille mange spheroider effektivt. Vi laver to typer: samskultur-spheroider af neuroblastomceller (ofte brugt som model for nerveceller) sammen med astrocytter (hjernens støtteceller), samt semi-syntetiske spheroider, hvor neuroblastomceller kombineres med gigantiske unilamellære vesikler (GUV'er), dvs. cellestørrelse lipidblærer. Efter at have dannet neurale spheroider undersøger vi, hvordan de modnes til mere modne neurale netværk under tre betingelser: (1) behandling med retinsyre, som er en kendt inducer af neuronal differentiering, (2) kontrolleret samskultur med astrocytter og (3) hybride spheroider af levende neuroner med minimale syntetiske "astrocytter" baseret på GUV'er, der bærer NrCAM-adhæsionsproteiner. Den sidste tilgang bruges som eksempel på, hvordan minimalistiske syntetiske celler kan anvendes til at undersøge specifikke differentieringssignaler og interaktioner mellem neuroner og gliaceller. For alle tre strategier vurderer vi morfologi og udtryk af differentieringsmarkører.

Three-dimensional cell cultures known as spheroids are increasingly used because they mimic tissues better than flat cell layers. Microfluidic devices allow spheroids to assemble in a precise, uniform microenvironment where cells sit closer together and interact more. In this thesis, we present and evaluate a scaffold-free microfluidic method that efficiently produces large numbers of spheroids. We generate two types: co-culture spheroids combining neuroblastoma cells (widely used as a neuronal model) with astrocytes (the brain’s support cells), and semi-synthetic spheroids that pair neuroblastoma cells with giant unilamellar vesicles (GUVs), which are cell-sized lipid bubbles. After forming neuronal spheroids, we examine how they mature into more developed neuronal networks under three conditions: (1) treatment with retinoic acid, a well-known trigger of neuronal differentiation; (2) defined co-culture with astrocytes; and (3) hybrid spheroids of living neurons with minimal synthetic “astrocytes” built from GUVs carrying NrCAM adhesion proteins. This last condition illustrates how minimal synthetic cells can be used to probe specific differentiation cues and neuron–glia interaction patterns. For all three approaches, we analyze spheroid morphology and the expression of differentiation markers.

[This abstract was generated with the help of AI]

spheroid ; neuroblastoma ; astrocyte ; GUV ; microfluidics

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