Title:

Plasmid DNA Hydrogels as a Scaffold for 3D Cell Culture

Three-dimensional (3D) cell cultures represent a significant leap in mimicking tissue microenvironments compared to conventional two-dimensional (2D) cultures. Unfortunately, current scaffolds are lacking in topological tunability, biocompatibility, and biodegradability. The utilization of plasmid DNA (pDNA) as a generic polymer to engineer hydrogel scaffolds for 3D cell cultures may combat these challenges. The practical application of pDNA in 3D culture necessitates efficient purification methods due to the large quantities required. Traditional purification protocols utilize anion exchange chromatography but often rely on RNase A or additional chromatographic steps, hindering scalability and cost-effectiveness. Furthermore, homogenous mixing and sterile filtration of lysate becomes increasingly challenging at larger scales. Specialized equipment is implemented in industry to overcome these limitations but are not accessible solutions for academic labs. Consequently, there is a need for new methodologies that allow for gram-scale purification of pDNA using materials and instruments that are available to academic researchers. Here, we present a simple, scalable, and unified method for pDNA purification. Our approach leverages anion exchange chromatography (AEX) with an optimized stepwise sodium chloride gradient to separate pDNA from contaminating RNA based on their net negative charge. Purity is assessed using agarose gel electrophoresis and UV-Vis spectroscopy. Our optimized purification method yields approximately 70% and can accommodate 100s of grams of biomass, promising significant improvements in scalability and cost-effective production of pDNA as a scaffold for 3D cell culture applications.

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