A Microfluidic System for Preparation and Digestion of Leaves to Isolate Protoplasts

Protoplasts are a prominent cell type for conducting gene transfection in the agricultural space in order to produce modified crops that can mature quicker or survive droughts. Traditional methods for isolation of protoplasts involve manually preparing leaves that are digested in an enzymatic solution containing cellulase and macerozyme for as long as six to eight hours. Understanding current techniques for isolation of protoplasts demonstrated there is prevailing need for a platform that can streamline this process, with quicker isolation times and less user interaction. Our lab has previously performed mammalian tissue digestion to isolate single cells with a microfluidic platform that utilizes mechanical shear forces in combination with enzymatic digestion. Initial tests were performed with the previously developed digestion device on tomato leaves, before being redesigned to suit leaf digestion by optimizing channel design and chamber holding volume. Tomato plants were used as our model because of their quick growth cycles, abundance and previous literature research. Multiple flow rates were tested to determine the ideal conditions for obtaining the highest yield and viability of protoplasts, through Trypan Blue staining. Protoplast yield and viability showed an increase over the digestion period, but a decrease with higher flow rates through the digestion device. Under optimal conditions the device yielded double the number of protoplasts in 2/3 the time compared to the traditional method, while maintaining equivalent viability. In order to streamline the preparation steps, a second preparation device was developed to prepare leaf samples in a quicker manner than the traditional method. The preparation device was then tested in conjunction with the digestion device. Cell yield and viability from using the leaf preparation device were shown to be equivalent to traditional preparation method with Trypan Blue staining. Cells were then cultured and tagged with calcein blue and calcofluor white, to demonstrate continued viability and cell wall formation