Taking individual cells and encapsulating them inside liquid droplets. The idea is simple, but powerful. It opens up a host of possibilities for high-throughput, single cell analysis ranging from genomics to screening for metabolite production. It has been used widely in analysis of bacterial communities and cell tissue cultures, but has not yet been adopted by plant scientists.
I have previously written about the process of extracting individual cells from leaves by enzymatic digestion. The next step is to encapsulate cells in microdroplets - this is achieved by flowing a cell suspension into a 'carrier solution', the two are immiscible so droplets of buffer containing cells are formed (Figure 1). If you have ever eaten an oily soup before you will have observed a similar phenomenon where circles of fat can be seen floating on the surface.
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| Figure 1: A diagram of encapsulation of cells in water in oil droplets |
However, protoplasts are extremely fragile as they lack a cell wall, and they are prone to rupturing. This can occur from excess agitation, shearing pressure from pipetting or even allowing protoplasts to drip from one solution to another (as during filtering). Although our yields have been okay, perhaps 30-50% of our cells rupture, so there is room for improvement. There is a recent method in Cold Spring Habour Protocols that suggests a means to remove ruptured protoplasts which we may be trying in the coming weeks.
Due to this fragility we were concerned that the pressures protoplasts encounter during passage through a microfluidic device might cause them to burst. So we needed to test whether it is possible to encapsulate cells without them rupturing. To help us do this we have been joined by Ziyi Yu on the project, who has previous experience working with algal cells.
The first attempts were unsuccessful, most droplets were empty, and the odd ones we did see encaspulated were irregular in shape, suggesting they were very unhappy or partially ruptured. Cell densities were too low, and we also experienced problems with protoplasts settling in the syringe used to inject the cell suspension into to the microfluidic device.
This project is split between the Plant Science and Chemistry Departments at the University of Cambridge. Transport of the protoplasts between the two caused a great deal of damage to the cells, so we instead performed the extraction in the chemistry department using wild type N. benthamiana leaves. W also added a product to alter the density of the buffer solution to stop cells pelleting in the syringe and made sure we loaded cells in the top of the syringe rather than sucking up through the needle to reduce shearing stress. After a couple of hours of fiddling we were able to effectively encapsulate cells as demonstrated in the following clip:
The droplets were collected and analysed under a fluorescence microscope (Figure 2).
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| Figure 2: Intact encapsulated protoplasts (20x magnification) showing bright field (left) and chlorophyll fluorescence (right). |
After our initial disappointments we were very pleased to see this! However, there is more work to be done; most droplets were still empty and we still had quite a large amount of cells rupturing. Over the coming weeks we will be tweaking the protoplast preparation procedure, and optimizing encapsulation to increase the number of droplets containing intact protoplasts (which can be achieved by varying cell density) before going on to test the ability to analyse fluorescent proteins in transformed plant cells.
I will also be uploading some protocols with a bit more detail for those who are interested asap, however in the OpenPlant project we are currently in the process of discussing the best way to do this. More soon.
