Encapsulating Cells

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.

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). 

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.

Introducing Protoplasts Part II

How do you isolate plant cells for high throughput analyses?

Under normal circumstances plant cells are held together in tissues by crosslinked pectin molecules. Enzymatic digestion of the cell wall releases individual cells, or “protoplasts” which can be genetically transformed and used for further analyses. Perhaps the most prominent proponent of protoplasts is Jen Sheen’s Lab at Harvard. Commendably on their website they provide detailed protocols about how to isolate protoplasts from Arabidopsis and maize, as well as answers to FAQs about preparing and transforming protoplasts and a very helpful video about how to isolate healthy Arabidopsis protoplasts.

In our initial trials (Figure 1) we have been relying heavily on these protocols; I am not going to repeat them here as I didn’t deviate from the Sheen Lab’s protocol for maize protoplast isolation. 

Figure 1: Protoplast isolation (A) Maize seed germination - 48 h in dH2O (B) Maize seedlings grown for 9 d under long day conditions (C) the middle 8 cm of the second leaves were cut into strips and incubated in enzyme mixture (D) vacuum infiltrate the enzyme mix into leaf sections (E) gentle shaking at 40 rpm for 90 minutes (F)  tissue was filtered through Miracloth and protoplasts harvested by centrifugation.   

For Marchantia, Christian has been using a different approach:

Marchantia polymorpha (Cam-strain) according to a protocol inspired by M. Bopp et al., 1988, Plant Cell Physiol.: 10 day old thalli were harvested and pre-incubated in 10 mL half strength Gamborg's B5 medium supplemented by 1 g/L casamino acids, 0.3 g/L L-glutamine, 20 g/L sucrose, and 60 g/L d-mannitol (Solution S1) for 30 min at room temperature. Subsequently, thalli were shaken in 10 mL S1 supplemented by 2% Driselase at room temperature for 3 h. Protoplast were then filtered through a nylon net of 80um mesh width, and washed four times in S1 (centrifugation: 100xg, 4 min). Protoplasts were shaken in S1 in light over night prior to processing.
 

As you can see in (Figure 2) the plant cell wall predominantly consists of cellulose microfibrils in a cross-linked polysaccharide network of pectins (a short review can be found here). To isolate Maize protoplasts we have been using a combination of Cellulase and Maceroenzyme R-10, a multi-enzyme mixture containing Cellulase, Pectinase and Hemicellulase from Rhizopus sp. (common saprophytic fungi). Likewise the Driselase used in Marchantia protoplast isolation is also a multienzyme mixture, this time from Basidiomycetes sp. (also fungi) that contains Cellulase, Pectinase and Hemicullase activity.

Figure 2: Diagram of the plant cell wall
Source: LadyofHats - Own work, Public Domain, https://commons.wikimedia.org/w/index.php?curid=2881078

Once digested for several hours protoplasts are harvested by filtering to remove residual tissue, followed by centrifugation. Our initial attempts were successful (Figure 3), although a fairly high amount of rupturing was observed in maize protoplasts, so the next few weeks will involve optimizing the procedure (if anyone has any tips we would love to hear from you), whilst trying to develop a transformation protocol.

Figure 3: Maize Protoplasts

PS: A tip for beginners – don’t use a glass slide with coverslip to view your protoplasts – they will pop leaving you with chloroplasts!

Introducing Protoplasts Part I - The Tortoise and the Hare

by Steven Burgess

‘There once was a speedy hare who bragged about how fast he could run. Tired of hearing him boast, Slow and Steady, the tortoise, challenged him to a race. All the animals in the forest gathered to watch.

Hare ran down the road for a while and then and paused to rest. He looked back at Slow and Steady and cried out, "How do you expect to win this race when you are walking along at your slow, slow pace?"  - Aesop’s fables

Working in plant science I sometimes feel like the tortoise in Aesop's fable. Animal biologists race on ahead making discoveries, before stopping to laugh, amazed plant science's ‘slow, slow pace’. Speed is a real issue when you work with plants, to illustrate the point a colleague of mine recently obtained some seeds that came with the following advice - ‘germination may take between two weeks and two years’. The challenge of time is one of the key reasons why a lot of research is still done in model plant Arabidopsis thaliana: it has a relatively short life cycle, so it can be quite quick to make a mutation and analyse the effect. However, even at 8 weeks to seed, as Arabiodpsis is diploid, so you need to do through the process of growing plants, collecting seed and select mutants three times in order to generate plants suitable for analysis – as a result it is months before you can carry out an experiment.

To speed things up, plant scientists can use transient assays, whereby it is possible to switch on or off a gene of interest for long enough to analyse the effect. The most common methods in plants include agroinfiltration (video), biolistic bombardment (video) or viral induced gene silencing (VIGS). Agroinfiltration uses agrobacterium to infect plants, this gram negative bacterium is able to transfer DNA from itself into a plant genome in a process that has been adapted to introduce a genes of interest. This process works great in Nicotiana benthamiana, but there are many species in which it is far from optimal, and we have not much success using either grasses such as maize, or our model species Gynandropsis gynandra. 

Biolistic bombardment involves coating metal particles with DNA, which are then introduced into plant cells using a genegun (sounds cool, but believe me it gets tedious after a while!). This works okay, and for my purpose it is probably the best option currently available, but biolistics is not really suited to high throughput analyses. VIGS can only be used for switching off genes. 

The alternative is to take an approach that is analogous to using cell lines, the use of which gives animal research its speed. 

Maize mesophyll protoplast

Techniques have been established to create plant cell tissue cultures as well as isolated plant cells known as protoplasts (which do not propagate) from mature leaves, or plant embryonic tissue (known as callus) (if you are interested in the history of protoplasts a personal account is provided by Prof. Edward Cocking who pioneered their use here). However, these approaches have their limitations, the most significant question is how representative of ‘real’ systems are these cells? Plant cell tissue cultures are suitable for biotechnological purposes, but as the gene activation patterns are so far removed from ‘normal’ systems they are not always suitable for scientific study. Protoplasting can introduce stress responses that must be considered when interpreting experiments, but protoplasts are a close enough to real cells to be a useful tool in preliminary analyses. 

Therefore, as previously mentioned, we hope to couple the use of protoplasts to developments in microfluidics to generate a system suitable for high throughput analyses and help give the Slow and Steady plant scientists their running shoes. In the next post we will be give a bit more information about protoplasts and provide an update on our initial attempts to isolate them, as well as preliminary tests about how they behave in a microfluidic system.

Building a Open Source Microfludic Device For Analysis of Plant Protoplasts

Day 0

This blog is an experiment in open science. I am a postdoctoral researcher in Prof. Julian Hibberd's lab at the University of Cambridge, and moonlight as a editor for the PLOS Synthetic Biology Community. In my day job I work as part of a team that is seeking to understand an adaptation possessed by high yielding crops like maize, with the aim that we may one day be able to use this knowledge to boost yields of species such as rice. Our lab is part of a couple of international consortia pursuing this goal including the C4 rice project and the 3to4project.

Our research is focused on identifying DNA regulatory elements which limit gene activity to specific regions of a leaf in order to aid the design of synthetic circuits. Conventional approaches involve the fusion of a reporter (often the E. coli uidA gene) to a promoter that is being tested. This reporter is then inserted into plants, and the regions where the promoter is active are visualized by staining (see Kajala et al. for an example).   

Plan for chip to sort protoplasts courtesy of Dr. Sara Abalde-Cela

The problem with these methods is that they are slow and low throughput. With genomic technologies starting to provide vast numbers of candidate promoters, novel methods are required to screen them. I am a molecular biologist, and so is my colleague Ivan Reyna-Llorens who works with me on this topic. We realized that to tackle the problem we needed to bring together a team of experts from different disciplines, so we managed to convince Christian Boehm, Dr. Sara Abalde-Cela, Dr. Paul Bennett to join us. The result was a proposal to use a combination of plant protoplasting, differential fluorescence analysis and microfludics to sort cells based on fluorescence intensity. 

We recently received funding from the Cambridge Strategic Research Initiative for Synthetic Biology to develop this device. This came with the stipulation that all outcomes are open source. Additionally, as my fellow colleague Richard Smith-Unna has been encouraging Cambridge scientists to start implementing open practices in their research, I though this project was an excellent opportunity to learn how to do science openly (hence the blog).

Funding for the project lasts for six months, and we will start in January 2016. We are very excited, and if this pilot project is successful we hope to utilize the device to tackle a range of additional problems. In the spirit of open science we will be reporting regularly and making all our data available online in public repositories. Additionally we recognize science is a collective effort, so we will be keen to hear from anyone who has any bright ideas or novel applications for our device that might be interested in getting involved, comments are welcome.