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Introducing the CES (2019)

We are happy to inform you that we will be introducing our new and improved version of the C. elegans Synchronizer (CES v2) a follow-up from the original LabTIE CES, at the 2019 International C. elegans conference in LA.

Based on your feedback during the meetings in Barcelona, Seoul and Ghent (2018) and the data from our beta-testers,  a less complex and smaller version of the original CES (2018) System has been developped. With a new and faster protocol the CES v2 can be manually operated without the need for a complex and large supporting hardware platform. However, despite the scale down the synchronization results are equal or even better compared to the former (larger) system and with better results compared to any prior art protocol for synchronization.

Don't take our word for it, just look at the below test results that have been generated by the Nagi Bioscience located at the Swiss Federal Institute of Technology (EPFL) using the new CES v2 system. The data shows better viability and much tighter synchronization, versus the sedimentation or bleaching protocol. 

The benefits of the CES v2 are clear:

• No bleach or chemicals
• Healthier worms
• Improved level of synchronization
• Removes variables
• Little or no training required
• Easy and fast

Below are the results with the new CES (v2) protocol versus sedimentation and bleaching protocols, as acquired by Nagi Bioscience located at the Swiss Federal Institute of Technology (EPFL). Nagi Bioscience is working on the first Organism-on-Chip technology using C. elegans, creating a technological platform that is fully automated in vitro handling, culture and analysis. Which will unlock endless possibilities for Toxicity testing, Drug discovery, Anthelmintics and many more applications. 

Figure A. Results showing the reproducibility of the CES to generate synchronous L1 population across 34 replicats.

L1 worms were injected in the SydLab platform developed by Nagi Bioscience and cultivate on chip during 5 days. Each channel (represented on the x-axis) corresponds to 3 to 8 microfluidic chambers (example on the right side of this panel) containing 1 to 4 worms. Each dots represented on the graphic correspond to the timing (in hours) when the first egg is observed in average in the corresponding channel. The error bars represent the standard deviation.

Figure B. Comparison of the percentage of fertile worms between three methods of L1 synchronization.

Each bars represented on the graphic correspond to the percentage of chambers (in average) with fertile adult worms. ‘n’ corresponds to the number of chambers analyzed. The error bars represent the 95% CI.

Figure C. Variation of the timing to reach the adult stage across single individuals.

Each dots represented on the graphic correspond to the variation of the timing for a single worms to reach the adult stage, compared to the whole population analyzed. n corresponds to the number of single worms analyzed. ‘sd’ corresponds to the value of the standard deviation.

A pdf version of these reults can be downloaded from our website at the following link:  PDF Nagi Bioscience CES results

If you are interested in the development by Nagi Bioscience of the the first Organism-on-Chip technology using C. elegans, you can reach them by sending an email at 

More information on the new CES v2 can be found on our website www.nemasync.com  or by sending an email at 

If you are attending the International C. elegans conference in LA (June 20 - June 24, 2019) then please come and visit us at booth 107, where we will be together with our North America distributor NemaMetrix. 

Regards,

Marijke Millenaar

How does it work

Let’s start with a quick overview and some observations from our side on what methods are available today to synchronize nematodes without using chemicals. As the objective is NOT to use any chemicals, we will not cover ‘bleaching’ in order to synchronize nematodes, besides that it is inherently unpredictable and its accuracy is far from perfect, let alone to cause ‘Transgenerational Effect’ effects from the bleaching chemicals. This leaves two other methods, not using ‘bleaching / chemicals’ to be considered:

• Sedimentation:

The ‘sedimentation’ protocol is a ‘gravity’ based method of cleaning and semi-staging nematodes based on the relative weight / size of the organisms. The method requires a setup with a 3 step gradient of layers of sucrose solutions, each with a different density in order to separate the nematodes. Avoiding mixing the different solutions requires some skills and practice. As it’s not an ‘exact’ method the end-results are by definition not predictable. Using sucrose, this protocol is certainly not without its downside as the nematodes are exposed to high levels of ‘sucrose’ that will to a certain extend influence / interfere with the results. The question remains as to what extend sucrose is considered an ‘environmental influence’ contributing to ‘Transgenerational Effect’.

• Sieving: (selecting on size)

The working principle of using sieves (mesh filters) is based on the assumption that there is a direct correlation between size and age in order to select a sub population. There are many issues with this assumption as external factors such as NGM versus Liquid cultures, sufficient feedstock, food arrest, different strains, temperature, etc less or more influence the development and therefor the physical dimensions of the nematodes. This method is far from accurate and there is no known ‘t=0’ reference to the exact time the L1’s hatched. It’s at best a ‘relative’ size sorting, where size is in fact the only ‘attribute’ of selecting a sub culture. Yes, there is a correlation between size and age, however at best ‘relative’ and for sure not ‘absolute’ in reference to the ‘t=0’ being the time of L1 hatching. Example: when food arrest is applied, there is almost no physical difference between a ~16 hours old L1 and one that only just hatched.

Over time, different systems using the ‘sieving principle’ have been invented / developed using mesh-membranes or microfluidics devices that have obstacles such as micro-pillars, micro-channels, etc to block the passage of nematodes above a certain size. In some cases, there are devices being promoted today that in fact need fluidic pressure (!) to push through the nematodes in these type of contraptions with an ‘advertised’ synchronization of approximately 80% !?. Not to mention ‘mechanical’ induced physical damage or stress …

Unless somebody proves us wrong, there are today NO known systems based on the principle of ‘sieving’ that have factually (!) demonstrated to be able to accurately (> 99%) select a L1 sub-population of nematodes with repeatable / predictable results. The ‘sieving principle‘ by itself simply does not allow this to happen.

Facing the challenges of ‘NO CHEMICALS’ being allowed in the process, plus the need for an ‘absolute’ Accuracy, Predictable, Repeatability and Ease of Use, a different working principle was needed for synchronizing nematodes in both high and low volume applications.

Early 2015 an innovative new working principle was invented, being able to synchronize nematodes, without the use of chemicals, with an 'absolute' working principle, assuring accurate, repeatable results and not the least of all very ease of use. In addition its scalable in case very large volumes are required. Sounds almost too good to be true, right?

So how does it work ?

The working principle of the NemaSync C. elegans  Synchronizer (CES) uses a 2 step process:

In the first step we ‘wash out’ all the debris and anything smaller than adults (most eggs will stay as they will clump/stick together and this is positive/intended). After the ‘wash’, we proceed with the second step being the actual ‘harvest step’. Adults and eggs are transferred after the washing step to the ‘Harvest’ filter. In this second stage, adults and egg’s cannot pass through, however as soon as the eggs hatch, the L1's will transfer through the filter with little or no delay. So you have L1’s where the ‘t=0’ is known with an error margin of less than a minute and depending on your total ‘harvest time window’ it will decide the ‘time spread’ between the first L1 and the last one L1 you harvest.

That’s all! Sounds simple and so it is!

For us (the NemaSync team) the real challenge turned out to be the cost, design and manufacturability of the micro filters as from the almost 200k elongated apertures on a 80mm diameter filter (CES 5000 model) not a single aperture is allowed to have a defect or deviate from a critical set of dimensions (ZERO defect is required as 99.999% is not good enough). Making these high precision micro-filters requires similar production and clean room technology as seen in today’s semiconductor industry.

It took us more than 4 years to solve the many challenges we faced, however today we have a system that not only works guaranteed ‘as advertised’, however it can now also be scalable manufactured.

Overcoming all the setbacks & challenges we faced, we were very excited and proud to introduced the first ‘production’ version of the ‘CES’ - C. elegans Synchronizer at the 2018 EMBO convention in Barcelona with multiple live demonstrations showing the unprecedented results for everybody to witness. The 2018 version of the CES (v1) was still rather large and heavy on ‘glass and metal’ and in the meantime a much smaller and simpler version has been introduced at the WORM#19 conference in LA, the ‘CES v2’. Not just much smaller, but also at 8x lower in cost.

We hope that this gives you some insight in the development and working of the truly unique ‘CES’ and answers the questions of ‘how does it work’ and why it is different from any other synchronization protocol we have seen so far.

Please feel free to visit the www.nemasync.com website for more information, including a short demo video of the CES protocol. Please do not hesitate to contact us if you have any questions or if you are interested in the CES itself.

Best regards,

Marijke

Why the CES filters are Special

It took 4 years to develop the CES System and the special micro-filters it relies on. The initial intend was to use commercial available membrane filters, however by ‘trial and error’ we found that commercial available membranes do not work for a number of reasons:

Most filters are so called depth filters and do a great job at retaining particles throughout the filter medium, rather than just on the surface of the filter medium. Reality is also that they are not ‘absolute’, despite sometimes being advertised as such. Depth filters are problematic for the CES working principle, as it is difficult to recover the adults after the first step of the synchronization protocol without applying (back washing) pressure and thereby damaging the nematodes. Most disc filters are in many cases semi-depth filters, they just happen to be thin. Just take a look under the microscope or see some of the pictures we added below. All of them are ‘membrane' filters of well-known brands, but none of them will accurately work in the CES protocol.

[images different type of Hydrophilic Sartorius Membrane filters]

The only type of filter that comes close to what we initially hoped could work are the so-called PET (membrane) filters, typically used in Cell Strainers.  See below.

[image PET  – pluriStrainer® ‘netted’ filter]

[image PET membrane - hydrophilic Polycarbonate Track-Etched (PC-TEM) ]

Unfortunately it turns out the PET filters do not work for our CES application for the following reasons:

1. Filters using a ‘netting’ made out of interlaced PET wires such as found in the pluriStrainers® didn’t work as the pores/apertures are not uniform when typically getting below 50um as the PET wires are about 40 um and pores/apertures are formed at the crossing point among the wires. Pores/apertures are not perfectly uniform and can vary up to 20-30 um in lateral direction and as such are considered ‘defects’ in the definition of what the CES System requires.
2. PET membrane (PC-TEM) pore size is typically limited to a pore size of max 8 um. The pore/aperture is also a round capillary hole, great for holding back cells, however the typical L1 nematodes will not transfer through these filters. 
3. PET membranes (PC-TEM) typically have so called ‘doublets’ and even ‘triplets’ and even the best high quality filters still have a tolerance of +/- 10% . In our CES application we need an absolute (100%) ZERO defect filter, i.e. no 'doublets’ or ‘triplets’.

Unlike traditional filters, the CES filters do not have 'round' or ‘semi-round' pores/apertures however feature a special elongated design (slit) in order to prevent the filters from clogging while allowing L1’s to pass through with ease. Below is a picture of the special metal alloy hydrophilic CES filter:

[image CES filter microscope and CAD image]

As mentioned before, a critical requirement for the CES to accurately work is an Absolute Zero Defect for every single aperture in order to get a perfect synchronization. Just imagine a single aperture (hole) to merge with an adjacent aperture (hole), and you have a so-called ‘doublet’ or ‘triplet’ and adult nematodes will also pass through and the system will not work as intended. For the CES application 99.999% is just not good enough!

It turns out there are NO commercial membranes available that meets the required specifications and therefor a different technology was needed. After several years of ‘trial and error’ prototyping, we were able to optimize the design and managed to manufacture a filter meeting all requirements in order to get a perfect (absolute) synchronization of nematodes.

Manufacturing the high precision CES micro-filters is done with a similar production process and clean room technology as found in the high-tech semi-conductor manufacturing. See the picture below of the actual manufacturing process of the CES filters.

[images CES filter manufacturing]

In addition, all CES glass parts are made from highest quality laboratory grade borosilicate glass and the high precision micro-filters are made from a very special high purity nickel alloy and are made to last. Did we mention that the CES system is developed and manufactured in the Netherlands, including the special micro-filters.

We hope this article explains ‘Why the CES Filters are Special’. The CES System is sometimes perceived as expensive compared to high volume, low cost, commercial filters. However if used with care the CES filters will last ‘forever’ and the real cost of ownership is in fact low. Taking into account all the benefits^* the CES provides including the long life span of the CES filters , the actual cost of ownership is lower compared to prior-art methods of synchronization.

(*) Healthy phenotype free worms synchronized without bleach, chemicals and starvation, improved level of synchronization with little variation, consistent results with little or no training required, less failed experiments!