NL2021482B1

NL2021482B1 - C. elegans synchronisation system

Info

Publication number: NL2021482B1
Application number: NL2021482A
Authority: NL
Inventors: Millenaar Arno; Millenaar Bas
Original assignee: A S B Millenaar
Priority date: 2018-08-20
Filing date: 2018-08-20
Publication date: 2020-02-26
Also published as: WO2020040636A1

Abstract

The present invention is in the field of a life cycle syn— chronization system for nematodes and a method for life cycle synchronization of such nematodes. In particular the present invention relates to an improved filter design for said system and an improved method of operating said filter, such that an efficient and effective synchronization can be established. An example of a nematode is Caenorhabditis elegans (C. el— egans). C. elegans is a small, free—living soil nematode (roundworm) that lives in many parts of the world. It feeds mainly on microbes, primarily bacteria. C. elegans is considered and used as an important model system for biological re— search in many fields including genomics, cell biology, neuro— science and aging.

Description

FIELD OF THE INVENTION

The present invention is in the field of a life cycle synchronization system for nematodes and a method for life cycle synchronization of such nematodes. In particular the present invention relates to an improved filter design for said system and an improved method of operating said filter, such that an efficient and effective synchronization can be established.

BACKGROUND OF THE INVENTION

The present invention is in the field of a life cycle synchronization system for organisms such as nematodes and a method for life cycle synchronization of such nematodes.

An example of a nematode is Caenorhabditis elegans (C. elegans). C. elegans is a small, free-living soil nematode (roundworm) that lives in many parts of the world. It feeds mainly on microbes, primarily bacteria. C. elegans is considered and used as an important model system for biological research in many fields including genomics, cell biology, neuroscience and aging (http ://www. wormbook.org/); the model system mainly relates to use of a nematode in well-defined and predictable settings and boundary conditions.

With reference to figure 7 a life cycle of the C. elegans nematode is presented, which is considered to represent a typical nematode life cycle, i.e. other nematodes have similar life cycles.

Some older articles and patent documents recite methods for obtaining nematodes. For instance Gandhi et al. In « A simple method for maintaining large, aging populations of C. elegans, in Mechanisms of ageing and Development, Elsevier Sequoia, Lausanne, Vol. 12, No. 2, (February 1980) pp. 137-150, and Patel et al. in Axenic and synchronous cultures of C. elegans in Nematologica, BRIL, Leiden, Vol. 24, No. 1 (January 1978) pp. 51-62, use centrifugation and glass wool or 0.4N NaOH, which may be considered equal to bleaching in terms of aggravation, respectively, for separating nematodes; such will inevitably not result in many viable nematodes and in addition will kill off many others. Gandhi reports on p. 141, 1. 7-10 that only 60-70% of the eggs hatch and further hatch relatively unsynchronized during 4-5 hours of incubation. The yield of larvae was even lower. In addition, contamination of eggs with worms was still an issue. It has also been found, upon precise evaluation, that organisms obtained still have stress symptoms (such as duaer stress) and differ in phenotypes, which makes them typically worthless for many scientific experiments and the like.

WO 2016/175658 Al describes a high-volume breeding and life cycle synchronization system and various aspects of the present invention; said document and its contents are incorporated by reference. Albeit the high-volume breeding is under good control and provides good results, in practice people using such a system would like to harvest a small amount of eggs or larvae at any given point in time, and synchronize said amount, which is rather difficult with said system. A further disadvantage is that the filter may get clogged, such as by formation of a biofilm. Further passage of species is not optimal. This invention is a further improvement thereof.

The present invention therefore relates to a life cycle synchronization system, and a method of operating said life cycle synchronization system, which solve one or more of the above problems and drawbacks of the prior art, providing reliable results, without jeopardizing functionality and advantages .

SUMMARY OF THE INVENTION

The present invention relates to an improved life cycle synchronization system according to claim 1, a method of operating said life cycle synchronization system, a population of nematodes obtainable by said method, and use of said population. The present invention uses a dedicated filter system characterized by certain properties that are required to allow passage of the organisms, which is rather unexpected. For instance, slits need to be provided, having a length that is substantially large (at least a factor 2) than its width. The width is typically precisely adapted to a size of the (largest) organisms allowed to pass through the membrane slits, such as from 7-40 pm. Circular openings, or mostly circular openings as ellipses, do not provide a good throughput. A length of a horizontal space between slits is typically 5-100% of a slit length mi (leading to a pitch of 105-200% of mi) such as 10-66% of mi, e.g. 20-50% of mi, and a length of a vertical (space between slits is typically 5-200% of a slit width m_w, such as 10-100% of m_w, e.g. 20-50% of m_w. The slits are typically evenly distributed in a vertical and/or horizontal direction of the membrane. Also, the membrane of the filters, typically located in a bottom part thereof, need not be too thick, as then also organisms are limited in passing through. In addition, the membrane need not be too thin, as it needs to provide some structural stability. The present filters may be regarded as plate membrane filters having at least one free standing membrane sheet (or foil). The membrane is at its periphery attached to a support or frame for providing structural integrity, such as a tube like structure. The present filters are similar to microfiltration filters in view of the organisms of 7-40 pm to be separated. In view of manufacturing the filter, the filter membrane is preferably as thin as possible, as then less material need to be removed for making slits. On the other hand, if the filter membrane is too thin it is found to be difficult to attach it to a supporting structure such as a cylindrical tube. A thickness of 5-100 pm is found to be adequate in this respect. And further it has been found that the membrane surface needs to be hydrophilic; if not the organisms simply do not pass through and over the membrane. It is noted that prior art filters, despite claims thereto, always have imperfections leading to a distribution in pore sizes which for the present invention is simply too large. Also, other type of filters, such as ceramic filters or hollow fibre filters, still have passages far larger than indicated by suppliers thereof, hence these are not absolute filters. Also, most of the prior art filters do not allow passage of organisms such as nematodes. Hence prior art filters have been found unsuitable for the present application.

The present life cycle synchronization (CES) system can provide relatively high volumes or likewise high amounts of nematodes, such as from 10^-4 litre up to 100 litres or more, in a continuous, semi continuous or batch wise mode of operation. These volumes comprise an aqueous solution forming typically >50% of the volume, and nematodes; a dry weight of the nematodes is typically 1-10% of the nematode volume. Instead of nematodes also other organisms having a life cycle similar to that of nematodes can be synchronized.

An advantage of the present system is that it produces as an output high amounts of synchronized eggs, and likewise synchronized nematodes, per equivalent amount of bio-mass used as feed supply. The biomass of a population may be in the order of 1-10 wt.%, relative to a total mass of the solution, such as 3-5 wt.%. The present life cycle synchronization system does not destroy the nematode population each and every time when synchronization is required, contrary to prior art methods using bleaching.

It is stressed that the present system does not use harmful or toxic chemicals, such as sodium hypochlorite or bleach, in order to separate the nematode eggs from the population of nematodes. As a consequence, it has been found that the present system produces e.g. healthy and synchronized nematodes and eggs, suitable for use in research and test applications. In addition, it is noteworthy to mention that during growth and development organisms may suffer from constraints, leading to so-called phenotypes that differ (or vary) significantly, at least to such an extent that for research said variation is often too large. There are no negative side effects such as of the prior art methods and use of bleaching chemicals therein on the vitality of the nematodes/eggs observed.

Throughout the description the term population refers to

i.c. nematodes of at least one life cycle stage being present therein. In a sub-population at least one life cycle stage is removed, and sometimes all but one life cycle stages are removed, leaving one life cycle stage left.

The present system as described in this application overcomes drawbacks of the prior art and in addition solves a number of challenges as mentioned below.

1) Synchronization is obtained without the use of chemical(s). In the present system a need for potentially harmful chemicals, such as bleach/leach, is absent. Therewith the nematodes can grow, reproduce, live, and be obtained under favourable conditions .

2) A consistent high quality of output is obtained, i.e. a nematode sub-population, especially eggs and nematodes hatchlings .

In comparison, prior art methods at the best generate yields of unhatched eggs between 70% and max 90% of eggs being in principle available. Assuming a nematode could produce 250 eggs, prior art method effectively only yields approximately 10 fertilized eggs that will hatch, i.e. only 4%. In addition, the vitality of the (hatched) nematodes obtained from prior art methods is very much depending on the protocol being used and, in many cases, a significant percentage is damaged but still 'lives', rendering the batch of nematodes unusable for the intended purpose. In contrast the present invention provides above 90% (relative to a total number of eggs), typically above 95%, and more typically above 99%, viable nematodes in a repeatable process environment. In addition, a variation in phenotype is minimal, as organisms are free of stress, which may be caused by chemicals (such as bleach), by food deprivation or starvation, by lack of oxygen, etc.

3) Synchronising is performed in a controlled environment. Such decreases a chance of contamination and it is found easy to monitor and control the growth of nematodes.

4) Scalability for High Volume use in for example High Throughput Screening applications or research that requires substantial bio-mass.

5) For some systems 24x7 availability 'ready to use' of synchronized nematodes.

In the present system at least one container or flask C1-C6 may be present. Each flask is in fluid connection with at least one further element. For instance, a flask Cl comprising a filtration buffer may be in fluid contact with the stabilization filter input. It may be in fluid contact with said stabilization filters output thereby receiving waste filtration buffer. Said waste may be passed over an in-line filter for removing organisms. A second container C2 may be connected similar to Cl, albeit without an in-line filter; Cl and C2 are typically not interconnected. A third flask C3 comprising buffered water may be in contact with a Pasteur pipette and/or venturi creating nozzle PPI, and a pressure source PSI, providing a pressure of 10-200 kPa. A fourth container C4 comprising a harvest buffer solution, which typically is autoclaved and therefore not comprising oxygen, is provided with an air inlet for adding oxygen, and in fluid contact with PSI. A fifth container C5, similar to C2 functions as waste container, may be provided with a waste solution, in fluid connection with a second pump P2 and via a second in-line filter

IF2 with the harvest filter F3a/b, wherein filter IF2 may function as a harvest filter. A sixth container C6 may be provided in fluid connection via the second in-line filter IF2 with the harvest filter F3a/b and via the second pump with the harvest filter. In addition, a fluid sparger SHI and SH2 may be provided per filter, respectively. For operation valves VIVll may be provided, which can be controlled manually, or by the controller. With respect to the present fluid connections it is noted that these may be combined; i.e. the term is mainly intended to indicate that two (or more) elements of the present system are in fluid connection.

In an exemplary embodiment the present system guarantees that organisms such as eggs or hatchlings can be harvested over the combined filters with minimum delay. This again provides a unique advantage to collect (harvest) a batch of organisms within a small time window or any given time window. By having a relatively small time-window of e.g. 15 minutes a perfect natural synchronization will have occurred without the need to withhold nutrients as in some prior-art. By not having to withhold nutrients when the eggs hatch, the nematodes will not be stressed for food and all types of unwanted stress related hormones (also causing variation in phenotype) will be avoided.

In an exemplary embodiment of system 100 no counter pressure over the micro filters is required and the organisms are found to pass through the filter by their own movement and possibly gravity. If a pressure is applied, the pressure is typically relatively small (< 50 kPa).

Thereby the present invention provides a solution to one or more of the above mentioned problems and drawbacks.

Advantages of the present description are detailed throughout the description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates in a first aspect to a life cycle synchronization system 100 according to claim 1.

In an exemplary errbodiment of the present system the width of the first slits may be from 17-25 pm, such as from 18-21 pm, adapted for letting larvae pass through.

In an exemplary embodiment of the present system the width of the second slits may be from 7-11 pm, adapted for letting hatchlings pass through.

In an exemplary embodiment of the present system the membrane layer of the first filter may be apart from slits fully intact (0 faults) and/or wherein the membrane layer of the second filter may be apart from slits fully intact. Such is found very difficult to establish and specific manufacturing techniques need to be applied. And even by doing so still a significant percentage (of up to 50%) still may have one or more defects, that is slits not according to specification or even openings, such as combined slits

In an exemplary embodiment of the present system a slit density may be from 10~⁶-10“⁴ /pm², preferably 2*10^_6-5*10'⁵/pm², providing 1-50% of a surface are of the membrane with slits, preferably 2-40%, more preferably 5-35%, such as 1633%. It is noted that slits hamper a structural integrity of the filter membrane, whereas to few slits make harvesting and stabilization to slow.

In an exemplary embodiment of the present system slits may be provided in alternating mode in at least one direction. Such improves the integrity of the membrane.

In an exemplary embodiment of the present system in at least one direction a size of at least one slit may decrease from a top side of the membrane to a bottom side thereof, such as could be obtained by under-etching. By using an exemplary manufacturing method such removing of material aside slits is obtained. It is found that organisms can pass through the membrane easier and faster, without a risk of getting stuck in a slit.

In an exemplary embodiment the present system may further comprise a Pasteur pipette and/or venturi creating nozzle (PPI), at least one of a container (C1-C6), a first and second container (C1-C2) in fluid connection with an output of the stabilization filter, a third container (C3) in fluid connection with the Pasteur pipette and/or venturi creating nozzle (PP), a fourth container (C4) in fluid connection with a pressure source PSI, optional containers (C5-C6) in fluid connection with an output of the harvest filter, a valve (Vl-Vll), preferably a valve per fluid connection, a pump (P1,P2), a first pump in fluid connection with containers (C1-C2) for providing pressure, optionally a second pump in fluid connec tion with optional containers (C5-C6) for providing pressure, a pressure source (PSI) for providing pressure to container (C3) and optional aeration to container C4, an optional sparger head (SH1,SH2) for providing sprayed liquid to stabilization filter and/or harvest filter and in fluid connection with pump (Pl) and optional pump (P2), and an in-line filter (IF1,IF2) provided in fluid connection with an output of a stabilization filter or harvest filter, fluid connections between containers, the pumps and pressure source adapted to provide fluid flow.

In an exemplary embodiment of the present system comprising at least two stabilization filters arranged in spatial series and/or at least two stabilization filters spatially arranged in parallel, such as 2³-2⁷ filters in series, such as 2⁴-2⁶ filters in parallel, and/or at least two harvest filters in series and/or at least two harvest filters in parallel, such as 2³-2⁷filters in series, such as 2⁴-2⁶ filters in parallel, such as in an array of 4-6144 filters, e.g. 8x12, 16x24, 32x48, and 64x96 (see ANSI SLAS 4-2004 and ASME Y14.5 2011). The filters may be operated in series (time) or concurrent.

In an exemplary embodiment the present system may further comprise at least one of a filtration buffer (Cl), a waste flush container (C2), a pressure flush container (03) comprising a liquid adapted to the nematodes, a first harvest buffer container (C4), a second waste harvest buffer container (C5), a third filtration buffer container (C6), a controller for regulation and controlling operation, and a fluid sparger (SHI,2).

In an exemplary embodiment of the present system for each filter independently mi>5*m_w, preferably wherein rru>10*m_w, more preferably wherein mi>20*m_w, such as wherein mi>30*m_w. It has been found that the larger the ratio is the better organisms pass through the membranes.

In an exemplary embodiment of the present system the filter membrane may be made from a metal, the metal preferably being selected from Ni, stainless steel, Ti, Cr, Si, W, Co, V, Al, and alloys thereof.

In an exemplary embodiment of the present system the filter can withstand a pressure of > 50 kPa, such as from 10-200 kPa. In an exemplary embodiment of the present system a uniformity in mi and m_w, respectively, is better than a standard deviation

3σ of <10% relative to an average of mi and m_w, respectively. Such a uniformity is difficult to obtain but is does support good selection of organisms by size.

In an exemplary embodiment of the present system a thickness of each membrane independently may be from 10-50 pm, preferably from. 15-40 pm, such as 20-30 pm.

In an exemplary embodiment of the present system at least one filter membrane may comprise a hydrophilic coating, such as a metal coating. Such a coating may be provided in addition to the hydrophilic membrane, or on a material from which said membrane is formed, such as a hydrophobic membrane.

In an example the micro filter membrane is attached to a cylinder.

For practical purposes, such as costs, volume, etc. various parts of the present system may be combined.

In a second aspect the present invention relates to a method according to claim 12, comprising providing at least one stabilizing filter (Fl), adding a population of organisms, such as nematodes, in a case of the population of nematodes (e.g. C. elegans) comprising at least two species selected from embryo's, such as E1-E6 embryo's, larvae, such as L1-L4, adolescents, young adults, and adults, on the stabilizing filter (Fl) , transferring the stabilization filter to a (open or closed) first receptacle (Rl), sub-merging the filter in an aqueous liquid or flushing the filter with said aqueous liquid therewith removing species through the filter slits into the receptacle, transferring remaining species to a harvest filter (F2,F3a,b), transferring the harvest filter to a second receptacle (R2), submerging the membrane of the harvest filter in an aqueous liquid, harvesting species that passed through the harvest filter. It has been found that without submerging the membrane of the harvest filter organisms do not pass through the membrane, or at least not in significant amounts and no or small yield of organisms is obtained. Such is not well understood. The method relates to a non-bleached production of organisms such as eggs, and hatchlings, with the possibility to use a very short time-window for harvesting. Using a short time-window will provide a synchronized population such as of nematode hatchlings, without having to withhold nutrient in or der to arrest the development of the hatchlings. It is therefore also no longer required to remove impurities such as nutrients in order to synchronize. As this is a much more natural process c.q. environment for the nematodes, the viability and condition of the hatched Ll nematodes will be optimal as a period of starvation (arrest) as necessary in the prior-art method is no longer required, assuming a relative short time-window will be used when harvesting the eggs.

Based on an example of 50 ml buffer fluid, stabilizing the system at a level of 1500 nematodes per millilitre a population of approximately 75000 nematodes will be available. Assuming each nematode lays between 2 and 6 eggs per hour, the output production rate of such system will be about 10⁵ eggs per hour. The output quantity ultimately will depend on the amount of fluid and the concentration of gravid nematodes used per millilitre and thereby the overall size of the reactor.

The quality of output of eggs or hatchlings will be close to perfect or sometimes perfect, i.e. 100% as the process and conditions produce the eggs or hatchlings without the influence of harmful chemicals and even offers the possibility of synchronization without having to apply a period of starvation. In addition, it has been found that the population has largely (>90%) the same phenotype.

By carefully selecting and adapting breeder system and method conditions the output may comprise > 99.9% of unhatched eggs or hatchlings, such as >99.99%.

In an example of the present method only eggs or hatchlings are collected; the present system provides an option of carefully and precisely selecting a sub-population of nematodes .

In an example the present method comprises the step of (7) harvesting eggs over a period of time in the range of 0.2-3000 minutes, preferably 0.5-240 minutes, more preferably 1-120 minutes, even more preferably 2-60 minutes, such as 5-30 minutes .

In an example of the present method the step of harvesting may be repeated over and over, as adults continue to produce eggs and hatchlings. Harvesting may be repeated e.g. 2-100 times, and/or during a period that the nematodes are producing eggs.

In an example of the present method only nematode hatchlings or eggs are collected; the present system provides an option of carefully and precisely selecting a sub-population of nematodes.

In an example of the present method th emembrane of the stabilization filter may have a thickness of 10-100 pm, and in the filter slits with a length mi of 20-800 pm, and a width m_wof one of 15pm, 20pm, 25pm, and 30pm, wherein a width varies less than 20%, preferably less than 10%, relative to the width m_w over the full filter (Fl), and/or wherein the membrane of the harvest filter has a thickness of 10-100 pm, and in the layer slits with a length mi of 20-800 pm, and a width m_w of one of 8pm, 10pm, and 25pm,, wherein a width varies less than 20% %, preferably less than 10%, relative to the width m_w over the full filter (F2,F3a,b), wherein the slits are provided in a hydrophilic layer.

In an example the present method may further comprise cleaning a filter (Fl,F2,F3a,b) before use with an alkaline aqueous liquid, such as comprising OH^-, and/or cleaning said filter with an acidic liquid to remove precipitates that may inhibit the flowthrough of the filter, such as a calcium comprising compound, such as CaO or CaCOi, such as acetic acid.

In an example of the present method harvested species are one of eggs, and L1-L4 nematodes.

In an example of the present method adult species are provided with nutrients, such as sucrose, before harvesting, therewith preventing adult species to pass through the harvest filter.

In a third aspect the present invention relates to a nonbleached and synchronized sub-population of nematode eggs according to claim 18, wherein a phenotype of nematodes is >95% the same. Under the term bleach also the term leach is considered to fall (treatment with e.g. NaOH), especially in terms of effect on the nematode population. The population does not comprise impurities, contrary to bleaching methods. 99% of the population is not damaged, typically 99.9%, more typically 99.99%; i.e. at the most there is very limited occasional damage. The sub-population typically comprises at least 10² nematodes, such as only one of eggs or hatchlings, sometimes at least 10⁴ nematodes, but at least 10⁶ nematodes is possible; likewise a volume of nematodes can be provided, such as of 0.001-2 litres, and typically 50-300 ml such as 200-250 ml; the quantity will depend on the amount of fluid and overall size of the present reactor. By carefully selecting and adapting the system and method conditions the sub-population comprises > 90% of only eggs or likewise only hatchlings ((relative to a total number of living organisms). It has been found that a size distribution of the sub-population can now be well controlled, and the population is healthy; prior art methods at the best generate a sub-population of nematodes of which, after using bleaching to separate the eggs, at the best yields between 70% and max 90% of expected eggs. In addition, the present population fully (> 99%) hatches, contrary to prior art methods wherein only 60-70% hatches. Assuming a single nematode could produce 250 eggs, prior art method effectively only yields approximately 10 eggs that will hatch per adult,

i.e. only 4% and in many cases will not be viable and thus not suited for further testing/research purposes, e.g. in terms of reliability of the outcome of such testing. The age of the eggs of the population varies only within a time needed to hatch, ±5% of said hatch time, if all synchronized nematodes are hatched. The average residence time of these non-flushed eggs or hatchlings is typically less than a minute, such as 0.5 minutes at the most. The variation in age is therefore typically within ±5% of the hatch time, or better. For example, if a synchronized population (eggs) would be harvested within a relative short time window of only 15 minutes, the harvested eggs have an age distribution of 0-15 minutes ±1 minute at the most, the variation merely caused by the time needed to harvest; in this example the age varies with 15-16 minutes at the most. Harvest times may vary from 5-1200 minutes, such as 10-60 minutes, or likewise 30 minutes. So all eggs or likewise nematodes have the same age, or put different when harvesting hatchlings they are born at exactly the same time, within a very small time window. When using a relatively short time window it also avoids unnecessary stress due to the withholding of nutrients as required in prior art method's (i.e. the need for arrest i.e. starvation in order to synchronize). As such having a potential large harvest within a short time window provides an unprecedented synchronization, not possible with prior art methods. Prior art methods use an incubation time of e.g. 4-5 hours at the best, leading to an age distribution of the same order, e.g. 4-5 hours.

In an example the present sub-population has a size distribution which is characterized by an average size (length) of the nematodes (C. elegans) and a standard deviation 3σ in size of < 30% relative, and typically 3σ in size of < 10% relative,

i.e. well defined.

In an example the present sub-population comprises at least 1000 organisms, preferably at least 10⁴ organisms, more preferably at least 10⁵ organisms, even more preferably at least 10⁶ organisms, such as up to 10¹⁰ organisms.

In a fourth aspect the present invention relates to a use of a sub-population of nematodes, obtainable by the present method, testing a medicine, for testing a chemical, for testing a substance, for testing toxicity, for testing an agrochemical, for providing a volume of nematodes, for genomics, for cell biology, for neuroscience, for aging, for phenotyping the population, for providing a population of nematodes, for studying DNA-changes over generations, or for high throughput screening.

The one or more of the above examples and embodiments may be combined, falling within the scope of the invention.

EXAMPLES

The below relates to examples, which are not limiting in nature.

Producing the filters:

Filters are made by either Laser Micro processing using a Pico or Femto Laser System or by a lithographic process named Electroforming. In both cases there is an absolute requirement for zero 'faults' that could lead to any of the critical dimensions being exceeded as even a single 'error' would cause a reduced yield of the system in the case of the stabilization filter and case of the harvest filter it would cause larger nematodes such as adults to pass the filter barrier.

Filter size: the diameter and therefor the area of the filter can vary depending on the configuration of the system.

I.e. for a 96 well configuration the individual filter diameter will approximately be 5mm and for a single or multiple filter High Volume system a filter or filters may be as large as 80mm diameter or even more if required.

Material: Micro Mesh Filter material is typically Stainless Steel or a Nickel alloy. However other materials such as polymers or silicon alike materials could also be used.

Filter opening size and shape of mesh opening:

The Stabilization filter (1st stage filter) will have a mesh opening no wider than required to let all hatchling up to 'L3/L4' size organism pass through, however retain the adults and if the system is configured for collecting hatchlings preserve as many eggs as possible. (For C. elegans this is typical 18pm-25pm, however may differ for different strains/organism).

The Harvest filter (2nd stage filter) in case of harvesting hatchlings will have a mesh size small enough to retain all Adults and eggs, however let the hatchlings pass through. (For C. elegans this is typical 8 pm-ll pm, however may differ for different strains/organism). In case of harvesting eggs only the mesh size will be in the range of 20-30 pm, however may differ for different strains/organism.

The shape of the mesh opening has a profound effect on the working and efficiency of the micro filter, where the absolute slit width has a very narrow tolerance (+/- < 2 pm) in achieving optimum performance and separation function. Slit length is less critical however together with the pitch (pitch is distance between slits/mesh openings) determines the % open area, hence the flow capacity of the filter.

Synchronization-harvest:

The Harvest filter will be placed in a receptacle, partly submerged such that the underside of the micro mesh filter will always be in contact with a receiving liquid media, which may be considered as wetting of the filter at a bottom side of the membrane thereof, and as required a few millimeters above the filter membrane where the Adults and eggs remain. The hatchlings will with no mechanical force and by gravity only pass through the filter and stay below the micro mesh in the media solution until the harvest time ends. If eggs are to be harvested, it is found to be helpful to have a mechanical induced flow/spray to flush the eggs through the filter.

The invention is further detailed by the accompanying figures and examples, which are exemplary and explanatory of na15 ture and are not limiting the scope of the invention. To the person skilled in the art it may be clear that many variants, being obvious or not, may be conceivable falling within the scope of protection, defined by the present claims.

EXAMPLES

The system can be used for harvesting synchronized eggs (Fig 5) or the system may be configured and used to harvest only synchronized hatchlings (Fig 4). As an example, in the latter case the hatchlings for the nematode C. elegans, are named 'Ll'. (Fig 7)

Harvesting synchronized Hatchlings :

In the example of the configuration (Fig 4) to be used for harvesting synchronized hatchlings 'Ll' the user will typically have prepared a culture of nematodes that consists mostly of adults and eggs, combined with other stages as well.

At the start of the protocol, all valves will be closed and the filtration buffer flask Cl will be filled with a fluid such as water and the Harvest Buffer C4 will typically be filled with a fluid consisting of water or any type of liquid buffer media such as; Basel or S media. Typically the culture will be added to the Pressure Flask C3 and through the applied pressure source transferred to the Stabilization filter. The shear force created by the venturi effect of the Pasteur pipette and/or venturi creating nozzle needle helps separate the organism(s) and improves the initial filtration. In addition the Pressure Flask C3 can be filled (again) with a fluid such as water, in case the user wants to further manually assist the first washing/stabilizing phase of the process.

The user optionally can aerate the content of the Harvest Buffer Fluid C4, by opening V6 using the air-pressure source. This is advised in order to increase oxygen levels in the liquid to increase quality/survivability of the organism(s) when the fluid is to be used in the final stage of the harvest process .

The user can start with the transfer of the organism(s) such as a culture of nematodes, such as C. elegans, onto the top of the Stabilizing Filter Fl. (Fig2-I) The Stabilizing filter Fl is placed in a closed receptacle and enough fluid is added to submerge the filter (Fig 2-II) and then left for a period of time, typically 20 minutes or more where most of the organism(s) smaller than the mesh size of the Stabilizing filter will transfer through the filter. This however can be accelerated while also getting a better separation result by actively washing the organism(s) with the filtration fluid from Cl, using a sparger head where the fluid is being circulated with a pump and all unwanted organism(s) that transfer through the filter will be retained in the in-line filter (Fig 2-II). This takes typically 5 minutes or less. If needed the user can manually assist this process using a hose with a needle Pasteur Pippete that provides a small pressurized stream of fluid coming from C3, that is pressurized by a pressure source.

After sufficient washing has taken place the content on top of the filter is considered stabilized, i.e. will mostly contain organism(s) of a size larger than the mesh size of the filter. In the case of the nematodes C. elegans it will contain mostly Adults and Eggs. More than 98% of other sized impurities and nematodes smaller than Adults and most Eggs will have been removed and retained in the in-line filter IF1 if active rinsing has been applied or collected at the bottom of the receptacle.

The user may then purge the Stabilizing fluid by using the pump to transfer the Stabilizing fluid to a waste Flask C2 with V3 and V5 open and pump running. This is when the user is ready for the transfer to the Harvest Filter F2 (Fig 2-III).

At this point in time the so called ΔΤ (delta time of the synchronization window) starts of the 'Ll' population. The user will typically start a timer to measure the time-window and in order to decide on how tight, or not, the synchronization window of the 'Ll' harvest will be.

Using the Harvest Buffer fluid from C4, the adult organising) and the eggs are washed of the stabilization filter and transferred onto the Harvest filter F2. (Fig 2-IV) The Harvest Filter is then placed in a receptacle and Harvest fluid from C4 is being added to submerge the filter itself, such that it completely covered. (Fig 2-V) The filter F2 is left in this position for at least 5 minutes or even up to 4 days or longer, depending on the amount of Ll hatchlings and the level of synchronization required. (Fig 2-VI) A prolonged harvest time will require additional food source for the Adult nematodes and the intermediate harvest of hatchlings at a regular inter val. If a food source is added, care should be taken that the hatchlings will also immediately start developing as food will be available.

After the user defined harvest time window, the harvest filter is removed from the receptacle in order to remove the hatchlings from the receptacle. (Fig 2-VII) Optionally this can be repeated a number of times, assuming the Adults are still capable of producing eggs. See above remark on the availability of a food source for the Adults as may be required for prolonged egg production. If needed the harvested and synchronized organism(s) can be further cleaned, impurities and optional food source removed and if needed sterilized using a Streptomycin & Nystatin solution.

Harvesting synchronized Eggs' :

In the example of the configuration (Fig 5) to be used for harvesting synchronized Eggs the user will typically have prepared a culture of nematodes that consists of mostly adults and eggs, combined with other stages as well.

At the start of the protocol, all valves will be closed and the filtration buffer flask Cl and C6 will be filled with a fluid (such as water) and the Harvest Buffer C4 will typically be filled with a fluid consisting of water or any type of liquid buffer media such as; Basel or S media. Typically the culture will be added to the Pressure Flask C3 and through the applied pressure transferred to the Stabilization filter. The shear force created by the venturi effect of the Pasteur pipette and/or venturi creating nozzle needle helps separate the organism(s) and improves the initial filtration. In addition the Pressure Flask C3 can be filled (again) with a fluid such as water, in case the user wants to further manually assist the first washing/stabilizing phase of the process.

The user can start with the transfer of the organism(s) such as a culture of nematodes, such as C. elegans, onto the top of the Stabilizing filter Fl. (Fig 3-1) The Stabilizing filter Fl is either placed in a closed receptacle (Fig 3-II ) and enough fluid is added to submerge the filter. If left for a period of time, typically 20 minutes or more, most of the organism(s) smaller than the mesh size of the Stabilizing Filter will transfer through the filter. This however can be accelerated while also getting a better separation result by actively washing the organism with the filtration fluid from Cl, using a sparger head where the fluid is being circulated with a pump and all unwanted organism(s) that transfer through the filter will be retained in the in-line filter. This takes typically 5 minutes or less. If needed the user can manually assist this process using a hose with a needle Pasteur Pippete that provides a small pressurized stream of fluid coming from C3, that is pressurized by a pressure source.

After sufficient washing has taken place the content on top of the filter is considered stabilized, i.e. will only contain organism(s) of a size larger than the mesh size of the filter. In the case of the nematodes C. elegans it will contain mostly Adults. Mostly all other sized impurities and nematodes smaller than Adults will have been removed and retained in the in-line filter IF1 if active rinsing has been applied or collected at the bottom of the receptacle.

The user will then purge the Stabilizing fluid by using the pump to transfer the Stabilizing fluid to a waste Flask C2 with V3 and V5 open and pump running. After purging the Stabilizing fluid the user transfers the Adults to the next stage (Fig 3-IV) where the Adults are put in receptacle.

Adults are left in the receptacle for a period of time to lay eggs. To stop the laying of eggs and to immobilize and swell the nematodes sucrose is added. At this point in time the so called ΔΤ (delta time of the synchronization window) will be set based on the time the adults have been allowed to produce eggs in this receptacle.

As soon as the nematodes are immobilized the content is then transferred from the receptacle to the Harvest Filter F2 (F3a/b) (Fig 2-V).

The still immobilized Adults and the eggs are transferred to the Harvest Filter F2 (F3a/b) (Fig 3-V) and are being placed in a closed receptacle with enough fluid being added to submerge the filter (Fig 2-VI) and then left for a period of time, typically 2 minutes or more where most of the eggs smaller than the mesh size of the Harvest Filter will transfer through the filter. However as there is little or no activity on top of the filter, the Adults are still immobilized, the eggs will hardly transfer and in many cases stick together and stay on top of the filter mesh. This can be overcome and also accelerated by actively washing with the filtration fluid from C6, optionally containing a detergent such as Tween or TritonX and using a sparger head where the fluid is being circulated with a pump and all eggs are collected in an in-line filter F4. (Fig 3-VI) If needed the user can manually assist this process using a hose with a needle Pasteur Pippete at the end that provides a small pressurized stream of fluid coming from C3, that is pressurized by a pressure source. After about 5 minutes of active washing/rinsing most of the eggs will have been transferred through the Harvest Filter and are collected in the in-line filter, leaving the Adults behind on top of the Harvest Filter.

The harvested and synchronized eggs can now be removed from the in-line filter, by applying a back-flush of the in-line filter cartridge or if a membrane filter has been used a simple reverse rinse will remove the eggs. If needed the harvested eggs can be further cleaned and if needed sterilized using a Streptomycin & Nystatin solution.

FIGURES

The invention although described in detailed explanatory context may be best understood in conjunction with the accompanying figures.

Fig. 1: Filter Mesh.

Fig. 2: principle with two filter stages, harvesting Hatchling ('Ll') .

Fig. 3 : principle with two filter stages, harvesting eggs.

Fig 4: schematic CES for Ll/Hatchling protocol /harvest ing/ synchronization .

Fig 5: schematic CES for eggs protocol /harvest ing /synchronization .

Fig. 6 shows symbols used.

Fig. 7 shows a life cycle of nematodes.

Fig. 8a,b shows populations of nematodes.

Fig. 9 shows an example of the present system.

Fig. 10 shows results of a harvesting experiment.

DETAILED DESCRIPTION OF THE FIGURES

In the figures:

100 life cycle synchronization system

Cl-6 container

Fl stabilization filter

F2 harvest filter

F3a,b harvest filter

IF1,2 in-line filter

Pl,2 pump

PPI Pasteur pipette and/or venturi creating nozzle

PSI pressure source

Rl,2 receptacle SH sparger head V_x valve

In figure 1 a top view of a filter membrane is shown (left) with slits having a length mi and width m_w, wherein the membrane is provided in a filter (top right). A cross section A-A' of the membrane is shown at the lower right. The membrane may be under-etched or the like, indicated by arrows, therewith proving a slit width m_w which at a bottom side may be up to two times as wide, typically 10-50% wider. The membrane may be provided with a hydrophilic layer, schematically shown.

In figure 2 in step I a stabilizing filter Fl with 20 pm wide slits is used. A mixed population of organisms is added on top of the micro mesh filter, optionally using a receptacle with an open bottom or output. In step II the stabilizing filter is a) used with a receptacle and a submerged filter with fluid/media as unwanted organism smaller than adults and/or eggs will transfer through filter over time, or is b) actively flushed to remove all unwanted organisms smaller than adults and/or eggs. Optionally one can use a receptacle with open bottom or output. In step III organisms remaining on the stabilizing filter are transferred to F2, the harvest filter. In step IV the harvest filter (7-11 pm) is used, wherein organisms are added on top of a micro mesh filter. A receptacle with closed bottom is used. In step V fluid/medium is added to the harvest filter to submerge the filter fully. A closed bottom receptacle is used. In step VI hatchlings/organisms of interest will transfer through the mesh filter in a user defined time period. In step VII one may remove the harvest filter after a time as required. The receptacle will contain the organisms of interest, such as hatchlings.

In figure 3 in step I a stabilizing filter Fl with 25-30 pm wide slits is used. A mixed population of organisms is added on top of the micro mesh filter, optionally using a receptacle with an open bottom or output. In step II the stabilizing filter is a) used with a receptacle and a submerged filter with fluid/media as unwanted organism smaller than adults will transfer through filter over time, or is b) actively flushed to remove all unwanted organisms smaller than adults. Optionally one can use a receptacle with open bottom or output. In step III organisms remaining on the stabilizing filter are transferred to F3a,b, the harvest filter. In step IV a) organism (Adults) are placed in a container and are allowed over a period of time to lay eggs. Remark: there is no Filter in place, b) After a user defined time period, add nutrients such as sucrose (in case of nematodes) to immobilize organisms (Adult nematodes) and have the adults swell. In step V the Adults and the eggs that have been produced are transferred to the harvest filter (25-30 pm), wherein organisms are added on top of a micro mesh filter. It is noted that in order to improve purity a 'F3b' filter may have a slightly smaller mesh size versus the 'F3a' filter. In step VI a harvest filter F3a,b is used (25pm- 30 pm). In a) a receptacle is used and the filter is submerged with fluid/media as organisms (eggs) smaller than Adults will transfer through filter over time; or in b) an active flush is applied in order to transfers organisms (eggs) smaller than Adults through filter. Optionally one can use a receptacle with open bottom or output. The output is filtered through an approximately 10-15 pm or smaller mesh size filter. Organisms (eggs) will be retained in this filter. In case of an in-line filter, backflush to harvest the organism (eggs) may be applied, or if a membrane filter has been used a simple reverse rinse will remove the eggs.

Figure 4 shows a simple layout of the present system for synchronizing and harvesting hatchlings.

Figure 5 shows a somewhat more complex layout of the present system for synchronizing and harvesting eggs.

Fig. 8a shows a population of C. elegans after the synchro nisation process, showing a perfect sub-population of onlyhatchlings , and fig. 8b shows the results after filtering over a 30 pm filter thereby letting all life cycles through.

Fig. 9 shows an example of the present system with manually controllable valves.

Figure 10 shows results of a harvesting experiment using the present system obtaining an average of 99.91% purity of Li's when using the LabTIE Ll C. elegans Synchronizer (CES). Using the CES, the amount of Li's present in the harvest samples compared to other stages (anomalies) was identified to determine the accuracy of the Synchronizer. A 50mL F3 generation S-medium culture was used, containing a C. elegans mixed population and was synchronized using the CES. The culture was washed for 15 minutes prior to 90 minutes of harvesting Li's in S-media without OP50. (left) After synchronizing, the total amonnt of worms was counted from 10 harvesting experiments, (right) Anomalies were counted in each harvesting experiment. Results were plotted against the total amount of worms. Example harvesting experiment 1: 6400 Ll worms vs 10 anomalies (7 x L2’s, 3 x L3's) = 100-(100/6400*10) = 99.84% Ll purity in harvest compared to other stages present. So with the present C. elegans Synchronizer freshly hatched Li's can be harvested without the use of chemicals, healthier Li's are obtained without the negative phenotypes, the level of synchronization is improved, larger yields than ever before are obtained, and user bias and training are eliminated. The same holds for organisms of other life cycles.

The figures have been detailed throughout the description.

The following section is provided for aiding search, and may be considered as an English version of the subsequent section.

1. Life cycle synchronization system (100) comprising at least one first stabilization filter unit for providing a first size sub-population of nematodes comprising a stabilization filter (Fl) and a stabilization receptacle (Rl), wherein optionally the membrane of the first filter unit is adapted to be submerged, wherein the first filter (Fl) comprises a hydrophilic membrane layer and a support for said membrane, wherein the filter membrane has a thickness of 5-100 pm, and in the membrane uniform slits with a length mi of 20-800 pm, and a width m_w in a range of 15-30 pm, with the proviso that mi>2*m_w, wherein a width varies less than 20% relative to the width m_w over the full filter (Fl), and at least one second harvest filter unit for providing a second size sub-population of the first sub-population of nematodes comprising a harvest filter (F2,F3a,b) and a harvest receptacle (R2), wherein the membrane of the second filter unit is adapted to be submerged, wherein the second filter comprises a hydrophilic membrane layer and a support for said membrane, wherein the filter membrane has a thickness of 5-100 pm, and in the membrane uniform slits with a length mi of 20800 pm, and a width m_w of 7-11 pm or 18-30 pm, with the proviso that mi>2*m_w, wherein a width varies less than 20% relative to the width m_w over the full filter (F2,F3a,b), wherein each filter has a surface area (SA) of 0.1-5000 cm².

2. System according to embodiment 1, wherein the width of the first slits is from 18-25 pm, and/or wherein the width of the second slits is from 8-11 pm.

3. System according to any of the preceding embodiments, wherein the membrane layer of the first filter is apart from slits fully intact (0 faults) and/or wherein the membrane layer of the second filter is apart from slits fully intact.

4. System according to any of the preceding embodiments, wherein a slit density is from 10“⁶-10^-4 /pm².

5. System according to any of the preceding embodiments, wherein slits are provided in alternating mode in at least one direction.

6. System according to any of the preceding embodiments, wherein in at least one direction a size of at least one slit decreases from a top side of the membrane to a bottom side thereof.

7. System according to any of the preceding embodiments, further comprising a Pasteur pipette and/or venturi creating nozzle (PPI), at least one of a container (C1-C6), a first and second container (C1-C2) in fluid connection with an output of the stabilization filter, a third container (C3) in fluid connection with the Pasteur pipette and/or venturi nozzle (PP), a fourth container (C4) for holding and optionally aerating a harvest fluid, optional containers (C5-C6) in fluid connection with an output of the harvest filter, a valve (Vl-Vll), preferably a valve per fluid connection, a pump (P1,P2), a first pump in fluid connection with containers (C1-C2) for providing pressure, optionally a second pump in fluid connection with optional containers (C5-C6) for providing pressure, a pressure source (PSI) for providing pressure to containers (C3) and optional aeration to container C4, an optional sparger head (SH1,SH2) for providing sprayed liquid to stabilization filter and/or harvest filter and in fluid connection with pump (Pl) and optional pump (P2), and an in-line filter (IF1,IF2) provided in fluid connection with an output of a stabilization filter or harvest filter, fluid connections between containers, the pumps and pressure source adapted to provide fluid flow.

8. System according to any of the preceding embodiments, comprising at least two stabilization filters arranged in spatial series and/or at least two stabilization filters spatially in parallel, such as 2³-2⁷ filters in series, such as 2⁴-2⁶ filters in parallel, and/or at least two harvest filters in arranged in spatial series and/or at least two harvest filters spatially in parallel, such as 2³-2⁷ filters in series, such as 2⁴-2⁶ filters in parallel.

9. System according to any of the preceding embodiments, further comprising at least one of a filtration buffer (Cl), a waste flush container (C2) , a pressure flush container (C3) comprising a liquid adapted to the nematodes, a first harvest buffer container (C4) comprising a liquid adapted to the nematodes, a second waste harvest buffer container (C5), a third filtration buffer container (C6), a controller for regulation and controlling operation, and a sparger (SHI,2).

10. System according to any of the preceding embodiments, wherein for each filter independently mi>5*m_w, preferably wherein mi>10*m_w, more preferably wherein mi>20*m_w, such as wherein mi>30*m_w.

11. System according to any of the preceding embodiments, wherein the filter membrane is made from a metal, the metal preferably being selected from Ni, stainless steel, Ti, Cr, Si, W, Co, V, Al, and alloys thereof, and/or wherein the filter can withstand a pressure of > 50 kPa, and/or wherein a uniformity in mi and m_w, respectively, is better than a standard deviation 3σ of <10% relative to an average of mi and m_w, respectively, and/or wherein a thickness of each membrane independently is from 1050 pm, preferably from 20-40 pm, such as 25-30 pm, and/or wherein at least one filter membrane comprises a hydrophilic coating, such as a metal coating.

12. Method of life cycle synchronization comprising providing at least one stabilizing filter (Fl), adding a population of organisms, such as nematodes, the population comprising at least two species selected from embryo's, such as E1-E6 embryo's, larvae, such as L1-L4, adolescents, young adults, and adults, on the stabilizing filter (Fl), transferring the stabilization filter to a (open or closed) first receptacle (Rl), sub-merging the membrane of the filter in an aqueous liquid or flushing the filter with said aqueous liquid therewith removing species through the filter slits into the receptacle, transferring remaining species to a harvest filter (F2,F3a,b), transferring the harvest filter to a second receptacle (R2), submerging the membrane of the harvest filter in an aqueous liquid, harvesting species that passed through the harvest filter, and optionally repeating the harvesting of species.

13. Method according to embodiment 12, when harvesting is performed over a period of 0.2 minute-48 hours, such as 30 minutes-9 hours.

14. Method according to any of embodiments 12-13, wherein the membrane of the stabilization filter has a thickness of 10-100 pm, and the layer slits with a length mi of 20-800 pm, and a width m_w of one of 15pm, 20pm, 25pm, and 30pm, wherein a width varies less than 20% relative to the width m_w over the full filter (Fl), and/or wherein the membrane of the harvest filter has a thickness of 10-100 pm, and in the layer slits with a length mi of 20-800 pm, and a width m_w of one of 8pm, 10pm, and 25pm, , wherein a width varies less than 10% relative to the width m_w over the full filter (F2,F3a,b), wherein the slits are provided in a hydrophilic layer.

15. Method according to any of embodiments 12-14, further com prising cleaning a filter (Fl, F2, F3a, b, IF1,2) before use with an alkaline aqueous liquid, such as comprising OH^-, and/or cleaning said filter with an acidic liquid to remove precipitates .

16. Method according to any of embodiments 12-15, wherein harvested species are one of eggs, and L1-L4 nematodes.

17. Method according to any of embodiments 12-16, wherein adult species are provided with nutrients, before harvesting, therewith preventing adult species to pass through the harvest filter .

18. Non-bleached synchronized sub-population of nematode eggs, in particular hatchlings or eggs, such as C. elegans eggs, obtainable by a method according to any of embodiments 12-17, wherein the sub-population comprises > 90% of synchronized hatchlings or eggs (relative to a total number of living organisms) , wherein an age of the hatchlings or eggs of the population varies within the harvest time ±30% thereof, wherein the sub-population has a vitality of >90% (relative to a total number of living organisms), and wherein a phenotype of nematodes is >95% the same.

19. Population according to embodiment 18, wherein the population has a size distribution which is characterized by an average size (length) of the nematodes (C. elegans) and a standard deviation 3σ in size of < 30% relative.

20. Population according to embodiment 18 or 19, comprising at least 200 organisms, preferably at least 1000 organisms.

21. Use of a population according to any of embodiments 18-20, for testing a medicine, for testing a chemical, for testing a substance, for testing toxicity, for testing an agrochemical, for providing a volume of nematodes, for genomics, for cell biology, for neuroscience, for aging, for phenotyping the population, for providing a population of nematodes, for studying DNA-changes over generations, or for high throughput screening .

Claims

CONCLUSIONS

A life cycle synchronization system (100) comprising at least one first stabilization filter unit for providing a subpopulation of the first size of nematodes comprising a stabilization filter (F1) and a stabilization receptacle (R1), optionally adapting the membrane of the first filter unit to being immersed, wherein the first filter (F1) comprises a hydrophilic membrane layer and a support for said membrane, the filter membrane having a thickness of 5-100 µm, and uniform slits of the length mi of 20-800 µm in the membrane, and a width m _w in a range of 15-30 µm, with the proviso that mi> 2 * m _w , wherein a width varies less than 20% from the width m _w over the full filter (Fl), and at least one second harvest filter unit for providing a subpopulation of the second subpopulation of the first subpopulation of nematodes, comprising a harvest filter (F2, F3a, b) and an eye catcher (R2), the membrane of the second filter unit being adapted to be submerged, the second filter comprising a hydrophilic membrane layer and a support for the membrane, the filter membrane having a thickness of 5-100 µm, and uniform slots of length in the membrane mi of 20-800 pm, and a width m _w of 7-11 pm or 18-30 pm, with the proviso that mi> 2 * m _w , where a width varies less than 20% from the width m _w across the full filter (F2, F3a, b), each filter having a specific surface area (SA) of 0.1-5000 cm ² .

The system of claim 1, wherein the width of the first slots is 18-25 µm, and / or the width of the second slots is 8-11 µm.

System according to any of the preceding claims, wherein the membrane layer of the first filter is completely intact apart from slits (0 errors) and / or wherein the membrane layer of the second filter is completely intact apart from slits.

4. A system according to any one of the preceding claims, wherein a slit density of 10 ^_s -10 ^{_, 4} / pm ^2.

System according to any of the preceding claims, wherein slits in alternating mode are provided in at least one direction.

The system of any of the preceding claims, wherein in at least one direction a size of at least one slit decreases from an upper side of the membrane to a lower side thereof.

System according to any of the preceding claims, further comprising a Pasteur pipette and / or venturi generating head (PPI), at least one of a container (C1-C6), a first and second container (C1-C2) in fluid connection with an output from the stabilizing filter, a third container (C3) in fluid communication with the Pasteur pipette and / or venturi head (PP), a fourth container (C4) for holding and optional aeration of harvesting liquid, optional containers (C5- C6) in fluid communication with an output of the harvest filter, a valve (V1-V11), preferably a valve per fluid connection, a pump (P1, P2), a first pump in fluid communication with containers (C1-C2) to provide pressure, optionally a second pump in fluid connection with optional containers (C5-C6) for supplying pressure, a pressure source (PS1) for supplying pressure to containers (C3) and optional aeration to container C4, an optional spray nozzle (SH1, SH2) for providing atomized flow fluid to stabilizing filter and / or harvest filter and in fluid communication with pump (PI) and optional pump (P2), and an in-line filter (IF1, IF2) providing fluid communication with a stabilizing filter or harvest filter output, fluid connections between containers, where the pumps and pressure source are adapted to provide fluid flow.

System according to any of the preceding claims, comprising at least two stabilizing flashes arranged in spatial series and / or at least two stabilizing filters spatially parallel, such as 2 ³ -2 ⁷ filters in series, such as 2 ⁴ -2 ⁶ filters arranged in parallel, and / or at least two harvest filters arranged in spatial series and / or at least two harvest filters arranged in spatial parallel, such as 2 ³ -2 ⁷ filters in series, such as 2 ⁴ -2 ⁶ filters in parallel.

System according to any of the preceding claims, further comprising at least one of a filtration buffer (C1), a waste rinse container (C2), a pressure rinse container (C3) comprising a liquid adapted to the nematodes, a first harvest buffer container (C4) comprising a liquid adapted to the nematodes, a second waste harvest buffer container (C5), a third filtration buffer container (C6), a controller for regulation and control, and a spray nozzle (SH1,2).

10. A system according to any one of the preceding claims, wherein, for each filter independently mi> 5 * m _w, preferably wherein mi> 10 * m _M, more preferably wherein mi> 20 * m _w such as, wherein m> 30 m * _w.

A system according to any preceding claim, wherein the filter membrane is made of a metal, the metal preferably being selected from Ni, stainless steel, Ti, Cr, Si, W, Co, V, Al, and alloys thereof, and / or where the filter withstands a pressure of> 50 kPa, and / or where a uniformity in mi and m _{w is} better than a standard deviation 3σ of <10% with respect to an average of mi and m _w , respectively, and / or wherein a thickness of each membrane is independently from 10-50 µm, preferably from 2040 µm, such as 25-30 µm, and / or wherein at least one filter membrane comprises a hydrophilic coating, such as a metal coating.

A method of life cycle synchronization comprising providing at least one stabilization filter (F1), adding a population of organisms, such as nematodes, the population comprising at least two species selected from embryos, such as E1-E6- embryos, larvae, such as L1-L4, adolescents, young adults, and adults, on the stabilization filter (Fl), transferring the stabilization filter to an (open or closed) first receptacle (Rl), immersing the filter membrane in an aqueous liquid or flushing the filter with the aqueous liquid and thereby removing the species through the filter slits in the container, transferring remaining species to a harvest filter (F2, F3a, b), transferring the harvest filter to a second tray (R2), immersing the membrane of the harvest filter in an aqueous liquid, harvesting species that have passed through the harvest filter, e n optionally repeating species harvesting.

The method of claim 12, wherein harvesting is performed over a period of 0.2 minutes to 48 hours, such as 30 minutes to 9 hours.

A method according to any one of claims 12-13, wherein the membrane of the stabilizing filter has a thickness of 10-100 µm, and the layer slits have a length ml of 20-800 µm, and a width m _w of one of 15 pm, 20 pm, 25 pm and 30 pm, with a width varying less than 20% with respect to the width m _w over the entire filter (Fl), and / or wherein the membrane of the harvesting filter has a thickness of 10- 100 µm, and in the layer slits having a length ml of 20-800 µm, and a width m _w of one of 8 µm, 10 µm and 25 µm, a width varying less than 10% with respect to the width m _w over the entire filter (F2, F3a, b), the slits being provided in a hydrophilic layer, the layer having a thickness of 10-100 µm.

The method of any of claims 12-14, further comprising cleaning a filter (F1, F2, F3a, b, IF1,2) prior to use with an alkaline aqueous liquid, such as 0H ~, and / or cleaning of said filter with an acidic liquid to remove precipitates.

The method of any one of claims 12-15, wherein the harvested species are one of eggs, and L1-L4 nematodes.

A method according to any one of claims 12-16, wherein adult species are supplied with nutrients prior to harvesting, thereby preventing adult species from passing through the harvest filter.

Unbleached synchronized subpopulation of nematode eggs, in particular larvae or eggs, such as C. elegans eggs, obtainable by a method according to any one of claims 12-17, wherein the subpopulation comprises> 90% synchronized larvae or eggs (relative to a total number of living organisms), where the age of the larvae or eggs of the population varies within the harvest time ± 30% thereof, with the subpopulation having a vitality of> 90% (relative to a total number of living organisms ), and where a nematode phenotype> 95% is the same.

The population of claim 18, wherein the population has a size distribution characterized by an average size (length) of the nematodes (C. elegans) and a standard deviation 3σ in size of <30% relative.

Population according to claim 18 or 19, comprising at least-

5 th 200 organisms, such as at least 1,000 organisms.

Use of a population according to any of the claims

18-20, for testing a drug, for testing a chemical, for testing a substance, for testing toxicity, for testing an agro10 chemical, for providing a volume of nematodes, for genomics, for cell biology, for neuroscience, for aging, for phenotyping the population, for studying DNA changes across generations, for delivering a population of nematodes, or for high-throughput screening.