FIELD OF THE INVENTION

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 in vention relates to an improved filter design for said system and an improved method of operating said filter, such that an effi cient and effective synchronization can be established.

BACKGROUND OF THE INVENTION

The present invention is in the field of a life cycle syn chronization 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. ele- gans) . C. elegans is a small, free-living soil nematode (round- worm) 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 typi cal 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, Lau sanne, Vol . 12, No. 2, (February 1980) pp . 137-150, and Patel et al. in "Axenic and synchronous cultures of C. elegans" in Nema- tologica, 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, respec tively, for separating nematodes; such will inevitably not re sult 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 dur ing 4-5 hours of incubation. The yield of larvae was even lower. In addition, contamination of eggs with worms was still an is sue. 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 A1 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 sys tem 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 bio film. Further passage of species is not optimal. This invention is a further improvement thereof.

The present invention therefore relates to a life cycle syn chronization 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 re sults, without jeopardizing functionality and advantages.

SUMMARY OF THE INVENTION

The present invention relates to an improved life cycle syn chronization system according to claim 1, a method of operating said life cycle synchronization system, a population of nema todes 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 pre cisely 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 pro vide 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 . 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 struc tural integrity, such as a tube-like structure. The present fil ters are similar to microfiltration filters in view of the or ganisms 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 needs 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 ade quate 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 im perfections 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 pre sent application.

The present life cycle synchronization (CES) system can pro vide relatively high volumes or likewise high amounts of nema todes, 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 nem atodes can be synchronized.

An advantage of the present system is that it produces as an output high amounts of synchronized eggs, and likewise synchro nized 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 de stroy 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 nema todes. As a consequence, it has been found that the present sys tem 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 phe notypes 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 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 re moved, 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 condi tions .

2) A consistent high quality of output is obtained, i.e. a nema tode 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 princi ple 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 mon itor and control the growth of nematodes .

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

5) For some systems 24x7 availability 'ready to use' of synchro nized 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 fil tration 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 organ isms. A second container C2 may be connected similar to Cl, al beit without an in-line filter; Cl and C2 are typically not in terconnected. A third flask C3 comprising buffered water may be in contact with a Pasteur pipette and/or venturi creating nozzle PP1, and a pressure source PS1, providing a pressure of 10-200 kPa. A fourth container C4 comprising a harvest buffer solution, which typically is autoclaved and therefore not comprising oxy gen, is provided with an air inlet for adding oxygen, and in fluid contact with PS1. A fifth container C5, similar to C2 functions as waste container, may be provided with a waste solu tion, 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 VI- Vll 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 in tended 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 natu ral synchronization will have occurred without the need to with hold 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 hor mones (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 cy cle synchronization system 100 according to claim 1.

In an exemplary embodiment 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 hatch lings 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 fil ter 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 den sity may be from 10 ⁶ -10 ⁴ /pm ² , preferably 2*10 ⁶ -5*10 ⁵ /pm ² , providing 1-50% of a surface are of the membrane with slits, preferably 2-40%, more preferably 5-35%, such as 16-33%. 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 increase from a top side of the membrane to a bottom side thereof, such as could be obtained by under-etching. By using an exemplary manufactur ing method such removing of material aside slits are 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 (PP1), at least one of a container (C1-C6) , a first and second con tainer (C1-C2) in fluid connection with an output of the stabi lization 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 PS1, 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 con tainers (C5-C6) for providing pressure, a pressure source (PS1) for providing pressure to container (C3) and optional aeration to container C4, an optional sparger head (SH1,SH2) for provid ing sprayed liquid to stabilization filter and/or harvest filter and in fluid connection with pump (PI) 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 2 ³ -2 ⁷ filters in series, such as 2 ⁴ -2 ⁶ filters in parallel, and/or at least two harvest filters in series and/or at least two harvest filters in parallel, such as 2 ³ -2 ⁷ filters in series, such as 2 ⁴ -2 ⁶ 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 (C3) comprising a liquid adapted to the nematodes, a first harvest buffer con tainer (C4), a second waste harvest buffer container (C5) , a third filtration buffer container (C6 ) , a controller for regula tion 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 mi>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 3s of <10% relative to an average of mi and m _w , respectively. Such a uniformity is difficult to obtain but is does support good se lection 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 cyl inder .

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 stabi lizing filter (FI), adding a population of organisms, such as nematodes, in a case of the population of nematodes (e.g. C. el- egans) 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 (FI), transferring the stabilization filter to a (open or closed) first receptacle (Rl), sub-merging the filter in an aqueous liq uid 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 liq uid, 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 organ isms is obtained. Such is not well understood. The method re lates 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 order 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 LI nema todes will be optimal as a period of starvation (arrest) as nec essary 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 10 ⁵ eggs per hour. The output quantity ultimately will depend on the amount of fluid and the concentration of gravid nematodes used per milli litre 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 synchro nization without having to apply a period of starvation. In ad dition, 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 care fully 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 the membrane of the sta bilization 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 _w of 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 clean ing 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 CaCOs, 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 pro vided with nutrients, such as sucrose, before harvesting, there with preventing adult species to pass through the harvest fil ter .

The present invention describes a non-bleached and synchro nized 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 impuri ties, 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 10 ² nematodes, such as only one of eggs or hatchlings, sometimes at least 10 ⁴ nematodes, but at least 10 ⁶ 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 se lecting and adapting the system and method conditions the sub population comprises > 90% of only eggs or likewise only hatch lings ((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 nema tode 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 relia bility 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 popu lation (eggs) would be harvested within a relative short time window of only 15 minutes, the harvested eggs have an age dis tribution 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 distribu tion which is characterized by an average size (length) of the nematodes (C. elegans) and a standard deviation 3s in size of < 30% relative, and typically 3s in size of < 10% relative, i.e. well defined.

In an example the present sub-population comprises at least 1000 organisms, preferably at least 10 ⁴ organisms, more prefera bly at least 10 ⁵ organisms, even more preferably at least 10 ⁶ or ganisms, such as up to 10 ¹⁰ organisms.

The present invention describes 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 popula tion 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 na ture . 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 dimen sions being exceeded as even a single 'error' would cause a re duced yield of the system in the case of the stabilization fil ter and case of the harvest filter it would cause larger nema todes such as adults to pass the filter barrier.

Filter size: the diameter and therefor the area of the fil ter 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 diame ter or even more if required.

Material: Micro Mesh Filter material is typically Stainless Steel or a Nickel alloy. However other materials such as poly mers 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 pre serve 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-11 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 be tween 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 mem brane thereof, and as required a few millimeters above the fil ter 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 nature and are not limiting the scope of the invention. To the person skilled in the art it may be clear that many variants, being ob vious 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 syn chronized 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 cre ating 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 FI. (Fig2-I) The Stabilizing filter FI is placed in a closed receptacle and enough fluid is added to sub merge 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 trans fer 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 un wanted organism(s) that transfer through the filter will be re tained 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 re moved and retained in the in-line filter IFl 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 *T (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 organ ism^) 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 com pletely covered. (Fig 2-V) The filter F2 is left in this posi tion for at least 5 minutes or even up to 4 days or longer, de pending on the amount of LI hatchlings and the level of synchro nization required. (Fig 2-VI) A prolonged harvest time will re quire additional food source for the Adult nematodes and the in termediate harvest of hatchlings at a regular interval. If a food source is added, care should be taken that the hatchlings will also immediately start developing as food will be availa ble .

After the user defined harvest time window, the harvest fil ter is removed from the receptacle in order to remove the hatch lings from the receptacle. (Fig 2-VII) Optionally this can be repeated a number of times, assuming the Adults are still capa ble 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 re moved 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 pre pared 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 organ ism^) and improves the initial filtration. In addition the Pressure Flask C3 can be filled (again) with a fluid such as wa ter, 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 . (Fig 3-1) The Stabilizing filter FI 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 trans fer 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 pres sure 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 •T (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, typi cally 2 minutes or more where most of the eggs smaller than the mesh size of the Harvest Filter will transfer through the fil ter. 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 ac tively washing with the filtration fluid from C6, optionally containing a detergent such as Tween or Triton-X 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 wash ing/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 fil ter cartridge or if a membrane filter has been used a simple re verse rinse will remove the eggs. If needed the harvested eggs can be further cleaned and if needed sterilized using a Strepto mycin & Nystatin solution.

FIGURES

The invention although described in detailed explanatory con text 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/harvesting/syn chronization .

Fig 5: schematic CES for eggs protocol/harvesting/synchroniza tion .

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

FI stabilization filter

F2 harvest filter

F3a,b harvest filter

IF1,2 in-line filter

PI, 2 pump

PP1 Pasteur pipette and/or venturi creating nozzle

PS1 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 FI 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/me dia 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/me dia 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 organ isms 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 organ isms (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 syn chronizing 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 synchroni sation process, showing a perfect sub-population of only hatchlings, 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 LI C. elegans Synchronizer (CES) . Using the CES, the amount of Li's present in the harvest samples com pared 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 with out OP50. (left) After synchronizing, the total amount 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 LI worms vs 10 anomalies (7 x L2's, 3 x L3's) = 100- (100/6400*10) = 99.84% LI 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 chemi cals, healthier Li's are obtained without the negative pheno types, 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.