NL2015884B1

NL2015884B1 - High volume breeding and life cycle synchronization system.

Info

Publication number: NL2015884B1
Application number: NL2015884A
Authority: NL
Inventors: Millenaar Arno
Original assignee: Labtie B V
Priority date: 2015-12-01
Filing date: 2015-12-01
Publication date: 2017-06-14

Abstract

The present invention is in the field of a high volume breeding and life cycle synchronization system for nematodes and a method for breeding 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, neuro science and aging.

Description

High volume breeding and life cycle synchronization system

FIELD OF THE INVENTION

The present invention is in the field of a high volume breeding and life cycle synchronization system for nematodes and a method for breeding such nematodes.

BACKGROUND OF THE INVENTION

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 2 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.

The life cycle of hermaphrodite nematode C.elegans takes about 3 days at 20 °C. It is divided into multiple distinguishable stages. In a first stage approximately 250 eggs per adult are laid. The development of the embryo's (embryo-genesis) starts at the moment the eggs leave the nematode. Development of the embryo can be divided into different stages, starting at El and continued until E6. So called hatching occurs when a fully developed embryo develops from the E6 stage into a LI larval stage (also referred to as juveniles in some nematode literature). In the case when there is no nutrition available for the LI larvae, its growth and development will come to a halt (also referred to as arrest) which can last for up to 48 hours (H). If nutrition is presented to the LI larvae it continues to grow into a L2 larvae, which growth takes about 12 hours. Further development from L2 into L3 takes about 8 hours, whereas development from L3 into L4 takes about 8 hours as well. When L4 larvae continue to grow for another 10 hours they become young Adults (yA). Development from young adult into Adult (A) takes about 8 hours. When the adult stage and form are reached the nematode eggs have developed into the El stage. At this point, C. Elegans is capable of laying its first eggs and the cycle may be considered as completed.

In order to obtain accurate and repeatable results in said model system a homogeneous population of the nematode, preferable in its first development stage (LI) or egg stage, is considered a pre-requisite. In order to acquire a homogeneous population of nematodes, e.g. a single stage LI population, a typical prior art process that is being applied involves bleaching a non-homogeneous population of egg-producing nematodes in order to separate the eggs from the nematode.

This process is typically referred to as 'synchronization' or 'staging' in that it is an objective to obtain one single stage (synchronized stage) of the population wherein nematodes have a similar age. Not only is the use of chemicals in the synchronization process a challenging and uncertain process, wherein only a small deviation in temperature, bleach concentration, time, etc. will lead to failure of the egg separation process. In addition the typical yield of the prior art bleaching process is low compared to natural egg producing capabilities of adult nematodes, In addition the LI nematode hatchlings vitality is negatively affected by the use of the bleaching chemicals, rendering many of the LI nematodes unsuitable for further research or testing application. In addition to the above drawbacks and rather disappointing yield and quality of the produced LI nematode, the prior art method is very time consuming and not scalable. Scalability, yield and constant quality are however, for example, a pre-requisite for using the C. elegans nematode in high throughput screening applications .

The present invention therefore relates to an improved breeding system, and a method of operating said breeding 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 a an improved breeding system according to claim 1, a method of operating said breeding system, a population of nematodes obtainable by said method, and use of said population.

The present high volume breeding and life cycle synchronization (HVBS) system can provide relatively high volumes or likewise high amounts of nematodes, such as from 10“4 litre up to 1 litre or more, in a continuous, semi continuous or batch wise mode of operation. The volumes comprise an aqueous solution forming typically >50% of the volume, and nematodes; a dry weight of the nematodes is typically 2-10% of the nematode volume. For sake of comparison prior art systems typically provide a few millilitres at the most.

An advantage of the present system is that it produces as an output at least twenty times more egg's, i.e. synchronized LI nematodes, per equivalent amount of bio-mass used as feed supply, compared to prior art methods wherein bleaching is used as a way for synchronization. The present HVBS 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 LI nematodes, suitable for use in research and test applications. There are no negative side effects of the prior art methods and use of bleaching chemicals therein on the vitality of the LI nematodes observed.

Throughout the description the term "population" refers to i.c. nematodes of at least one life cycle stage being present up to all life cycle stages being present. In a "subpopulation" 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 High Volume Breeder and Synchronizing (HVBS) system as described in this application overcomes draw backs 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 chemicals, other than used for growing the nematodes, 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 LI nematodes.

In comparison, prior art methods at the best generate yields of unhatched eggs between 70% and max 90% of eggs being in principle available. Of this reduced fraction of unhatched eggs only between 4% and 8% will hatch. 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%, typically above 95%, and more typically above 99%, viable nematodes in a repeatable process environment. 3) Breeding, and 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 C. elegans. 4) Scalability for High Volume use in for example High Throughput Screening applications or research that requires substantial bio-mass.

Thereto the present system uses a breeding reactor Rl, and incorporated in the reactor at least one micro filter over which the content of the breeding reactors is filtered. Filter operation is done in a semi-constant mode, wherein the fluid content of the breeding reactor is kept in fluid motion over the filter. The fluid-motion may be halted/interrupted for filtration and settling purposes. Nematode eggs can then be harvested in an external filter cartridge, which can be easily exchanged. A system may have a number of parallel operating bio-reactors, connected to a control tower. In this respect the present system is considered modular.

The present reactor comprises at least one inlet arranged to receive fluids, and at least one outlet for removing nematodes, a first space between the MF and at least one outlet of the reactor arranged to receive nematodes, at least one first fluid recirculation connection (FRC), the recirculation connection comprising a nematode harvest micro filter (HF) with a mesh size smaller than the mesh size of the MF, wherein the nematode harvest filter is removably attached to the recirculation connection, wherein the recirculation connection comprises at least one valve (QC1,QC2), preferably a so-called quick coupler with an integrated shut-off, arranged for opening and closing the connection, a nutrient feedstock container (SI) in fluid connection with the breeding reactor, at least one oxygen supply (GS) in fluid connection with the breeding reactor, and a controller (Cl) for regulation and controlling operation, such as fluid flow, temperature of the reactor, im-pellor speed, valve opening and closing, and on/off switching. In an example the male side of the quick coupler is provided at a harvest filter side, or back-up filter side, respectively; an example hereof is a Parker Hannifin Push Button Quick coupler .

In the breeding reactors nutrients and gas, comprising oxygen, are provided in order to have the nematodes grow, and producing eggs for further generations of nematodes. Thereto a gas supply and a nutrient feedstock are provided.

The nutrient feedstock typically comprises bacteria, such as E. Coll.

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.

It has been found important to control flow over the MF, in order to have adequate filtering, to prevent clogging of the filter, and in order not to damage nematodes in any life stage. Also the flow of fluid(s) comprising nematodes needs to be controlled, e.g. in terms of throughput. As over the filter fluids are removed from the breeder reactor(s) it is (e.g. in a semi-constant mode) at least sometimes necessary to replenish the amount of fluid in the breeder reactor(s).

Thereto a fluid flow through the recirculation connection may be controlled. Thereto a controller is provided.

In an exemplary embodiment a part (1-10% relative) of the fluid is removed from the system; such is repeated regularly. It has been found that by removing part of the fluid a dauer stress hormone is kept at acceptable concentration levels in the fluids, such that it is prevented that nematodes enter a dauer stage. Thereto a removal conduct is provided, comprising a valve VI.

In an exemplary embodiment a high flow rate, will guarantee that eggs can be harvested in the external filter with minimum delay. This again provides a unique advantage to collect (harvest) a batch of eggs within a small time window. An example thereof is that a new filter cartridge can be added and already removed after 15 minutes. By having a relatively small time-window of only 15 minutes, the eggs will all hatch at approximately the same time (within 15 minutes) and 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 will be avoided.

It is preferred to use relative to the environment an over-pressure in the present system, in order to prevent air or contaminants from entering the present system.

The system may optionally add a UV sterilization unit, which may be switched on/off, in the return feed in order to help prevent undesired bacteria, fungi, or even viruses, from entering/remaining in the reactor without the use of chemicals. Using such an UV sterilization device, requires the use of inactivated (not alive) nutrients.

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 breeding system according to claim 1. The present breeding system comprises at least one bio-reactor with an integrated micro-filter. A storage (nutrient feedstock) container will hold nutrients and is connected to the bioreactor, e.g. by means of a controllable peristaltic pump, or likewise a mechanical pump, allowing precise administration of the required amount of nutrient. It is noted that pumping action can be reversed, if required, at least for some of the pumps provided.

In an example at the conical bottom of the bioreactor vessel and output will lead the filtered fluid output towards an exchangeable filter cartridge. The mesh size of the external filter is chosen such that the nematode eggs are retained, and the nutrients will pass through. A system may typically be started, also referred to as inoculated, with a small number of eggs. In the first start-up phase the system operates with the external filter(s) being removed. After a number of days, depending on the number of eggs being used to inoculate the system, the system will reach a level at which the system will contain a sufficient number of nematodes to start the process of harvesting.

In order to pre-pare the system for harvest mode, it typically is stabilized first, i.e. a homogeneous population of adult nematodes is created. Essentially only eggs will be passing through the internal micro filter. In order to reach this point, an external filter will be placed for a period of time in the recirculation connection. The external filter is used for collecting all life-stages of nematodes and eggs, passing through the internal filter, until all nematodes still residing in the bio-reactor are at a life-stage that prevents them from passing through the micro-filter (in view of their size) .

When this stage is reached, the output will only contain eggs and no longer contain any other life-stages of the nematodes. The system is now ready for operating in harvest mode. An external container that may have been used for stabilizing the system, can be disposed or prepared and stored for future inoculation.

The moment the system has stabilized, i.e. only eggs and nutrient being present in the output fluid, an operator, may place an external filter cartridge for harvesting the eggs. Depending on intended use and the quantities c.q. vol- umes required, the harvest time may be adjusted.

In order to reduce the risk of eggs returning into the bio-reactor, e.g. as a result of operator error, an optional backup filter cartridges may be placed in the return connection, when operating in harvest mode.

After a number of days, typical 2 to 3, the production of eggs will come to a halt as the adult population will have exhausted it capacity to lay eggs. The period the present system will be able to produce eggs may also depends on factors such as temperature and the composition of the different life stages at the time the system was considered stabilized and switched to harvest mode.

When the system is coming to the end of its production cycle, an operator has a choice to re-inoculate the system by harvesting a final batch of eggs or to use a previous harvest of eggs. Typically the existing population of nematodes will be flushed out, before the system will be inoculated again. Inoculation is considered to be a fairly simple process, where a filter cartridge containing previously harvested eggs may be mounted in reverse direction, using a sterile and fresh buffer fluid to flush out the eggs and return them into the bioreactor, while at the same time filling the bio-reactor to the required fill level.

In an example of the present breeding system the recirculation connection comprises at least one nematode back-up micro filter with a mesh size smaller than the mesh size of the MF, wherein the at least one second nematode filter is preferably removably attached to the recirculation connection.

In an example of the present breeding system the recirculation connection comprises at least one valve per filter arranged for opening and closing the connection, the at least one valve being adjacent to the first nematode filter or to the second nematode filter, respectively.

In an example of the present breeding system a second space is arranged between the micro filter and a side wall of the reactor in order to allow fluid flow in said second space, and comprising a support for the micro filter.

In an example of the present breeding system the breeding reactor comprises at least one of an air sparger AS to meet biological oxygen demand, an impellor IM, an air filter AF, a conical bottom, a fill level sensor, a dissolved oxygen sensor DOS, a pH sensor pHC, a control unit 101, and a temperature sensor TS. With the air sparger biological oxygen demand is provided. If an impellor is used preferably a "marine type' blade impellor is used, having a rounded shape, preferable custom designed to be close to the contour of the Micro Filter surface, and operating at a low RPM, e.g. in order to prevent damage to nematodes. The impellor may operate in alternating Clock Wise and Counter Clock Wise rotation, depending on operating modus, and may be controlled by the controller. The present reactor preferably has a conical shape, wherein the conical shape is at a bottom thereof. It is preferred to grow nematodes at about 20 °C, thereto a temperature controller including a sensor and not preferred also a heat-er/cooler is provided. With the air filter an overpressure in the system is feasible, whereas at the same time microbes are prevented from entering the system; an example of the air filter is a bidirectional micro filter with an appropriate mesh size, typically smaller than 1 ym. The air sparger may be placed inside the reactor vessel or integrated in the return fluid connection.

In an example of the present breeding system the at least one micro filter comprises uniform openings (0) with an average opening diameter (d) of 10-100 ym, preferably with an average opening diameter (d) of 40-90 ym, preferably 50-80 ym, more preferably 50-70 ym, such as 63-67 ym. The opening diameter may be adapted in view of a desired population size (cross sectional diameter). Examples are 10-30 ym for only LI nematodes, 50-80 ym for retaining young adults and adults, and so on up to 100 ym. It has been found that a standard deviation 3σ of <10% relative to the average size is sufficient in this respect, whereas even better results are obtained with a 3σ of <5% relative. The surface area of the MF may vary in view of throughput and/or in view of breeder reactor sizes. Typically the micro filter has a surface area (SA) of 10-5000 cm2, such as 50-2000 cm2, per plate.

In an example the micro filter mesh plate is attached to a cylinder, with a cylinder diameter smaller than the inner dimension of the bio-reactor vessel. The fluid level within the bio-reactor is typically maintained at a lower level that the top of this cylinder.

At the top of the bio-reactor, nozzles may spray the returning buffer fluid inside the micro filter cylinder and also partially in-between the micro filter cylinder and the bio-reactor vessel. The fluid that flows on the outside of the micro filter cylinder prevents any eggs or nematodes to get trapped in-between the micro filter cylinder and the bioreactor vessel and also creates a flow near the bottom of the micro filter cylinder, supporting the eggs to be transported to the output of the bio-reactor.

In an example of the present breeding system at least one micro filter comprises a cylindrical section, such as from glass, wherein the cylindrical section extends upwards to a level above a maximum fluid level, and wherein an external diameter of the cylindrical section is 0.5-20 mm smaller than an internal diameter of the reactor, taken at a similar height, The present internal micro filter may have a polygonal shape, such as hexagonal and octagonal, a circular shape, and an ellipsoidal shape. The micro filter and its filter function may be supported by a rotational movement of the cylinder filter.

In an example the present breeding system comprises a plate section at the bottom of the cylindrical section with openings. Optionally, filter openings may also be present on the vertical section of the cylindrical section, typically towards the lower halve of the cylinder section.

In an example of the present breeding system the plate section is made from a metal, the metal preferably being selected from Ni, stainless steel, Ti, Cr, Si, W, Co, V, Al, and alloys, oxides, and nitrides thereof, and plastic, wherein the plate section preferably has a curvature.

In an exemplary embodiment the external harvest filter cartridge has a filter mesh size, small enough to retain the nematode eggs, however large enough for letting the nutrients pass through. A typical mesh size between 5-10 pm will suffice. It has been found that a standard deviation of <10% relative to the average size is sufficient in this respect, whereas even better results are obtained with a standard devi ation of <5% relative. The surface area of the external harvest filter may vary in view of throughput and/or in view of breeder reactor sizes. The harvest filter may be made of a polymer, such as PTFE. It may have the form of an exit and inlet tube being connected to a central disc shaped part. The disc shaped part comprises the actual filter; an example hereof is a Whatman polydisc. All components, or some of them, may be disposables. The disposables and likewise the whole system may be cleaned and freed from microorganisms by γ-rays.

In an exemplary embodiment the external harvest filter cartridge may be connected with push button quick coupler connectors in order to facilitate easy change of the filter cartridge and the filters typically will be considered disposable .

In an example the present breeding system further comprises further operational elements, such as one or more pumps, a fill level detector, a dissolved oxygen sensor, a pH sensor and controller, a temperature sensor and temperature controller, and a UV sterilization unit. Some of the elements are deemed optional such as the UV sterilization unit and for example the temperature and pH controller. The pumps may provide pumping action between various elements of the present breeding system. The sensors may be used to determine a status of the present system, e.g. in terms of detection of the fluid level within a reactor. Such information is typically fed back to the controller.

In a second aspect the present invention relates to a method according to claim 12, comprising the steps of (la)loading the breeding reactor (Rl) with an egg producing nematode species, such as C. elegans, (lb) loading nutrients and oxygen,(2) providing nutrients and oxygen to the breeding reactor (Rl),(3)flowing part of the fluid of the breeding reactor through the micro filter (MF) to an outlet of the reactor, thereby separating early life stage (egg-L4) of the nematode species from (young)adult nematodes thereby creating a homogeneous population of nematodes, and (4) collecting at least one of eggs and other life stages of nematodes in the harvest filter. The method relates to a non-bleached production of eggs, with the possibility to use a very short time- window for harvesting the eggs. Using a short time-window will provide a synchronized populations of LI nematodes, 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 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 1000 ml buffer fluid, stabilizing the system at a level of 1500 nematodes per millilitre a population of approximately 1.5 million nematodes will be available. Assuming each nematode lays between 2 and 6 eggs per hour, the output production rate of such system will be between 3*106 and 9*106 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 will be close to perfect, i.e. 100% as the process and conditions produce the eggs without the influence of harmful chemicals and even offers the possibility of synchronization without having to apply a period of starvation.

By carefully selecting and adapting breeder system and method conditions the output comprises > 99.99% of unhatched eggs .

In an example of the present method only eggs 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 0.2-540 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 the present method comprises the step of providing a back-flush over the at least one micro flow filter .

In a third aspect the present invention relates to a non-bleached population of nematode eggs according to claim 18. 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 population typically comprises at least 102 nematodes, such as eggs or Li's, more typically at least 104 nematodes, even more typically at least 106 nematodes, such as 107 nematodes; likewise a volume of nematodes can be provided, such as of 0.001-50 litres; the quantity will depend on the amount of fluid and overall size of the present reactor. By carefully selecting and adapting breeder system and method conditions the population comprises > 90% of eggs. It has been found that a size distribution of the population can now be well controlled, and the population is healthy; prior art methods at the best generate a population of nematodes of which, after using bleaching to separate the eggs, at the best yields between 70% and max 90% of expected eggs. Thereof only between 4% and 8% will hatch and in many cases will not be viable and thus not suited for further test-ing/research purposes, e.g. in terms of reliability of the outcome of such testing.

In a fourth aspect the present invention relates to a use of a 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, 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.

The invention is further detailed by the accompanying figures, 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 obvious or not, may be conceivable falling within the scope of protection, defined by the present claims.

FIGURES

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

Fig. 1 shows an illustration of the present high volume breeding and life cycle synchronization system (HVBS) (100) .

Fig. lb shows an illustration of the Controller Input and Output control signals (101) .

Fig. 2 shows a schematic overview of the life cycle of C. Elegans. DETAILED DESCRIPTION OF THE FIGURES In the figures:

100 HVBS 101 controller AF air filter AS air sparger BF back-up filter DOS dissolved oxygen sensor FLS fluid level sensor GS gas supply HF harvest (cartridge) filter IM impellor MF micro filter PP peristaltic (or mechanical) pump pHC pH sensor and controller QCX quick coupler with integrated valve RV regulator valve R1 breeding reactor SI nutrient feedstock container TS temperature sensor

Vx valve

Fig. la shows an illustration of the present high volume breeding and life cycle synchronization system (100).

Fig. lb shows an illustration of the Controller Input and Output control signals (101).

Fig. 2 shows a schematic overview of the life cycle of C. Elegans. HVBS working example description:

After preparation of the HVBS system, loading an initial batch of nematodes, especially eggs, into the breeding reactor, and allowing time for the nematodes to multiply under optimal and controlled conditions (sufficient nutrients, oxygen, etc.) a next step is to start filtering the content of the breeding reactors .

In the beginning the breeding reactors will contain a non-homogeneous population mix of all levels (stages) of development of nematodes. Filtering is done by mounting an external filter cartridge 'HC', also referred to as the external harvest filter cartridge.

As soon as the external filter cartridge is mounted, the circulation pump will continue and commence filtering. Any nematode life stage small enough to pass through the internal micro filter, will be collected in the external filter cartridge .

After some time, typically within 30 hours, the output of the system will only contain eggs and the system is considered, stable and ready for harvesting eggs.

The filter cartridge used to stabilize the system can be removed and replaced by a new filter cartridge. Depending on the type of synchronization and the output volume required, the filter cartridge will be removed after a set time and the harvested eggs can be used for further processing and use.

The figures have been detailed throughout the description .

Claims

A large volume nematode culture and life cycle synchronization system (100) comprising a culture reactor (R1), wherein the culture reactor has at least one inlet for receiving liquids, and at least one outlet for removing nematodes, included in the reactor comprises at least one micro filter (MF) with mesh size filter openings, a first space between the MF and at least one outlet of the reactor arranged to receive nematodes, at least one first fluid recirculation connection (FRC), the recirculation connection comprising a nematode pick microfilter (HF) with a mesh size smaller than the mesh size of the MF, wherein the nematode pick filter is removably attached to the recirculation port, the recirculating port comprising at least one valve (QC1, QC2) adapted to open and close the connection , a nutrient supply container (S1) in fluid communication with the culture reactor, at least one suction dust supply (GS) in fluid communication with the culture reactor, and a controller (C1) to control and control operation, such as fluid flow, reactor temperature, pH level, paddle speed, valve opening and closing, and switching on / off.

The culture system of claim 1, wherein the recirculation port comprises at least one second nematode backup microfilter (BF) with a mesh size smaller than the mesh size of the MF, the at least one second filter preferably being removably attached to the recirculating port.

A culture system according to any one of the preceding claims, wherein the recirculation connection comprises at least one valve (QC) per filter arranged to open and close the connection, the at least one valve adjacent to the first nematode filter or the second nematode respectively filter.

A culture system according to any one of the preceding claims, wherein a second space is arranged between the microfilter and a side wall of the reactor to allow a liquid flow in the second space, and comprising a support for the microfilter.

A culture system according to any one of the preceding claims, wherein the reactor has a conical bottom.

A culture system according to any preceding claim, wherein the culture reactor comprises at least one of an air distribution device (AS), an air filter (AF), a paddle wheel (IM), a fill level sensor, a dissolved oxygen sensor (DOS), a pH sensor (pHC), a control unit (101), and a temperature sensor (TS).

A culture system according to any one of the preceding claims, wherein the at least one micro filter comprises uniform openings (O) with an average opening diameter (d) of 10-100 µm, and a standard deviation 3σ of <10% relative to the average wherein the microfilter has a surface area (SA) of 10-5000 cm 2.

A culture system according to any one of the preceding claims, wherein the at least one microfilter comprises a cylindrical part, such as of glass, the cylindrical part extending upwards to above a maximum liquid level, and wherein an outer diameter of the cylindrical part 0.5 -50 mm smaller than the internal diameter of the reactor, taken at the same height, and a plate section at the bottom of the cylindrical section with openings.

The culture system of claim 8, wherein the plate portion is made of a metal, the metal preferably being selected from Ni, stainless steel, Ti, Cr, Si, W, Co, V, Al, and alloys, oxides, and nitrides thereof, and plastic, the plate portion preferably having a curvature.

The culture system of any one of claims 8-9, wherein a microfilter surface comprises openings with an average opening diameter (d) of 15-80 µm.

The culture system of any one of the preceding claims, wherein the at least one gas supply provides oxygen.

A method of operating a system according to any one of the preceding claims, comprising the steps of (1a) loading the culture reactor (R 1) with an egg-producing nematode species, such as C. elegans, (1b) loading nutrient and oxygen, (2) providing nutrient and oxygen to the culture reactor (R1), (3) flowing a portion of the fluid from the culture reactor through the microfilter (MF) to an outlet of the reactor, causing early life phase (egg-L4) separation of the nematode species from (young) adult nematodes, creating a homogeneous population of nematodes, and (4) collecting at least one of eggs and L1-L4 nematodes in the harvest filter.

Method according to claim 12, comprising the step of (5) harvesting nematodes from the harvest filter, for example by removing the filter.

A method according to claim 12 or 13, comprising the step of (6) continuing the operation until only eggs are produced.

The method of claim 14, comprising the step of (7) harvesting eggs over a period of time in the range of 0.2-540 minutes.

The method of any one of claims 12-15, comprising the step of providing a back-flush via at least one micro stream filter.

The method of any one of claims 12-16, wherein only eggs are collected.

An unbleached population of nematode eggs, such as C. elegans eggs, obtainable by a method according to any of claims 13-18, wherein the population comprises> 90% of the eggs, and wherein the population has a viability of> 90% has.

Use of a population according to claim 18, for testing a drug, for testing a chemical, for testing a substance, for testing toxicity, for testing an agrochemical, for providing a volume of nematodes, intended, for cell biology, for neuroscience, for aging, for phenotyping of the population, for providing a population of nematodes, or for high throughput screening.