NL2014734A

NL2014734A - High volume breeding and life cycle synchronization system.

Info

Publication number: NL2014734A
Application number: NL2014734A
Authority: NL
Inventors: Millenaar Arno
Original assignee: Labtie B V
Priority date: 2015-04-30
Filing date: 2015-04-30
Publication date: 2016-11-07
Also published as: NL2014734B1

Abstract

The present invention is in the field of a high volume breeding and life cycle synchronization system for nematodes and a method for synchronizing a life cycle 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 re-search in many fields including genomics, cell biology, neuroscience 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). In the young adult phase in the nematode eggs start to grow and develop towards the El stage, i.e. they are ready to reproduce. 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), 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 very difficult 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 of nematodes, such as from 0.1 litre up to 100 litre or more, in a continuous, semi continuous or batch wise mode of operation. For sake of comparison prior art systems typically provide a few millilitres and a few litres 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. If the present HVBS system continues to operate during a number of consecutive days, the before mentioned efficiency will further increase to a factor of at least 40-50. 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 reduces fraction of unhatched eggs only between 25% and 35% 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) 24x7 availability 'ready to use' of LI nematodes. 4) 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. 5) Scalability for High Volume use in for example High Throughput Screening applications.

Thereto the present system uses at least two breeding reactors R1,R2, and at least one micro flow filter over which the contents of the breeding reactors is filtered. Filter operation is done is a cyclic mode, wherein the fluid content of a first breeding reactor is flushed over the filter to a second breeding reactor. Two or more breeding reactors may be used, as well as one or more micro filter. In this respect the present system is considered modular.

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. Coli.

In order to provide a counter pressure over the micro flow filter and to balance an amount of fluid a collector reactor is provided. The collector is also in fluid connection with at least one breeder reactor; the collected fluid and e.g. in case of restarting the present system also nematodes and eggs are returned to the breeder reactor. In order to filter the nematodes properly the collector provides a counter pressure to the micro flow filter. Therewith a pressure over the micro filter can be controlled precisely. The pressure is typically relatively small e.g. to prevent clogging of the filter .

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 pressure differences over the MFF, 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-continuous mode) at least sometimes necessary to replenish the amount of fluid in the breeder reactor(s). Thereto a fluid flow between the collector reactor and the breeder reactors may be controlled. Thereto a controller is provided.

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.

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.

In an example the present breeding system further comprises at least one storage container S2 in fifth fluid connection with the at least one micro flow filter MFF. In a mode of operation the mff can be deactivated by e.g. closing valves towards the collector. At the same time also flow from a first to a second breeder reactor can be stopped. As a consequence the nematodes in the MFF may consume the nutrients and especially oxygen rather quickly and may starve and/or create a toxic environment. In order to prevent starvation in the MFF, the MFF and its fluid connections can be flushed with a sterile fluid from container S2. As such a steady state situation can be maintained for a few hours, allowing the population of nematodes to further develop into a next stage, or for example allow a homogeneous population of adult nematodes to lay eggs. Thereafter fluid flow from a first to a second breeder reactor can be continued, and filtering over the filter can be effected.

The fluid from the storage container S2 can also be used during the cyclic filtering stages for rinsing the breeder reactors when they are near empty in order to improve the efficiency of the system.

In an example the present breeding system further comprises at least one separator for removing impurities. The separator is intended for purifying the nematode population, especially eggs, in terms of removing impurities. Typically impurities present are bacteria, nutrients, and cell residues. The impurities may be separated using a centrifuge, a micro filter, or the like. Typically a 5-20 pm pore micro filter is used, allowing impurities to pass through, and maintaining nematodes, especially eggs.

In an example the present breeding system further comprises at least one hatcher reactor and an oxygen supply for the hatcher reactor. The oxygen supply may be the same as for the breeder reactors. In the hatcher reactor e.g. a population of eggs can develop into LI nematodes in a controlled and precise manner. In addition, by arresting growth, a well-defined population of nematodes and especially Li's may be obtained .

In an example the present system the separator and hatcher reactor are combined into one single device (separator and hatcher), using an integrated mesh filter, allowing rinsing out the impurities, while maintaining the eggs. After rinsing, the eggs remain in the combined device in order to hatch. In addition or as an alternative eggs may after removing impurities be transferred to the final storage containers for hatching and freeing the HVBS for the next batch process; a cycle time may thereby be reduced to an average of about 4 hours .

In an example the present breeding system further comprises a sixth fluid connection between the collector and at least one breeding reactor. The connection provides a return flow opportunity, of which use can be made when stabilizing the present system, for replenishing a fluid amount of the breeder reactor(s), for flushing/cleaning nematodes out of the breeding reactors when in filtering mode, and for returning a mixed population of nematodes and eggs when restarting the system cycle.

In an example the present breeding system further comprises a cleaning unit in fluid connection with at least one element of the present system. In between operations and before operation the present system may need to be cleaned, e.g. into a sterile system. Thereto a cleaning unit is provided. The cleaning unit can clean parts of the present system, e.g. breeding reactors, the MFF, or the full system. The system is preferably equipped with flexible tubes, which may be considered as disposables. The tubes can be removed in between operation and before operation and be replaced. It is preferred to have an integrated cleaning unit, as opening and cleaning of e.g. reactors is cumbersome and time consuming, and opening per se may introduce a risk of (cross-)contamination. As a cleaning medium an alcohol, such as isopropyl alcohol, an alkaline solution, such as NaOH, and combinations thereof can be used. It may be preferred to use a heated solution, e.g. of 60 °C or more. After cleaning and optional replacement of fluid connection tubes the present system is ready for producing nematodes again.

In an example of the present breeding system the at least two breeding reactors comprise at least one of an air sparger to meet biological oxygen demand, an impellor, a coni cal bottom, a fill level controller, a pressure sensor, a pressure controller, a dissolved oxygen sensor, a pH sensor, a control unit, and a temperature controller. With the air sparger biological oxygen demand and turbulence are provided. If an impellor is used preferably a "marine type' blade impel-lor is used, having a rounded shape and operating at a low RPM, e.g. in order to prevent damage to nematodes. The present reactors preferably have a conical shape, wherein the conical shape is at a bottom thereof. It has been found that complete emptying of the breeder reactor is improved by such a shape.

In order to monitor and control an amount of fluid in the breeder reactor a fill level controller is provided. Typically the reactors are filled from 0% (when empty) to approximately 30%. During the transfer of fluids and nematodes from one reactor R1/R2 to the other R2/R1 the combined volume will add-up to approximately 60% and leave sufficient head-space in order to deal with potential issues such as foaming, etc. A pressure sensor is provided for controlling pressure between the two reactors and over the mff. It is preferred to grow nematodes at about 20 °C, thereto a temperature controller including a sensor and not preferred also a heater/cooler is provided.

In an example of the present breeding system the at least one micro flow filter comprises uniform openings (0) with an average opening diameter (d) of 10-100 pm. The opening diameter may be adapted in view of a desired population size (cross sectional diameter). Examples are 10-20 pm for only LI nematodes, 55-75 pm for retaining young adults abd adults, and so on up to 100 pm. 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 mff 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 side In view of maintaining a uniform flow across all parts of the mff, thereby avoiding areas where nematodes and alike build-up (clog), certain areas of the filter mesh plate are free from openings, especially corner and side sections thereof. Therefore preferably at least 10% of the surface area does not comprise openings, more preferably at least 20%, and specifically 5-20% relative to a length/width/diameter of the surface at each side thereof is left free from openings. In view thereof the present MFF may have a polygonal shape, such as hexagonal and octagonal, a circular shape, and an ellipsoidal shape.

In an example of the present breeding system the at least one micro flow filter comprises a first plate, the first plate having at least one predefined fluidic path, and at least one inlet and at least one outlet, a filter plate in contact with the first plate, a third plate in contact with the filter plate, the third plate having at least one predefined fluidic path, and an inlet and an outlet, and means for fixing the plates together. Thereto a first plate is provided, typically made of acrylic glass, Poly(methyl methacrylate) PMMA, or the like, having a thickness of 10-30 mm. In the first plate a fluidic path is provided, typically having a depth of 0.2-5 mm. Through inlets the fluid, comprising nematodes and eggs, flows equally over the filter plate. A part of the fluid flows from the first to the second breeder reactor, and a part flows through the filter. Only small nematode subpopulations flow through the filter openings. The small nematodes sub-populations and fluid flow through a fluidic path of a third plate towards an outlet thereof. The third plate has similar characteristics as the first plate. The plates are fixed together using fixing means, therewith providing a liquid tight sealing.

In an example the present breeding system further comprises at least one impurity remover, which may be separate or combined with the hatching bioreactor. In order to arrest growth it is required to remove any nutrients from the LI hatchlings thereby preventing their further development and as a result creating a synchronized homogeneous population of LI nematodes. Therefore the impurity separators function is to remove any impurities that could provide nutrients to the hatchlings, i.e. the LI nematodes after hatching from the eggs .

In an example the present breeding system further comprises further operational elements, such as one or more pumps (MP1-P10), at least one sensor (LS1-LS7) in a fluid con nection for detecting the end of the filter cycle, i.e. a fill level of the breeder reactor, and at least one storage reactor (R10,Rll,Rx) for storing a sub-population, especially LI nematodes, preferably a removable storage reactor. 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 empty reactors. Such information is typically fed back to the controller. For practical purpose one or more storage reactors are provided. It is preferred to have a removable reactor R10-Rx; breeding can than take place at a first location, and the use of the sub-population for research and testing and the like at a second location.

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 14. First the system is loaded such as the one or two breeder reactors, depending on the need of utilization of the system. Then the nematodes are allowed to grow and reproduce during a sufficient period of time. At some point flowing of the breeder reactor fluid over the mff towards a second reactor starts. The contents of the first breeder reactor are thereby transferred to the second breeder reactor and some fluid is transferred over the filter to e.g. the collector. A differential controlled pressure between the at least two breeding reactors of 1 kPa-200 kPa, preferably 2-100 kPa, more preferably 5-50 kPa, such as 10-20 kPa, is applied. A differential controlled pressure between at least one breeding reactor and collector (R3) of 0.1 kPa-50 kPa, preferably 0.2-20 kPa, more preferably 0.5-10 kPa, such as 1-2 kPa, is applied; such a pressure is considered relatively low. A less preferred alternative one or more mechanical pumps may be used. Therewith an early life stage (egg-L4) of the nematode sub-populations is separated from (young)adult nematodes. A healthy and homogeneous population of adult nematodes is thereby created in the breeder reactors. If the process is continued the population in reactors R1 and R2 becomes more and more homogeneous over time. Using either the sampling output valve SO to determine the output content or by relying on a pre-determined number of filter cycles, the output of the mff will consist mainly (>95%) of eggs. This would be the appropriate moment to switch the output selector valve SW1 towards the filter/impurity separator device IS. The impurity separator could in fact be a combined device with the Egg Hatcher R4. The main purpose of filter IS is to remove the nutrient (s) and other impurities from the eggs. It is considered that when the eggs hatch the LI stage nematodes should be without nutrients and be prevented from further development. This is referred to as 'arrest'. Than at least one of eggs and L1-L4 nematodes can be collected in the separator.

In an example the present method comprises (6) after removing nutrients and impurities storing the eggs for hatching, e.g. for a period of time < 10 hours. Thereafter the hatchlings, as a homogeneous population, of nematodes (LI), can be stored in at least one storage reactor (RIO, Rll, Rlx). In addition or as an alternative eggs after removing impurities may be transferred to the final storage containers for hatching and freeing the HVBS for the next batch process. As there are no nutrients available for the LI nematodes to feed on, they will remain 'arrested' and maintain their LI life cycle development stage. I.e. the nematode population is now synchronized and ready for use.

In an example the present method comprises the steps of (lc) loading a sterile buffer in the buffer container (S2), and after step (3) and before step (4) steps (3ai) wherein the flow is interrupted during a period of 10-600 minutes, during which a sterile buffer is provided to the at least one micro flow filter and the fluid connections between the reactors Rl, R2 and R3 respectively, and thereafter repeating step (3), followed by steps (4) and (5). As such the population is further controlled in terms of size distribution and life cycle stage .

In an example the present method comprises the step of providing a back-flush over the at least one micro flow filter from e.g. the collector to at least one breading reactor, thereby cleaning the filter.

In an example of the present method only LI nematodes or 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 steps of (6) moving to and maintaining the eggs in a hatcher for up to 10 hours under sterile conditions in the absence of nutrients, providing oxygen, and (7) hatching Li nematodes. In addition or as an alternative eggs after removing impurities may be transferred to the final storage containers for hatching and freeing the HVBS for the next batch process.

In a third aspect the present invention relates to a non-bleached population of nematodes according to claim 19.

The population does not comprise impurities, such as residues of nematodes. 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 104 nematodes, such as eggs or Li's, more typically at least 105 nematodes, even more typically at least 106 nematodes, such as 107 nematodes; 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 LI. 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 25% and 35% will hatch 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.

In an example the present 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 a fourth aspect the present invention relates to a use of a population of nematodes, obtainable by the present method, for testing a medicine, for testing a chemical, testing a substance, for testing toxicity, for testing an agro- chemical, for providing a volume of nematodes of 0.1-50 litres, for genomics, for cell biology, for neuroscience, for aging, 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. la-b 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.

Fig. 3 shows an illustration of the Micro Flow Filter . DETAILED DESCRIPTION OF THE FIGURES In the figures:

100 HVBS 101 Controller AS Air Sparger

Cl controller CCI Controller Computer Interface DOS Dissolved Oxygen Sensor FLS Fluid level sensor GS gas supply IM Impellor IS Impurity separator LS light sensor

Ml Filter Support Plate with flow channels M2 Micro Mesh Plate M2 Filter Support Plate with flow channels MFF micro flow filter MP Mechanical pump pHC pH sensor and controller PS pressure sensor PR pressure regulator R1,R2 breeding reactor R3 collector reactor R4 hatcher reactor RIO Storage reactor

Rll Storage reactor

Rlx storage reactor lx 51 nutrient feedstock container 52 storage container 53 Cleaning Fluid Storage container/unit SO Sample output valve SW Switch valve TS Temperature Sensor VS Valve Switch

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

Fig. 2 shows a schematic overview of the life cycle of C. Elegans.

Fig. 3 shows an illustration of the Micro Flow Filter with two support plates (top + bottom) holding the Micro Mesh Plate in-between. For clarity the assembly is shown in an 'exploded' view without bolts, washers and gaskets. HVBS working example description:

After preparation of the HVBS system, loading an initial batch of nematodes, especially eggs, into the breeding reactors R1,R2, 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 alternate flushing the content of the two reactors (R1 and R2) over a micro flow filter MFF. By flushing smaller subpopulations, and in a specific example life stages of nematodes smaller than a typical adult size, are filtered out and subsequently collected in collector reactor R3. Multiple filtering cycles, in alternating direction (back and forth), using a controlled pressure difference between the two reactors, are executed. During filtering cycles a controlled counter pressure provided by the collecting third reactor (R3) is maintained. These filtering cycles result in a homogeneous population of adult nematodes in the main reactors R1 and R2, filtering out all smaller sub-populations, such as LI up to L4 nematodes and eggs, which smaller sub-populations are collected in the R3 collector reactor.

After a number of filter cycles, such as 5-100, depending on the volume of the reactors and the characteristics of the micro flow filter used, the main R1 and R2 reactors contain nematodes that have a cross section that is larger than the perforations (or openings) in the micro flow filter. The micro flow filter may contain a mesh. An (average) perforation size of the mesh as used in the micro flow filter is chosen such that only adult and some young adult nematodes will remain in the R1 and R2 reactors.

When the required level of homogeneous population is reached, which is established by little or none LI, L2, L3 and L4 stage nematodes being detected by sampling an output side of the micro flow filter, the micro filter output is switched using a valve switch SW towards an impurity filter system IS. Just before or just after the switch SW a sample tap SO and/or dedicated light sensor may be present, e.g. for determining the content status of the output flow. The IS filter separates the smaller sub-populations, such as the eggs, from any impurities and nutrients, bacteria, and cell residues, before the smaller sub-populations, especially eggs, are being transferred to a hatcher reactor R4. In the hatcher reactor R4 the eggs will be allowed to hatch and evolve into LI nematodes. A combination of the filter system IS and the hatching reactor R4 is also a feasible option. In addition or as an alternative eggs may after removing impurities be transferred to the final storage containers for hatching and freeing the HVBS for the next batch process; a cycle time may thereby be reduced to an average of about 4 hours.

After a period of time (up to 10 hours) most of the smaller sub-populations, e.g. eggs, in the hatching storage reactor R4, or optionally in the final storage reactors R10,Rll,Rlx, will have hatched into the first life stage 'Ll' of the nematodes. As there are no nutrients available for the LI nematodes to feed on, they will remain 'arrested'' and maintain their LI life cycle development stage; i.e. the nematode population is now synchronized and ready for use.

In a final stage the LI nematodes may be transferred and stored into 'storage bioreactors' RIO, Rll, Rx. Therein the LI nematodes can be kept without food (nutrients) for a limited amount of time, while the HVBS system may restart its breeding and/or filtering cycle. In addition or as an alternative eggs may after removing impurities be transferred to the final storage containers for hatching and freeing the HVBS for the next batch process; a cycle time may thereby be reduced to an average of about 4 hours. Depending on conditions, such as temperature, the 'shelf life' for the LI nematodes contained in the storage bioreactors is up to 48 hours. In order to maintain a continuous supply of 'fresh' LI nematodes, the HVBS system is setup to run in a continuous batch-process mode. Therein at regular intervals batched of 'fresh' eggs and/or ready to use LI nematodes are harvested and transferred to ready to use 'storage bioreactors'.

As mentioned above the HVBS system batch-process is setup and maintained in accordance with a required volume and scheduled use. Multiple final storage bioreactors (RIO, Rll, Rlx) may be required to assure a continuous availability of LI stage nematodes.

The figures have been detailed throughout the description .

Claims

A high volume culture and life cycle synchronization system (100) comprising at least two culture reactors (R1, R2) in at least one first fluid connection, in at least one of the at least one first fluid connection at least one micro stream filter ( MMF) with filter openings, a nutrient feedstock holder (S1) in second fluid communication with the at least one of the two cultures reactors, a collector reactor (R3) in the third fluid communication with the at least one microfilter, at least one oxygen supply (GS) in fourth fluid compound with at least one of the two culture reactors and with the collector reactor. (R3), and a controller (C1) for controlling and controlling fluid flow and pressure difference, such as (i) at least one fluid flow, (ii) a pressure difference over the at least one micro-flow filter, (iii) a pressure difference between at least two of the at least two culture reactors, and (iv) a fluid flow between the collector reactor and at least one culture reactor.

The culture system of claim 1, further comprising at least one supply container (S2) in fifth fluid communication with the at least one microcurrent filter (MMF).

A culture system according to any one of the preceding claims, further comprising at least one separator (IS).

A culture system according to any preceding claim, further comprising at least one incubator reactor (R4) and an oxygen supply for incubator reactor.

A culture system according to any preceding claim, further comprising a sixth fluid communication between the collector (R3) and at least one culture reactor (R1, R2).

A culture system according to any preceding claim, further comprising a cleaning unit (S3) in fluid communication with at least one element of the present system.

7. Cultivation system according to one of the preceding claims. wherein the at least two culture reactors comprise at least one of an air distribution device, an impellor, a conical bottom, a fill level controller, a pressure sensor, a pressure controller, a dissolved oxygen sensor, a pH sensor, a control unit, and a temperature controller.

A culture system according to any preceding claim, wherein the at least one microcurrent filter comprises uniform openings (0) 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.

The culture system of claim 8, wherein the at least one microcurrent filter comprises (ml) a first plate, the first plate having at least one predetermined fluid path, at least one inlet, and at least one outlet, (m2) a filter plate in contact with the first plate, (m3) a third plate in contact with the filter plate, the third plate having at least one predetermined fluid path, and an inlet and an outlet, and means for holding the plates together.

The culture system of any one of claims 8-9, wherein at least 10% of the microcurrent filter surface has no openings.

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

The culture system of any one of the preceding claims, further comprising one or more pumps (MP1-P10), at least one sensor (LS1-LS7) in a fluid connection for detecting a fill level of the culture reactor.

A culture system according to any one of the preceding claims, further comprising at least one storage reactor (R10, R11, R1x).

A method of operating a system according to any one of the preceding claims, comprising the steps of (1a) loading at least one culture reactor (R1, R2) with an egg-producing nematode species, such as C. elegans, (lb) loading of nutrients and oxygen, (2) providing nutrients and oxygen to at least one culture reactor (R1, R2), (3) flowing the fluid from the at least one culture reactor over the microcurrent filter (MMF) to at least one second culture reactor, wherein the at least one culture reactor is emptied, and reversing the flow at least once from the at least one second culture reactor to the at least one culture reactor, wherein the second culture reactor is emptied, under a controlled pressure difference between the at least two culture reactors of 1 kPa-200 kPa and a differential regulated pressure between at least one culture reactor and collector (R3) of 0.1 kPa-50 kPa causing early life phase (ei-L4) of the nematode speci es from (young) adult nematodes are separated, creating a homogeneous population of nematodes, (4) collecting at least one of eggs and L1-L4 nematodes in the separator, and (5) removing nutrients and contaminants from the collected at least one of eggs and L1-L4 nematodes.

A method according to claim 14, comprising (6) after removing nutrients, storing the eggs for incubation, and optionally (7), after a period of time, storing the population of nematodes (L1) or eggs in at least 10 hours at least one storage reactor (R10, R11, R1x).

The method of any one of claims 14-15, comprising the step of providing a back flush over the at least one microcurrent filter.

A method according to any of claims 14-16, wherein only eggs are collected, further comprising the steps (6a) moving and maintaining the eggs in an incubator for 2-12 hours under sterile conditions in the absence of nutrients, and providing oxygen, and (7a) incubating L1 nematodes.

An unbleached population of nematodes such as C. elegans obtainable by a method according to any one of claims 13-17, wherein the population comprises> 90% of L1, and wherein the population has a viability of> 90%.

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

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 agrochemicals, for providing a volume of 0.1-50 liter nematodes, for genomics, for cell biology, for neuroscience, for aging, or for high throughput screening.