HIGH VOLUME BREEDING AND LIFE CYCLE SYNCHRONISATION SYSTEM

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 neuroscience and aging.

Claims

1-20. (canceled)

21. A high-volume nematode breeding and life-cycle synchronization system comprising: at least one breeding reactor (R1), the at least one breeding reactor having at least one inlet arranged to receive fluids, and at least one outlet for removing nematodes, incorporated in the system, such as in the reactor, at least one micro filter (MF) with filter openings having a mesh size, a first space between the MF and at least one outlet of the reactor system arranged to receive nematodes, at least one first fluid recirculation connection (FRC) for collecting fluid from the breeding reactor and replenishing fluid to the reactor, the recirculation connection preferably comprising a nematode harvest micro filter (HF) with a mesh size smaller than the mesh size of the MF, wherein the recirculation connection comprises at least one valve (QC1,QC2) arranged for opening and closing a connection, at least one oxygen supply (GS) in fluid connection with the breeding reactor, and a controller (C1) for regulation and controlling operation.

22. The breeding system according to claim 21, additionally comprising a nutrient feedstock container (S1) in fluid connection with the breeding reactor.

23. The breeding system according to claim 21, wherein the controller (C1) regulates and controls one or more of the group consisting of fluid flow, temperature of the reactor, pH level, impellor speed, valve opening and closing, and on/off switching.

24. The breeding system according to claim 21, wherein the at least one breeding reactor comprises at least one of an air sparger (AS), an air filter (AF), an impellor (IM), a conical bottom, a fill level sensor, a pressure sensor, a pressure controller, a dissolved oxygen sensor (DOS), a pH sensor (pHC), a control unit, and a temperature sensor (TS).

25. The breeding system according to claim 21, wherein the at least one micro filter comprises uniform openings (O) with an average opening diameter (d) of 10-100 ?m, and a standard deviation 3? of <10% relative to the average, wherein the micro filter has a surface area (SA) of 10-5000 cm.sup.2.

26. The breeding system according to claim 21, wherein the at least one gas supply provides oxygen.

27. The breeding system according to claim 21, wherein the recirculation connection comprises at least one second nematode back-up micro filter (BF) with a mesh size smaller than the mesh size of the MF.

28. The breeding system according to claim 21, wherein the recirculation connection comprises at least one valve (QC) 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.

29. The breeding system according to claim 21, wherein 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.

30. The breeding system according to claim 21, wherein at least one micro filter comprises a cylindrical section, wherein the cylindrical section extends upwards to a level above a maximum fluid level, and wherein an external diameter of the cylindrical section is 0.5-50 mm smaller than an internal diameter of the reactor, taken at a similar height, and a plate section at the bottom of the cylindrical section with openings.

31. The breeding system according to claim 30, wherein a micro filter surface area comprises openings with an average opening diameter (d) of 15-80 ?m.

32. The breeding system according to claim 21, additionally comprising: at least two breeding reactors (R1,R2) in at least one first fluid connection, within at least one of the at least one first fluid connection at least one micro flow filter (MFF) with filter openings, and a collector reactor (R3) in third fluid connection with the at least one micro filter, wherein the controller (C1) is for regulation and controlling fluid flow and differential pressure.

33. The breeding system according to claim 32, further comprising at least one of: a storage container (S2) in fifth fluid connection with the at least one micro flow filter (MFF), a separator (IS), a hatcher reactor (R4) and an oxygen supply for the hatcher reactor, a sixth fluid connection between the collector (R3) and at least one breeding reactor (R1,R2), a cleaning unit (S3) in fluid connection with at least one element of the present system, a pump (MP1-P10), a sensor (LS1-LS7) in a fluid connection for detecting a fill level of the breeder reactor, and a storage reactor (R10,R11,R1x).

34. The breeding system according to claim 32, wherein at least one micro flow filter comprises: (m1) a first plate, the first plate having at least one predefined fluidic 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 predefined fluidic path, and an inlet and an outlet, and means for fixing the plates together.

35. The breeding system according to claim 24, wherein the impellor (IM) comprises one or more blades (B), preferably 1-6 blades, and an axis having a cross-section (cs) and centre point (cp) adapted for connection to a drive, wherein at least one out of mechanical balance blade (Bob) having a longitudinal axis (LA) that does not coincide with the centre point (cp) and wherein the at least one blade (Bob) extents in a direction parallel to the longitudinal axis at least over the cross-section (cs) of the central axis.

36. A method of operating a system according to claim 21, comprising the steps of: (1a) loading the at least one breeding reactor (R1) with an egg producing nematode species, (1b) loading nutrients and oxygen, (2) providing nutrients and oxygen to the at least one breeding reactor (R1), (3) flowing part of the fluid of the at least one breeding reactor through/over the micro filter (MF), thereby separating early life stage (egg-L4) of the nematode species from adult nematodes thereby creating a homogeneous population of nematodes, and (4) collecting at least one of eggs and L1-L4 nematodes.

37. The method according to claim 36, further comprising at least one step selected from the group consisting of: (5) harvesting nematodes from the harvest filter, (6) continuing operation until only eggs are produced, (7) harvesting eggs over a period of time in the range of 0.2-540 minutes, (8) providing a back-flush over the at least one micro flow filter, (9) removing nutrients and impurities from the collected at least one of eggs and L1-L4 nematodes, (10) after removing nutrients storing the eggs for hatching, and (11) after a period of time <10 hours storing the population of nematodes (L1) or eggs in at least one storage reactor (R10, R11, R1x).

38. The method according to claim 36, wherein (3) flowing the fluid of the at least one breeding reactor over the micro flow filter (MFF) is to at least one second breeding reactor, thereby emptying the at least one breeding reactor, and at least once reversing the flow from the at least one second breeding reactor to the at least one breeding reactor, thereby emptying the second breeding reactor, under a differential controlled pressure between the at least two breeding reactors of 1 kPa-200 kPa and a differential controlled pressure between at least one breeding reactor and collector (R3) of 0.1 kPa-50 kPa.

39. The method according to claim 36, additionally comprising the step of (1c) loading a sterile buffer in the buffer reactor (S2), and after step (3) and before step (4) step (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 thereafter repeating step (3), followed by steps (4) and (5).

40. The method according to claim 36, wherein only eggs are collected, and additionally comprising the steps (10a) moving to and maintaining the eggs in a hatcher for 2-12 hours under sterile conditions in the absence of nutrients, and providing oxygen, and (11a) hatching L1 nematodes.

Description

FIGURES

[0093] The invention although described in detailed explanatory context may be best understood in conjunction with the accompanying figures.

[0094] FIG. 1a shows an illustration of the present high volume breeding and life cycle synchronization system (100).

[0095] FIG. 1b shows an illustration of the Controller Input and Output control signals (101).

[0096] FIG. 1c shows a cross-section of the present high volume breeding and life cycle synchronization system (100).

[0097] FIG. 2 shows a schematic overview of the life cycle of C. Elegans.

[0098] FIG. 3a shows an illustration of the present high volume breeding and life cycle synchronization system (200).

[0099] FIG. 3b shows an illustration of the Controller Input and Output control signals (201).

[0100] FIG. 4 shows an example of an MFF 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.

[0101] FIG. 5a-j show schematics of the present impellor.

DETAILED DESCRIPTION OF THE FIGURES

[0102] In the figures:

[0103] 100 HVBS (single reactor)

[0104] 200 HVBS (multiple reactor)

[0105] 101/201 controller (single/multiple reactor)

[0106] AF air filter

[0107] AS air sparger

[0108] BF back-up filter

[0109] C1 controller

[0110] CCI Controller Computer Interface

[0111] DOS dissolved oxygen sensor

[0112] FLS fluid level sensor

[0113] GS gas supply

[0114] HF harvest (cartridge) filter

[0115] IM impellor

[0116] IS Impurity separator

[0117] LS light sensor

[0118] M1 Filter Support Plate with flow channels

[0119] M2 Micro Mesh Plate

[0120] M3 Filter Support Plate with flow channels

[0121] MF micro filter

[0122] MFF micro flow filter

[0123] MP Mechanical pump

[0124] PP peristaltic (or mechanical) pump

[0125] pHC pH sensor and controller

[0126] PS pressure sensor

[0127] PR pressure regulator

[0128] QC.sub.x quick coupler with integrated valve

[0129] RV regulator valve

[0130] R1,R2 breeding reactor

[0131] R3 collector reactor

[0132] R4 hatcher reactor

[0133] R10 Storage reactor

[0134] R11 Storage reactor

[0135] R1x storage reactor 1x

[0136] S1 nutrient feedstock container

[0137] S3 Cleaning Fluid Storage container/unit

[0138] SO Sample output valve

[0139] SN Spray Nozzle

[0140] SW Switch valve

[0141] TS temperature sensor

[0142] V.sub.x valve

[0143] VS Valve Switch

[0144] In FIG. 1c certain elements of the present system are indicated, such as a spray nozzle SN for distributing fluids evenly, an impellor IM, the micro filter MF, a peristaltic pump PP, a UV light source UVS, the reactor R1, and the harvest filter HF.

[0145] In FIG. 5a a schematic side view of the present impellor is shown. Therein an axis, having a central virtual axis, is shown. A blade Bob is attached to the axis, typically at a bottom end thereof. The blade is preferably rotated over an angle 20-70? relative to the central virtual axis. The blade has a (virtual) longitudinal axis.

[0146] In FIG. 5b a schematic bottom view of the present impellor is shown. Therein an axis having a central point cp, a blade Bob having a (virtual) longitudinal axis, a second blade B attached in a symmetric manner to the axis, and a cross section cs. The blade Bob may be attached such that the cross section is relatively small, or may be attached close or above the cp, thereby having a large cross section.

[0147] In FIG. 5c a front view is shown wherein it is shown that the blade B and/or Bob may be rotated an angle 20-70? relative to the central virtual axis.

[0148] In FIG. 5d a bottom view of an impellor with a larger blade Bob and a smaller blade B is shown. The Bob blade is asymmetrical. Both blades extent downwards with respect to the lowest point of the axis. Both blades have a flattened section at a side closest to the bottom (in operation).

[0149] In FIG. 5e a side view of FIG. 5d is given. In FIGS. 5f and 5g a perspective view of FIG. 5d is given. In FIG. 5h a perspective view of the impellor of FIG. 5d is given; similar views are given in FIGS. 5i and 5j, having four and seven blades respectively.

HVBS Working Example (1) Description

[0150] After preparation of the HVBS system 100, loading an initial batch of nematodes 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

HVBS Working Example (2) Description

[0155] After preparation of the HVBS system 200, loading an initial batch of nematodes 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 sub-populations, 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 be-tween 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 L1 up to L4 nematodes and eggs, which smaller sub-populations are collected in the R3 collector reactor.

[0156] After a number of filter cycles, such as 5-100, de-pending 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 (aver-age) 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.

[0157] When the required level of homogeneous population is reached, which is established by little or none L1, 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 L1 nematodes. A combination of the filter system IS and the hatching reactor R4 is also a feasible option.

[0158] 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,R11,R1x, will have hatched into the first life stage L1 of the nematodes. As there are no nutrients available for the L1 nematodes to feed on, they will remain arrested and maintain their L1 life cycle development stage; i.e. the nematode population is now synchronized and ready for use.

[0159] In a final stage the L1 nematodes may be transferred and stored into storage bioreactors R10, R11, Rx. Therein the L1 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. De-pending on conditions, such as temperature, the shelf life for the L1 nematodes contained in the storage bioreactors is up to 48 hours. In order to maintain a continuous supply of fresh L1 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 L1 nematodes are harvested and transferred to ready to use storage bioreactors.

[0160] 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 (R10, R11, R1x) may be required to assure a continuous availability of L1 stage nematodes.

[0161] The figures have been detailed throughout the description.