WO2006097749A1

WO2006097749A1 - Fluidic devices for cell and embryo culture

Info

Publication number: WO2006097749A1
Application number: PCT/GB2006/000969
Authority: WO
Inventors: John Robert Dodgson; Malcolm Austen
Original assignee: Robio Systems Ltd
Priority date: 2005-03-16
Filing date: 2006-03-16
Publication date: 2006-09-21
Also published as: GB0505378D0

Abstract

A device for culturing/maturing cellular entities comprises a substrate having one or more wells, the one or more wells being adapted to hold a cellular entity in fluid, one or more fluidic channels for fluidic communication with the wells and one or more restriction means for restricting the movement of the cellular entity. The restriction means is porous and may comprise a hydrophilic or hydrophobic sintered polymer formed by in-situ polymerization.

Description

FLUIDIC DEVICES FOR CELL AND EMBRYO CULTURE

[001 ] This invention relates to a system and method for culturing cells, oocytes, embryos, maturing ova or other cellular structures in vitro. It also relates to means for transportation of cells, ova, embryos, oocytes or other cellular structures or entities.

[002] Various apparatus and methods are known for maturing ova and culturing embryos in vitro. In standard practice these processes are achieved using conventional tools such as pipettes for manipulation of an ovum or embryo, and Petri dishes to contain the ovum or embryo and maturation or culture medium. The ova or embryos are usually cultured in an incubator in conditions of controlled temperature and gas environment. They may be cultured singly or in groups, and for ova in particular, may be cultured in the presence of other cells, such as cumulus cells. Maturation or culture is often done in microdrops of medium in a Petri dish, the medium covered by an inert oil, the dish having gas access to the environment in the incubator. In some conventional maturation or culture procedures the volume of the medium environment in which the ovum or embryo is contained is important - there is evidence in some methods that maturation and culture is more successful if several ova or embryos are present together in a small volume of medium. This autocrine effect is thought to result from trace chemical substances produced by a first ovum or embryo affecting the development of a second. However, it is also advantageous in certain circumstances to track the identity of individual ova or embryos and conventional apparatus in general does not allow the embryos or ova to be kept separate while allowing exchange of chemical substances between them. The well-of-wells (WOW) method of Vajta et al. as disclosed in WO 0 102 539 allows this to be done, but does not close the wells against exit of the embryos and so is not suitable for use in a transportable device. [003] The medium is usually buffered against changes in pH; this buffer may be based on bicarbonate / CO₂, in which case the partial pressure of CO₂ in the external gaseous environment is important, and it may be based in whole or part on other buffer systems, for example HEPES, in which case the gaseous environment may be less closely controlled or in some circumstances not controlled at all. The medium may be of nominally constant composition during maturation or culture, or may be changed, for renewed media of the same nominal composition, or a new medium to modify the medium conditions in order for example to assist or control the process of maturation or culture. In particular, in certain methods for culture of embryos it is known to be advantageous to culture the embryos initially in serum-free medium, changing to medium containing serum (often fetal calf serum, FCS) later in culture. In the case of maturation of ova, it is known that the progress of maturation may be controlled by addition of species to the maturation medium or their removal from it by replacing the medium with fresh medium. This may be particularly advantageous if the ova or embryos are to be transported during the maturation or culturing process, for example from a location at which the ova are harvested or the embryo created, and a second location where the ova might be used or the embryo implanted. Conventionally medium is changed by moving the ovum or embryo by pipetting from one medium to another, for example from one microdrop to another in a common culture dish. This uses simple apparatus but suffers from several disadvantages: the ova and embryos are delicate and can be damaged by pipetting; an amount of medium is necessarily transferred from one medium environment to another, which is significant especially in the small volume of a microdrop, and gives the possibility that substances from the old medium active at very low concentrations may be transferred into the new medium, unless sequential washing steps are used; the transfer process is slow and requires skilled personnel; and the transfer cannot be done remotely, so cannot be done in transit or outside a fully equipped laboratory setting. [004] In the description that follows reference will be made to culture of embryos as an example of the function of apparatus and description of the method. Many of the processes can also be applied to maturation of ova and culturing of cells or other cellular entities and it will be apparent to those skilled in the art how this application can be made, with appropriately chosen dimensions for the different size scales of embryos, ova and cells. Therefore the terms maturation and culturing, and ova and embryos and cells, are used interchangeably in the following and where convenient referred to collectively as Objects'. Where specific features of the invention apply to maturation of ova, or to culturing of embryos, this will be noted.

[005] A number of apparatus and methods have been proposed to alleviate these and other problems in the conventional art.

[006] Beebe et al. US 6 193 647, US 6 695 765 have proposed a system of approximately embryo-sized microchannels in which the embryos reside, being located at a constriction within the microchannels by entrainment in flow along the channels, that flow causing them to roll along the channel in contact with one of the channel walls. This apparatus achieves close control of the medium environment of the embryo, but suffers from the disadvantages, among others, that it does not provide a means of positive location of the embryo against flow of the medium in the reverse direction, which tends to move the, embryo away from the constriction; it does not provide ready means of gas exchange between the medium and an external gas environment, and does not provide a ready means of storage of a number of embryos in individual locations while tracking their identity - i.e. it is possible in the apparatus and method of US 6 193 647 for the embryos to move from one retention position to another, so losing information as to their identity. No adaptation is disclosed which will make the apparatus suitable for use in transportation, in which potential problems of the embryos moving under gravity or motion will arise. [007] Campbell et al. US 2002 0 068 358 have proposed an apparatus for embryo culture which is adapted for transportation, in which the embryo is retained in a well which is capable of being closed in such a way that the embryo is positively retained, and which has a supply of medium and flow generating means which allows the medium in the well to be replaced under remote or automatic control. US 2002 0 068 358 also discloses means to monitor and/or control parameters in the medium or the well, such as temperature; pH, and chemical constituents, though details of the apparatus showing exactly how this is to be achieved are not disclosed. The apparatus and method of US 2002 0 068 358 are poorly adapted to shipping a number of embryos in a controlled chemical environment while keeping track of their identity - there is no means of segmenting embryos in a common well or wells; the well is considerably larger than the embryo, so giving poor control of the medium environment and a long time and large volume of medium for complete exchange of a first medium for a second; access to the well is down a long inlet tube or by entrainment in a microchannel and cannot readily be achieved using conventional pipettes; the design is not suitable for use with conventional microscopy.

[008] Thompson et al., US 6 673 008, disclose a method and apparatus for culturing of embryos in which the embryo is cultured in medium in a tank, the tank being supplied with medium from one or more reservoirs, and optionally provided with sensors for, for example, temperature, pH, dissolved O₂, ions in solution or metabolic products from the respiration of the embryo, allowing the medium around the embryo to be changed in response to conditions in the medium or to a programme stored in a control unit. The apparatus as disclosed in US 6 673 008 comprises macro-scale devices enclosing a significant volume of solution, and the tanks of the invention are of large volume (10-50 ml), so requiring an even larger volume of medium in order to replace a first medium with a second. The device is not self-contained, in that it uses separate reservoirs and flow system components external to the apparatus and is not adapted for transportation. No means of gas (CO₂, air) perfusion of the embryos inside the tank is disclosed, except by means of flow of newly gas- enriched medium from the reservoir. In a practical transportation apparatus, the size of the apparatus and hence the volume of medium surrounding the embryo is advantageously smaller than specified in US 6 673 008, and so a means to allow gas equilibration with the medium around the embryos is preferred.

[009] Van den Steen et al., US 2004 0 234 940, disclose a micro-chamber arrangement for development of embryos that allows flow of medium through a chamber based on a stacked array of sieve-like components that retain embryos in individual compartments. The embryos are located in the compartments and the stack of sieve-like components is then assembled to enclose them. The compartments are illustrated as being approximately embryo-sized, but the illustration in US 2004 0 234 940 is purely schematic and no means is disclosed of fabricating such a structure. No lid or other means of closure is disclosed that will allow transportation of the apparatus.

[0010] Vajtaet al. WO 0 102 539 disclose a method of culturing embryos in an array of small wells located at the base of a larger well (known as the well-of-wells method). This allows embryos to be located separately in a common medium, but does not include means to retain the embryos in situ if the medium or the device comprising the well is disturbed. Consequently it is unsuitable for transport of embryos outside the laboratory environment. Also, as the method is based on an open well, it relies on exchange of gas from, and heating by, the environment in an incubator. Further, no means is disclosed of changing the composition of the medium other than by pipetting the medium into and out of the larger well.

[001 1 ] Seidel et al. US2004 0 132 001 disclose transport of ova or embryos in capillary-like straws, as used for embryo transfer, the straw having optionally sealed ends, and in which the maturation or culture process can take place during transport, but this does not allow for exchange of gas or medium during transport.

[0012] Transport devices for embryos or ova are known, for example as manufactured by Cryologic Pty (Australia) (www.cryologic.com, www.bioqenics.com) which maintain constant temperature during transport over a period of hours or days, but which can not maintain a constant gaseous environment for exchange with medium in the inner containment. The inner containment is typically in the form of vials, straws or capillaries and again there is no means for exchange of medium during transport.

[0013] A further feature of devices of the prior art in which the environment of the objects is to be changed by flow of medium around them is that they require a form of containment which holds the objects in place in the flowing medium. Conventionally this is done by e.g. a constriction in a microchannel, as in Beebe et al US 6 193 647 or a mesh or filter in the base of a well, as in Campbell et al. US2002 0 068 358 and van den Steen et al US2004 0 234 940. The constriction as shown in Beebe et al is sized to prevent a large cellular object such as an embryo or oocyte from passing, but the fabrication methods disclosed, such as etching into a step into a silicon substrate, are difficult to adapt for smaller objects such as individual cells. , Additionally there is no means disclosed in that work to locate the objects against back-flow or sedimentation under gravity away from the constriction should the device be moved or held away from horizontal. The mesh or filter base in a well as in Campbell et at, Thompson et al US6673008 is well known in the art of microwell plate-based cellular assay and will work to retain objects of all sizes; however, it has the disadvantage that it does not allow good visibility from below, which is a prerequisite for accurate visual inspection of the objects in a number of biotechnical operations, for example in embryology. Further, no ready means is disclosed in these references for fabrication of the mesh-based well. [0014] The disadvantages with the devices of the prior art described above can be overcome by an apparatus of the invention, designed so as to include a porous element in a fluidic pathway, the pores being sufficiently small that the object cannot enter them, the porous element being located so as to prevent the object from leaving the well wherein it is placed, while allowing flow of medium along the fluidic pathway through the pores. Various embodiments will be described below which illustrate the principle, and a method of culturing objects based on the principle will be described.

[0015] According to the invention, there is provided a device as specified in claims 1 to 15.

[0016] According to a further aspect of the invention there is provided a device for culture of a cell, embryo or other cellular structure (hereinafter referred to as an 'object') comprising:

a base comprising one or more wells open to a surface of the base;

a fluidic pathway comprising: a well, at least one fluidic channel in fluid communication with the well;

an inlet port;

a porous material forming part of the fluidic pathway, located so that the object can not move along the fluidic pathway away from the well;

a lid which seals against the base and closes the well to prevent the object from leaving the well.

[0017] The porous material may be formed from polymer, ceramic, metal, glass or a mixture of these. It is preferably a solid material containing pores but may alternatively be formed from an accumulation of particles held in place in a given location in the device, in the manner of a filter bed. Preferably the porous material is a hydrophilic polymeric porous material, for example formed from sintered particles, such as sintered polypropylene for example material marketed by Porvair Ltd., Wrexham UK as 'VYON' TM. Alternative suitable materials may be made by co-polymerisation of two or more materials followed by further post-polymerisation treatment, leaving pores in the solid mass, which may optionally be done in-situ in the device (see for example Mutlu S. et al., Micromachined porous polymer for bubble-free electro- osmotic pump, Proc. IEEE MEMS2002, Las Vegas, pp.19-24). A variety of solid polymeric filter materials is available, many of which may be used in the device of the invention by the fabrication methods to be outlined below or by adaptations of them, within the scope of the invention.

[0018] In alternative embodiments the porous material is hydrophobic, for example 'VYON' ™ without treatment to render it hydrophilic. A hydrophobic material will wet under liquid pressure and will tend to pass any gas bubbles in a liquid media easily. The degree of hydrophilicity / hydrophobicity of the porous material is chosen for each embodiment to give a preferred balance between ease of initial filling and ease of passing gas bubbles through the material.

[0019] Preferably the well is bounded in whole or in part by a porous element and a flow channel is in fluidic communication with the opposite side of the porous element, the porous element acting to allow flow of medium into or out from the well while retaining the object.

[0020] More preferably the well base is formed from a transparent material, so allowing observation from below using an inverted microscope.

[0021 ] Preferably the well is defined by a porous element in ring form in the base of the device, the bottom of the well closed by a transparent material, the top of the well closed with a removable lid, a fluidic channel in fluid communication with all or part of the outer circumference of the porous element so that medium may flow from the well, through the porous element and along the channel, or vice-versa. In use the objects will rest on the transparent base and cannot exit the well into the channel.

[0022] Alternatively the well is formed as a depression in a porous element, the objects then resting on the porous material in the bottom of the well.

[0023] In preferred embodiments the device comprises two fluidic channels forming fluidic pathways into the well, both pathways comprising a porous element, so allowing medium to flow through a first channel, through a first porous element, through the well and into the second channel, which comprises a second porous element. In use, the first and second porous elements act to prevent movement of objects from the well along either the first or the second fluidic channels.

[0024] In some embodiments the well is formed wholly or partially within a porous element, one region of the porous element appearing in the fluidic pathway from the first channel, another region appearing in the fluidic pathway from the second channel.

[0025] In embodiments of this type the well is preferably defined by a porous element in ring form in the base of the device, the bottom of the well closed by a transparent material, the top of the well closed with a removable lid, with the first and second fluidic channels disposed in the base so that in use medium may flow into the well through one region of the ring and out of the well through another.

[0026] Alternatively the well is formed as a depression in a porous element, the objects then resting on the porous material in the bottom of the well. The first and second channels may then be in fluidic communication with regions of the circumference and/or the base of the well. [0027] Preferably the first and the second fluidic channels are in fluidic communication with the well near the bottom of the well, the fluidic pathway between the first and the second channels through the well being substantially perpendicular to the axis of the well.

[0028] Alternatively the first fluidic channel is in fluidic communication with the well near the bottom of the well, the second fluidic channel is in fluidic communication with the well near the top of the well, the fluidic pathway between the first and the second channels through the well being substantially parallel to the axis of the well.

[0029] Preferably the well is partially defined by two porous elements, each in fluidic communication with a flow channel, one disposed near the base of the well and one near the top.

[0030] Preferably the lid comprises a porous element and when in position on the base completes a fluidic path from the well through the porous element to an inlet port.

[0031 ] The base of the device may be formed by co-moulding a non- porous polymer around or over a porous element such as a subcomponent of porous polymer, or a region of porous polymer on or within a main component of non-porous polymer.

[0032] The porous elements are preferably formed as one or more separate subcomponents that are inserted into the material of the base during assembly, that material being wholly or predominantly non-porous, and which defines the fluidic channels. The porous elements may be formed from a deformable material that may be held in place by compliance after a push-fit, or by adhesive bonding, heat bonding, ultrasonic welding or similar bonding means as known in the art. The device may be designed so that the porous element may be inserted into an open recess into a body component of the base, the recess then being closed with a further component. Many polymeric porous materials are compliant and so can be inserted in this way. If necessary the material may be heated to increase its deformability, subject to the pores not becoming occluded. Alternatively the body component of the base in the vicinity of the well may be formed from a compliant material, for example PDMS, into which a less-compliant porous material may be inserted and held in place. The whole body component of the base may be formed from the same material, or a compliant material may be provided surrounding a well location, which acts to hold the insert in place.

[0033] The insert comprising the porous material may be of uniform composition, or may have regions of greater and lesser porosity and hence capability to pass fluid flow, or may have regions which are impermeable. The insert may be formed by co-moulding porous and non-porous polymer; the process of formation of the porous polypropylene VYON TM as referred to above allows this to be done readily. Therefore in some embodiments a portion of a fluidic channel in communication with a porous element is defined in part by a non-porous region of the insert, or may be defined wholly by it, for example as a channel running through the insert.

[0034] Preferably, the insert is formed of co-moulded porous and non- porous regions, the non-porous regions acting in use to limit access of liquid medium into regions of the insert away from the fluidic pathway.

In a preferred embodiment the portions of the insert which are adjacent to fluidic channels in the body component of the base are left porous , allowing medium to flow through them, while the rest of the insert is either formed from a non-porous polymer, either co-moulded or bonded to the porous polymer, so limiting the flow region within the insert.

[0035] Preferably, the material of the insert may be treated or processed so as to vary its properties from one region to another, for example to block the porosity in some regions or to render some regions more or less hydrophilic or hydrophobic, so limiting access of aqueous medium to certain regions of the insert.

[0036] Preferably the well is partially defined by a porous element that acts as a passageway for gas molecules to reach the contents of the well. This porous element may be in addition a further element that acts as a fluidic pathway for medium, or may be a region of a porous element other regions of which act as a pathway for the medium. The gas passageway may be a portion of a porous material element left hydrophobic while the remainder is treated to render it hydrophilic, or rendered hydrophobic while the remainder is hydrophilic. In a similar way, such a porous element may be provided to act as a gas passageway for gas to exit the fluidic pathway, for example to remove gas bubbles during initial filling of the pathway with liquid medium or later operation if a gas bubble should enter entrained in flowing medium.

[0037] In an alternative embodiment, the insert is designed to be a self- contained unit which comprises a well adapted to contain the object, the well wholly or partially defined by a porous material which is chosen so that when the insert is filled with medium and removed from the device, the pores of the insert act as a capillary stop and so the medium is held within the well. This arrangement allows the inserts to be removed from the device and handled separately from it, for example for microscopy or other analytical procedures. In this embodiment the design differs from the prior art design of Thompson et al. US6673008 in which an embryo is housed in a container within a tank containing medium, in which the container is significantly smaller than the tank and so controls exchange of medium between the contents of the tank and the contents of the container. In the present embodiment the insert is intended to be a close fit to the device, so there is little or no medium surrounding the insert and the predominant effect is flow through it, rather than interdiffusion between the media. [0038] In all of the above embodiments, where more than one well and associated flow channel are present, the porous elements forming part of each fluidic pathway, or may be formed in common, that is a common region of porous material might function as the porous element in a number of fluidic pathways. In particular, a porous component might have more than one well formed within it, providing one or more fluidic pathways into each well and preventing objects in each well from moving along those pathways.

[0039] In preferred embodiments the common region of porous material is formed from a substantially planar subcomponent that is mounted on or bonded to the base, the base and the subcomponent together defining one or more fluid flow pathways through the device, at least one pathway comprising a region of the porous subcomponent. The subcomponent may be formed wholly or substantially from the porous material, and may be processed, for example machined, treated, filled, embossed, compressed or hot-blocked, so as to define regions of greater, lesser or essentially zero porosity or altered hydrophilicity. Such regions might themselves form wells, channels etc. as described above or might define these in combination with the base component.

[0040] In alternative embodiments the device comprises a first component comprising a non-porous substrate material on which one or more regions of porous material are formed or mounted, and a second component which is mounted on or bonded to the first, the first and second components together defining fluid flow pathways through the device, at least one pathway comprising one or more porous elements formed from a portion of a porous material region. Preferably the porous material is in the form of one or more raised regions formed or mounted on the substrate material, and around which the second component is formed or fitted. The fluid flow pathways might comprise channels formed in the first component, the second component, or both. [0041 ] In preferred embodiments the porous material is moulded onto the non-porous base material so as to form regions that will define the one or more wells and flow pathways. Alternatively the porous material is moulded, laminated or bonded to the non-porous substrate as a planar sheet to form an assembly of two or more layers, and the assembly processed, for example machined, treated, filled, embossed, compressed or hot-blocked, so as to form such regions.

[0042] In preferred embodiments, the porous subcomponent or first component as above are detachable from the base or second component, so as to allow handling of the subcomponent or first component separately from the rest of the device. This has application in e.g. filling the device with medium, locating entities within the wells, microscopy and the like.

[0043] In further preferred embodiments, the fluid flow pathways are substantially within the porous regions, and include regions of porous material shaped so as to direct flow preferentially through the porous material. In preferred embodiments the flow channels leading to the one or more wells comprise porous material, the porous material being either bounded by an impermeable barrier such as a treated edge to the material, by a non-porous material adjoining one or more faces, or may be unbounded on one or more faces, the fluid being retained within the porous material by capillarity.

[0044] In all of the above embodiments, where a fluid pathway is referred to through the porous element, it is to be understood that the fluid pathway may comprise either flowing fluid or stationary fluid, in which case the fluidic pathway allows diffusion of species through the pores. For example, in embodiments in which a fluid flow path is provided leading to a well, it is within the scope of the invention that the flow path might cause substantially all or part of the fluid to flow past or around the well, so allowing diffusion of species from the fluid in the flow path, through the porous element and into the well and vice versa, while retaining the contents of the well against flow. [0045] The wells for the cellular entity can be of any form provided that they form a designated area for retaining the cellular entity.

[0046] Figure 1 shows a partial vertical cross section of a first embodiment of a device according to the invention.

[0047] Figure 2 shows a partial vertical cross section of a second embodiment of a device according to the invention.

[0048] Figure 3 shows a partial vertical cross section of a third embodiment of a device according to the invention.

[0049] Figure 4 shows a partial vertical cross section of a fourth embodiment of a device according to the invention.

[0050] Figure 5a shows a partial vertical cross section of a fifth embodiment of a device according to the invention.

[0051] Figure 5b shows a plan view of the bottom of an insert which forms part of the embodiment of figure 5a.

[0052] Figure 6 shows a partial vertical cross section of a sixth embodiment of a device according to the invention.

[0053] Figure 7 shows a partial vertical cross section of a seventh embodiment of a device according to the invention.

[0054] Figure 8 shows a plan view of the bottom of an insert which forms part of the embodiment of figure 7.

[0055] Figure 9a shows a partial vertical cross section of an eighth embodiment of a device according to the invention.

[0056] Figure 9b shows a plan view of the bottom of an insert which forms part of the embodiment of figure 9a. [0057] Figure 10 shows a plan view of a ninth embodiment of a device according to the invention.

[0058] Figure 1 1 shows a vertical cross section of a the embodiment of figure 10.

[0059] Figure 12a shows a partial vertical cross section of a tenth embodiment of a device according to the invention.

[0060] Figure 12b shows a partial plan view of some components of the embodiment of figure 12a.

[0061 ] Figure 13 shows a partial vertical cross section of an eleventh embodiment a device according to the invention.

[0062] Figure 14 shows a partial vertical cross section of a twelfth embodiment a device according to the invention.

[0063] Figure 15a shows a first partial vertical cross section of a thirteenth embodiment a device according to the invention.

[0064] Figure 15b shows a plan view of some components of the embodiment of figure 15a.

[0065] Figure 15c shows a second partial vertical cross section of the embodiment of figure 1 5a.

[0066] Figures 16 a, b and c and 17 a, b and c show further embodiments according to the present invention.

Detailed description of preferred embodiments

[0067] Figure 1 shows a first embodiment of a device according to the invention, which comprises a base 12 and a lid 14, the base comprising a well 20 open to the upper surface of the base, adapted to contain one or more objects 24. A fluidic channel 30 opens to the base of the well, and forms a fluidic pathway from a port 40, through the channel 30 to the well 20. The base 12 comprises a body part 15 and a substrate 13 bonded to it, which acts to close the base of the well 20 and the channel 30, though other constructions are possible. The substrate 13 is preferably transparent to allow observation of the object in the well. A porous element 50 is disposed so that it forms part of the fluidic pathway and acts to prevent the object from leaving the well along the channel 30. The well 20 comprises an inner portion 22 of smaller minimum dimension and an outer portion 26 of larger dimension, though the well may be of any other shape in either cross-sectional plane. In this embodiment the lid 14 is advantageously of stepped profile so that it occupies part at least of the outer portion of the well, so reducing the volume of the well and acting in use to reduce the volume of medium bathing the object(s) and to speed up exchange of one medium by a second medium when flow is applied. Optionally a further recess region 28 is provided in the base 12 to assist fitting of the lid in the case that the region 26 is too small to give positive location. Equally, the lid may be formed differently and in some embodiments may comprise a planar surface that seals to the major surface of the base. The port 40 is shown in figure 1 in a form suitable for forming a fluidic connection with an external connection means 60, comprising a fluidic channel 62 such as the bore of a capillary or pipette on a robotic pipettor, and a location means 64 that locates the fluidic channel 62 in communication with the port and forms a seal to a surface 66 of the device surrounding the inlet port. This arrangement allows positive and negative pressure to be applied at the port 40 in order to move fluid in one or the other direction on the device. Other connection means are within the scope of the invention, and it is within the scope of the invention that fluidic connections might be made in a similar way to the well 20 itself once medium comprising an object has been pipetted into the well. [0068] The porous element 50 may be formed or mounted in the base by a number of means. In a preferred embodiment it is formed as an insert which fits into a recess in the body part 15, the insert being held in place by a mechanical fit, assisted by one or both of the insert and the body part 15 being compliant to a degree and sized to be a tight fit. Substrate 13 is then bonded to the body part 15. In some embodiments the insert is held in place by substrate 13. At least in the case of larger objects 24, it is not necessary that the insert have a very tight fit to the recess; merely that any gap is small enough that the objects cannot pass through it. The insert might be held in place by adhesive bonding, ultrasonic bonding, heat bonding, solvent welding or other joining methods.

[0069] In the case that the device 12 comprises more than one well and associated fluidic channel 30, each well might open to a separate port, or might open to a common port by means of additional fluidic channels in fluid communication with the channels.

[0070] Figure 2 shows a further embodiment of the invention where parts are labelled in common with figure 1 , comprising a well 20 in fluidic communication with two fluidic channels 30, 32, each forming a fluidic pathway into the well and in fluid communication with a port 40, 42. The fluidic pathway through channel 30 comprises a porous element 50, and the fluidic pathway through channel 32 comprises a porous element 52. In this embodiment a porous insert 54 defines the inner region 22 of the well and the porous elements 50, 52. In a preferred embodiment the insert 54 is in the form of a ring, optionally with a tapered upper inner diameter as shown, the ring fitting into a matching recess in the body component, and sized so that when the substrate 13 is bonded to the body component there is no, or a suitably small, gap between the insert and the substrate. Connection to the port 40 is shown using a connection means as in figure 1 , except that in this case it is clarified that a pressure seal (if present) may be formed to the major surface of the base as shown at 66.

[0071 ] In use, liquid medium may be dispensed into the well 20, comprising one or more objects. The insert 54 preferably comprises hydrophilic porous material, which will then wet spontaneously with the medium. Under suitable circumstance the medium may spontaneously move into the fluidic channels 30, 32, but this is not necessary for operation of the device. Negative pressure applied to the port 40 or 42, or positive pressure applied to the well 20, will then force medium along one or both channels 30, 32 and to the ports 40, 42. The lid 14 is adapted for use to close the well, preferably displacing excess medium from the well as it is applied, into one or both of the ports. The device is then ready for use, and medium may be flowed from one port to another by means of appropriate fluidic connections, medium and pressure or pumping means. Alternatively the insert 54 comprises hydrophobic porous material, and preferably medium is flowed through channels 30 (and 32 where present) to wet the porous elements before medium and objects are dispensed into the well.

[0072] Figure 3 shows a further embodiment similar to those in figures 1 and 2, but now with two separate inserts 50, 52, disposed in fluidic pathways through channels 30, 32 as before. The same assembly processes as before may be used. Additionally, different fluidic connections to the channels 30, 32 are provided, in the form of inlet and outlet tubes 301 , 302 to which flow lines may be connected.

[0073] Figures 4-9 show partial diagrammatic views of the well and the porous element of embodiments of the invention in which the lid 14 is shown as a simple planar structure sealing to the base 12. The embodiments may have features in common with other embodiments disclosed herein. In all of these the porous elements may be formed as integral part of the base 12, or as regions in an insert, or in more than one insert either mounted together before assembly into the base or mounted separately.

[0074] Figure 4 shows a further embodiment in which the second fluidic channel 32 opens to the upper region 26 of the well 20. The device comprises a second insert 70, preferably in the form of a ring, which provides a porous element in the fluidic pathway from channel 32 as before. The second insert can be mounted on the base 12 in one of the ways mentioned earlier for the first insert.

[0075] Figures 5a and 5b show a further embodiment in which the device comprises an insert 54 which defines a greater part of the well than in figure 4, and in a preferred embodiment may define the whole of the side walls of the well, the top and the bottom of the well being closed by the lid and the substrate 13 of the base component respectively. This has the advantage that the insert is a larger component, more easily fabricated and assembled into the device. Here the channels 30, 32 communicate with the lower portion of the insert and through it to the lower region of the well. The porous element in the fluidic pathway from channel 30 into the well may be the full thickness of the insert, as shown by portions 72 plus 74, or the insert may be formed with an open channel across part of its base in the region of 74, so limiting the porous element to portion 72, and hence reducing the pressure drop through the insert.

[0076] Likewise, the porous element in the fluidic pathway from channel

32 may be the full thickness of the insert as shown by portions 76 plus 78 or may be formed with an open channel across parts of its base in the region of 78.

[0077] The upper part 80 of the insert does not form part of a flow pathway, but may have the tendency to act as an elongated part of the fluidic pathway or as a fluid reservoir. A gas channel (not shown) might communication with part of the insert to provide gas access to medium in the well. The porosity in the upper region 80 might be blocked with filler or by deformation or coated in order to reduce this tendency. A preferred approach is to define different areas of porous and less- or non-porous material in the same insert, for example by comoulding or laminating the materials. This is readily achieved if the porous and the non-porous material are the same, for example both are polyethylene or polypropylene.

[0078] Figure 6 shows an embodiment as in figure 5, but with the second channel 32 communicating with the upper region of the insert, part of the wall of the insert forming the porous element 52 in the fluidic pathway.

[0079] Figure 7 shows an embodiment in which the lower region 82 of the insert 54 is porous polymer and the upper region 80 is non-porous; the fluidic pathway then passes only through the lower region. A further embodiment is to limit the portion of the lower region 82 that is porous; figure 8 shows the bottom of an insert 54 in which the porous material 86 is shaped to define the fluidic pathway to the well and the non-porous material 84 occupies the region away from the desired pathway.

[0080] Figures 9a and 9b show a preferred embodiment in which the porous material 86 is moulded only in regions of the fluidic pathways, to form porous elements 88, 90 and in which a channel 92 is provided leading to each as at 74 in figures 5 and 6.

[0081 ] In all the above embodiments with inserts that open to the upper major face of the base, the insert may be inserted from that face of the base. It may contact the substrate 13 of the base or may seat on a layer of other material, for example a layer of the material of the body component 15, or a seal material formed on the substrate 13 surrounding or over the bottom of the well. In this way the insert may be placed after the body component and the substrate have been bonded. [0082] Figure 10 shows a plan view of the underside, assuming a transparent base substrate 13, and figure 1 1 a cross-section at E-E in figure 10 of an embodiment in which multiple wells are fed from a common first channel 30 and have outlets to a common second channel 32 (or vice versa), with the fluidic pathways from the first channel through each well to the second channel comprising a porous element as described for the previous embodiments. The device 100 comprises a number of wells (four are shown but any number may be provided), each partially defined by an insert 54, each well in fluid communication with a first channel 30 through a first porous element 88, and the second channel 32 through a second porous element 90. Each well is closed by a lid such as that shown at 14 in figure 1 1 . The first channel is open to one or more ports 40, 41 , and the second channel to ports 42, 43 the bases of which are shown in figure 10. In a preferred embodiment either the first porous element 88 defines a region of the wall of the first channel 30 or the second porous element 90 defines a region of the wall of the second channel 32, or both. The porous elements may be formed separately or may be formed together, for example on the substrate 13.

[0083] One method of operation of this embodiment is as follows: first the wells are closed with the lids 14, then the first channel 30 is filled from a medium source at port 40 to a valved outlet 41 . The valve at 41 is then closed and the medium at port 40 pressurised to cause it to flow through the porous elements and the lower regions of the wells below the lids and into the second channel 32. Air bubbles in channel 32 from the priming process are flushed from port 42 to port 43 and the port 43 closed. Then medium may be flowed from port 40 to port 42 through all the wells in parallel. At this point the lids may be removed and objects added to the wells. The operational details, and behaviour of air bubbles in the system, will depend to an extent on the degree of hydrophilicity or hydrophobicity of the porous elements. If hydrophobic, they are more likely to clear bubbles from the wells preferentially, but the device will be harder to prime with pressure. Hydrophilic material will wet with aqueous medium and will give advantageous priming characteristics, but may be less able to clear bubbles trapped in the wells and so careful removal and replacement of the lids is needed. In practice, by keeping the level of medium slightly above the fitting point of the lid means medium will be displaced on fitting the lid and air bubbles can be avoided. The choice of the porous material depends on the precise design and application, but all types are claimed to be within scope of the invention.

[0084] The embodiment in figures 10 and 1 1 avoids problems in priming which are often encountered with prior art devices that arrange parallel flow paths that rejoin into a common manifold channel: that of trapping an air pocket in one of the branches, which then cannot be cleared as the parallel branches act as a fluidic 'short-circuit' to the input pressure. The porous elements of the embodiment in figures 10 and 1 1 act as pressure drop elements and so control liquid flow through each branch of the network. This allows the pressure drop for liquid flow to be kept higher than the pressure drop across any capillary stop points in the system, for example where the porous element 88 opens into the well 20 or the porous element 90 opening into the second channel 32. Therefore each channel in the device can be primed and those already filled with flowing liquid will not short-circuit the pressure driving the meniscus in the branches being primed. Hydrophilic porous elements 88, 90 have another useful feature in that they will wet with aqueous medium and form a capillary stop at the junction between the elements and the channel, for example between elements 90 and channel 32. This allows all the branches to be primed simultaneously up to this capillary stop; the channel 32 can then be filled to complete the liquid circuit and to remove air from the system.

[0085] The embodiments described so far comprise at least a first fluidic pathway from a port to a well, comprising a porous element, the at least one pathway primarily intended to be liquid-filled once the device has been filled initially. A further pathway might be provided into the well through a further porous element, which might be part of a common component with one or more of the porous elements in the other pathway(s), this further pathway being for gas, leading through a hydrophobic porous element into fluid communication with the interior of the well. In some embodiments this might be a means of supply of gas to equilibrate with the medium in the wells. In a further preferred embodiment this further pathway in use acts to vent gas from the well, for instance air trapped in the well when the lid 14 is closed.

[0086] The fluid channel or channels leading to the well may be formed anywhere within, or on the open surface of, the device, and may be defined in part by the lid and in part by the body of the device itself, in certain embodiments by other fluidic systems or components on or within which the device is mounted.

[0087] Figure 12a shows a partial cross-section and Figure 12b a partial plan view of the well and channels of a further embodiment, in which the channels 30 and 32 and the well 20 are formed at the upper surface of the base 12, so that the channels are closed in part by the lid 14. One channel only, or more than two may be provided in this embodiment as described for previous embodiments. Porous elements 50, 52 are provided in the fluidic pathways from the channels to the wells. Observation may be from below with an inverted microscope as shown at 102, in which case the device may have an observation region shown diagrammatically at 104 with improved optical characteristics of the base 12, for example a reduction in thickness of the base. Alternatively observation may be from above as shown at 106. The porous elements may be formed as by polymerisation, co-moulding with the base 12, or as inserts, either as separate bodies or as two regions of a larger insert as described before. In a preferred embodiment the porous elements define portions of the walls of the well, and are regions 86 in a ring-shaped insert as shown in plan in figure 12b. The remainder of the insert may be of the same material, optionally differently treated, or may be of a non-porous material bonded to or co-moulded with the porous material, shown as 84.

[0088] Figure 13 shows a partial cross-section of an embodiment in which the base 12 comprises material that might not be optimal for observation, such as an opaque material or a material whose properties are such that it does not have sufficient rigidity if made thin enough for good optical quality. Some grades of PDMS fall into this category. In this case a viewing window 108 may be mounted on the base, for example a glass window plasma-bonded to the PDMS base.

[0089] Figure 14 shows an alternative embodiment in which flow of medium is provided through the base of the well. Channel 32 might now be the inlet, and channel 30 the outlet. The porous element in the pathway from channel 32 comprising part of the base of an insert 54 and the porous element 50 part of the sidewall leading to channel 30. The insert 54 in this embodiment may have porous and non-porous regions as in previous embodiments. In a further embodiment in which more than one well is present, the porous element 52 of the base of the well may be in fluid communication with, or define a region of the wall of, a manifold channel 32 as in the embodiment in figure 10.

[0090] Figure 15b shows a partial plan view of a further embodiment, in which the device comprises a third porous element in fluid communication with the well, which is intended to give gas access to or from the well. Figure 15a shows a cross section at F-F and Figure 15c a cross-section at G-G figure 15b. Channels 30 and 32 are in fluidic communication with the well 20 via porous elements 50 and 52 as before. An additional fluidic pathway extends from the well to, in one embodiment, a breather 1 10 open to gas, and in another embodiment a gas access channel 1 16, optionally via a fluidic channel 1 14. The fluidic pathway comprises a porous element 1 12, in preferred embodiments formed from hydrophobic material that allows passage of gas but resists passage of liquid. In a preferred embodiment the porous element 1 12 forms a region of a common insert component 54 together with the other porous elements, and which insert 54 optionally comprises non-porous regions 84 also. Alternatively porous element 1 12 might be a separately formed region of the base, or a separate insert mounted in the base. The breather 1 10 is shown diagrammatically as a hole through the lid, but may be any other form of access to the outside of the device. In use, gas bubbles trapped in the well will tend to vent through the porous element 1 12, which can be assisted by a differential pressure across 1 12, for example by positive pressure in the channels 30 and/or 32 or negative pressure in the gas access channel 1 16. Of course, gas access channel 1 16 may additionally form an access route for gas such as CO2/air for equilibration with the contents of the well.

[0091 ] The embodiments of figures 12 to 15 may be used with the fluidic network design of figures 10 and 1 1 , with appropriate rearrangement of the common fluidic channels.

[0092] Figures 16a-c show a further embodiment, in which the device comprises a common porous component which acts to provide porous elements in a multiplicity of fluidic pathways. The device 150 comprises a base 1 52 and a removable lid 154, together defining a fluidic space 156. Space 156 defines a fluidic pathway through the device, between fluidic connections 158. The device comprises a well-defining component 160, comprising well-forming subcomponents 162 and spacer components 164, 168 which together act to define an array of wells 166 within the space 156. When the lid is in place, the wells are closed against movement of the embryo or other cellular entity out of the well. In preferred embodiments at least part of the well-forming subcomponents are formed from porous material such that a fluidic pathway exists from the space 156 through the subcomponent 162 to the interior of the well 166. In particularly preferred embodiments the porous material is hydrophilic in the region of the desired fluidic pathway. In operation the space 1 56, the wells 166 and the wetted portions of component 160 are filled with liquid medium and the wetted portions of component 160 form porous elements in the fluidic pathway connecting the inlets 158, the space 156 and the wells 166. The liquid medium may flow through the porous elements, and hence through the wells, or may be wholly or substantially stationary within the porous elements, in which case diffusion will result in transport of chemical species between the medium in space 156 and the contents of the wells. Embryos or other cellular entities are introduced into, or removed from the wells by pipetting with the lid removed.

[0093] It will be seen from figure 16a, b that the flow pattern within the space 156 is controlled by the conformation of the well-defining components 162 and the spacers 164, 168. The spacers 164, 168 are preferably of lower height than the wells, so allowing the flow pattern to be substantially uniform in the space 156. In some embodiments the spacers 164 and 168 may be designed so as to impede or direct flow. In preferred embodiments the spacers 164, 168 or both may extend to the walls of the space 156, and may act to locate the well-defining component 160 within the space. In preferred embodiments the lid 154 seals to the base 152 by means of a sealing region, for example comprising an elastomeric layer or component, around the periphery of the space 156, the dimensions being so that the wells are closed when the lid is in place. In other embodiments the lid may also seal to the individual wells. The geometry of the lid and base are shown schematically in figures 16a-b and it will be understood that any configuration which achieves closure of the fluidic space 156 and the wells 166 is within the scope of the invention.

[0094] The device 150 preferably comprises a multiplicity of wells and these may be arranged geometrically in any appropriate manner. Preferably the wells are closely packed, so increase the number of wells in a given space 156, and different arrangements of wells from that in figure 16b may be provided to achieve this, such as shown in figure 16c. The wells are shown as being round, but may be of any other shape, for example asymmetrically shaped to as to direct flow in their vicinity.

[0095] The well-defining component 160 may be formed by any appropriate means as known in the art, such as moulding from VYON ™ porous polypropylene, then treating to render it wholly or partially hydrophilic, or stamping, cutting, machining from an initial uniform sheet of material so as to form the wells and optionally to compress the material to form the spacers 164, 168. The moulding or formed sheet may then be treated to define regions of greater or lesser porosity or hydrophilicity to define further the desired fluidic pathways within the material.

[0096] Figures 17a-c show a further embodiment, in which the device

200 comprises a base 202 and a lid 204, defining a fluidic space 206 as before, with fluidic connections 208. One or more wells 216 are defined within the space 206 by well-defining features 212 formed or mounted either on the base 202 or the lid 204. The features 212 are preferably formed from a hydrophilic porous material, such as VYON ™ and are sized so that when the lid is in place the wells are closed against exit by an embryo or other contents of the wells.

[0097] In operation the wells 216 are filled with liquid medium and embryos or other desired contents pipetted into them. The lid and base are closed together, and liquid medium is flowed through the space 206. A fluidic pathway is established through the porous feature 212 connecting the liquid in the fluidic space 206 and the interior of the well, allowing exchange of species between the contents of the well and the liquid flow while retaining the contents against flow.

[0098] Figure 17c shows a further embodiment, in which a device of the invention comprises a first component comprising a planar substrate in the form of a lid as shown as 204 in figure 17b, comprising on its surface an array of well defining components 212, porous in whole or part, and a second component 220, comprising one or more recesses 222 sized to accommodate the well-defining components 212 and flow channels 224 linking the recesses 222 to fluidic connections 208. When the device is assembled, the first component seals to the second, at least around the periphery of the device, and the well-defining components fit within the recesses 222. Preferably the recesses 222 are a close fit to the well-defining components, so as to direct flow preferentially through the porous elements and the wells 216. However, in alternative embodiments the fit is less close, to allow easy assembly of the device, and a proportion of the flow bypasses the well.

[0099] In further preferred embodiment, the well-defining components are mounted on the base 202 and second component 220 is permanently mounted around them, and optionally mounted on or bonded to the base. The lid 204 is then a substantially planar component and acts to close both the wells 216 and the flow channels 224.

[00100] The well-defining components 212 may be moulded onto the lid or base by co-moulding, for example VYON ™ onto non-porous polypropylene, or by bonding or mounting individually moulded components 212 onto a substrate. Alternatively the components 212 might be formed by processing a layer of porous material as described above.

Claims

1 . A device for handling cellular entities, the device comprising:- a substrate having one or more wells, said one or more wells being adapted to hold a cellular entity in fluid; one or more fluidic channels for fluidic communication with the one or more wells; and at least one movement restriction means for restricting the movement of the cellular entity from the well while allowing said fluidic communication.

2. A device according to claim 1 wherein the movement restriction means is an accumulation of particles or an element comprising a porous portion.

3. A device according to claim 2 wherein the element is at least partially formed from a hydrophilic sintered polymer, a hydrophobic sintered polymer or a mixture thereof.

4. A device according to either claim 2 or 3 wherein the porous element is formed from a resilient material.

5. A device according to any of claims 2 to 4 wherein the element is held in place by adhesive bonding, heat bonding or ultrasonic welding.

6. A device according to any of claims 2 to 5 wherein element is formed from in-situ polymerisation.

7. A device according to any of claims 2 to 6 wherein the element has varying degrees of porosity.

8. A device according to claim 7 wherein the element contains at least one of the following portions: a portion which is permeable to liquid, a portion which is impermeable to liquid and/or gas and a portion which is impermeable to liquid but permeable to gas.

. A device according to any of claims 2 to 8 wherein the element is annular.

10. A device according to any preceding claim wherein the movement restriction means is positioned at least in one of the following ways: in the one or more f luidic channels; integral with at least one of the one or more wells; or forms at least one of the one or more wells.

1 1 . A device according to claim 10 wherein when the movement restriction means is in the fluidic channel there is a gap between the movement restriction means and the fluidic channels which allows flow of fluids past the restriction means but which is too small to allow passage of the cellular entity.

12. A device according to claim 10 wherein when the movement restriction means forms at least one of the one or more wells it is removable from the device.

13. A device according to any preceding claims further comprising a lid means for preventing entry or exit of the cellular entity from the one or more wells.

14. A device according to claim 13 wherein the lid is the movement restriction means in the form of an element comprising a porous portion.

15. A device according to any preceding claim wherein the movement restriction means is not a polymer mesh.