WO2006097740A1

WO2006097740A1 - Capillary devices for cell and non-human embryo culture

Info

Publication number: WO2006097740A1
Application number: PCT/GB2006/000957
Authority: WO
Inventors: John Robert Dodgson
Original assignee: Robio Systems Ltd
Priority date: 2005-03-16
Filing date: 2006-03-16
Publication date: 2006-09-21
Also published as: GB0505377D0

Abstract

An apparatus for culturing embryos and/or other cellular entities comprising a flow channel adapted to hold one or more cellular entities, the flow channel having an inlet through which a cellular entity may be introduced and a source of fluid for generating a flow of fluid in the flow channel towards a constriction passed which the cellular entity cannot move in use. The apparatus is adapted such that the cellular entities interaction with the channel wall is minimized. The constriction may comprise a moveable porous plug provided loaded with a magnetic material which can be moved by applying a magnetic field.

Description

CAPILLARY DEVICES FOR CELL AND NON-HUMAN EMBRYO CULTURE

This invention relates to apparatus and methods for culturing cells, maturing ova and culturing embryos and other cellular structures in vitro, and means of transportation of cells, ova, embryos and other cellular structures.

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 auto/paracrine 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 WO0102539 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.

The medium is usually buffered against changes in pH; this buffer may be based on bicarbonate / C02, in which case the partial pressure of C02 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.

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" or cellular entities. Where specific features of the invention apply to maturation of ova, or to culturing of embryos, this will be noted.

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

Beebe et al. US6193647, US6695765 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 US6193647 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.

Campbell et al. US20020068358 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. US20020068358 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 US20020068358 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.

Thompson et al., US6673008, 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 02, 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 US6673008 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 (C02, 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 US6673008, and so a means to allow gas equilibration with the medium around the embryos is preferred.

In the absence of a means of gas access to the medium in the vicinity of the object, this may be done using a steady flow of gas-equilibrated medium itself, in which case the volume surrounding the embryo is necessarily small.

Van den Steen et al., US20040234940, 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 US20040234940 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 .

Vajta et al. WO0102539 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.

Seidel et al. US 2004/0132001 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 medium during transport.

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

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 US6193647 or a mesh or filter in the base of a well, as in Campbell et al . US20020068358 and van den Steen et al US20040234940. 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.

It is an object of the present invention to mitigate or provide a solution to these and other difficulties in the design and operation of culture devices of the prior art, and a method of culture of biological objects that uses the devices advantageously to provide culture conditions which allow exchange of medium around the objects without handling of the objects themselves.

According to a first aspect, the invention provides a method for culturing embryos and other, cellular entities (Objects') comprising: - providing a medium-filled tube or channel comprising a constriction, introducing a object into the tube and causing it to move to the constriction, making fluidic connection to at least one end of the tube and flowing medium along the tube to control or modify the conditions around the object

Optionally the object moves through the medium under gravity, for example by sedimentation, or angular acceleration, for example using a centrifuge. The cellular entities are typically more dense than the medium in which they are matured.

Optionally the object is moved by flow down the tube. Preferably the tube or channel is substantially vertical in use (or at least makes a significant angle to the horizontal) in order to minimize interaction between the cellular entity and the channel wall. Preferably the tube or channel makes an angle of greater than 20 degrees to the horizontal. Very preferably the tube or channel makes an angle of greater than 40 degrees to the horizontal.

The apparatus is advantageously adapted so that the tube or channel can be returned to substantially horizontal once the entity has reached its desired resting place adjacent the constriction, if desired.

Optionally the method comprises observation or optical analysis of the object while inside the tube

Optionally the method further comprises providing a number of tubes, optionally arranged in an array.

According to a further aspect, the invention provides a method for culturing embryos and other cellular entities or " "objects" comprising: providing a medium-filled fluid channel comprising a constriction, introducing a object into the channel and causing it to move to the constriction in such a way that the objects do not interact with a wall of the channel so as to cause a rolling motion of the object along the wall, making fluidic connection to at least one end of the channel and flowing medium along the tube to control or modify the conditions around the object.

Optionally the object moves through the medium under gravity or angular acceleration.

Optionally the object is moved by flow down the channel, in a preferred embodiment while the channel is inclined away from horizontal.

Optionally the method comprises observation or optical analysis of the object while inside the channel.

Optionally the method further comprises providing a number of channels, optionally arranged in an array.

According to a further aspect, the invention provides a method for culturing embryos and other cellular entities (>objects=) as either of the previous methods, additionally comprising: introducing one or more further objects into the tube or channel, introducing between at least two of the objects at least one spacer adapted to fit the tube or channel so as substantially or wholly to separate two objects or groups of objects .

In a preferred method according to this aspect, the spacer acts to separate individual objects and is adapted to fit the tube or channel so that objects cannot pass it, so maintaining the sequence of the objects as they were introduced into the tube or channel.

Optionally the spacers comprise beads, for example inert glass or polymer beads. The spacers are optionally loaded with ferromagnetic material to allow magnetic actuators to be used to move them in use. This can be achieved by casting a shaped polymer spacer with ferromagnetic inclusions.

Optionally the spacers are designed to stack so as to reduce contact between them and the neighbouring objects when placed adjacent to one another in the tube or channel.

Optionally the spacers are adapted to remain at a given location within the tube or channel against normal flow of medium unless they are moved by a greater flow (for example, above a threshold value) or an external force, such as an external magnet.

In a further aspect, the invention provides a method comprising any of the above steps, additionally comprising:

Providing a second, removable constriction which acts to hold the objects in a region of the tube or channel between itself and the first, fixed constriction, while allowing flow of medium past it.

Optionally the removable constriction is a sliding friction fit to the tube or channel and so will move within the channel in response to an external force.

Optionally the removable constriction is moved by gravity or angular acceleration

Optionally the removable constriction is moved by a magnetic field.

Optionally the removable constriction is moved by flow of medium.

Optionally the removable constriction is moved by a force on a part of the constriction, such as by pulling on a handle, filament, or other tool.

In a further aspect the invention provides a method as any above, with the additional steps of:

Providing a container which ' fits within the tube or channel, adapted to contain one or more objects while allowing exchange of medium between the inside and the outside of the container.

Placing one or more objects into a container and introducing the container into the tube or channel

A preferred method according to this aspect additionally comprises :

Providing more than one container as above, the containers adapted to stack within the tube or flow channel so as to reduce contact of the object (s) within a first container with the second container

Optionally the containers are moved within the tube or channel by an external force such as gravity, angular acceleration, magnetic field or force from e.g. a tool introduced into the tube or channel. The containers may be loaded with a ferromagnetic material as described above for the spacers.

Optionally the containers are moved within the tube or channel by flow of medium.

Optionally the containers are adapted to remain at a given location within the tube or channel against normal flow or medium unless they are moved by a greater flow or an external force.

In a further aspect the invention provides a method according to any of the above, additionally comprising: providing an apparatus comprising a first tube or channel and a second tube or channel branching from the first, along which fluid may be flowed into or out from the first tube or channel.

In a further aspect the invention provides a method according to the above, additionally comprising: providing an apparatus comprising the tube or channel and one or more actuators adapted to move the containers or spacers within the tube or channel, introducing the objects and the spacers, or one or more containers containing one or more objects, into the tube or channel, positioning or moving an object within the tube or channel by means of a force from one or more actuators on one or more spacers or containers, or causing it not to move in response to another force.

A preferred method according to this aspect further comprises :

Sequentially operating the or each actuator to move or control the movement of one or more spacers or containers within the tube or channel.

According to a further aspect the invention provides a method for handling objects such as embryos and other cellular entities comprising the steps of: providing an apparatus comprising: a first tube or channel comprising a port at a location between its ends. one or more spacers or containers adapted to retain one or more objects between them or within them inside the tube or channel. moving one or more spacers or containers within the channel so as to locate the object adjacent to the port introducing or removing the object from the tube or channel via the port .

A further method according to this aspect additionally comprises: providing a cylindrical tube or channel, providing a substantially cylindrical container for objects, the container comprising an orientation means such as a permanent magnet which allows it to be rotated about its axis, with an opening on one side of the cylinder through which the object is introduced, rotating the container so that the opening faces the port, introducing the object into or removing it from the container through the port.

According to a further aspect the invention provides a method for handling objects such as embryos and other cellular entities comprising the steps of: providing an apparatus comprising a first tube or channel an at least a second tube or channel intersecting the first, the second channel comprising a port, one or more spacers or containers adapted to retain one or more objects between them or within them inside the tube or channel, moving one or more spacers or containers to a location within the channel where flow through the second tube or channel will act to move an object from between two spacers or from within a container, and cause it to move into the second channel, introducing the object into or removing it from the second channel via the port. According to a further aspect the invention provides an apparatus for culturing cells, embryos or other cellular entities (objects) comprising: a tubular flow channel adapted to contain one or more objects, a fitting to a first end of the tubular flow channel through which an object may be introduced to the flow channel, a constriction within the tubular flow channel past which the object cannot move, a removable fluidic connection to at least one end of the tubular flow channel, a source of fluid which can generate flow through the flow channel.

In a first embodiment the constriction comprises a porous element located within the tubular flow channel.

In a second embodiment the constriction comprises a narrowing of the flow channel, optionally the channel comprises a second flow component of smaller diameter than that of the object, such as a tube within a tube.

According to a further aspect the invention provides an apparatus comprising: a device comprising a fluid channel comprising a constriction, an inlet port in fluid communication with the channel a fluidic connection means which connects to the fluid channel to provide fluid flow through the channel, the device being adapted to facilitate movement of the object to the constriction by force from gravity or angular acceleration. In a preferred embodiment, the fluid channel is oriented at a significant angle to the horizontal to minimize interaction between the cellular entities and the channel wall.

In a preferred embodiment the inlet port and channel form part of a fluidic pathway from the inlet to the constriction, that pathway having a maximum angular deviation of less than 90 degrees, more preferably less that 60 degrees.

In a further preferred embodiment, the fluidic pathway is as above but at least one route from the inlet to the constriction which involves no angular deviation in the path of a moving object. In other words, the fluidic pathway or channel is adapted to allow the cellular entities to move from the inlet to the constriction in a substantially straight line.

According to a further aspect the invention provides an apparatus as above, further comprising: one or more spacers which are adapted to separate one or more objects within the channel, the spacers being of a size and shape that the objects cannot pass the spacer within the channel but medium can flow pass them.

In a preferred embodiment the spacers comprise beads, for example inert glass or polymer beads.

In an alternative embodiment the spacers are specially formed, for example so as to substantially occupy the cross- sectional area of the channel, while being for example porous, hollow or fluted so as to allow medium to flow past them.

In a further embodiment the spacers are designed to stack so as to reduce contact between them and the neighbouring objects when placed adjacent to one another in the tube or channel.

In a further embodiment the spacers are adapted to remain at a given location within the tube or channel against normal flow or medium unless they are moved by a higher flow (for example above a threshold value) or an external force.

According to a further aspect the invention provides an apparatus as above, additionally comprising a second, removable constriction which acts to hold the objects in a region of the tube or channel between itself and the first, fixed constriction, while allowing flow of medium past it.

Optionally the removable constriction is moved by gravity or angular acceleration.

Optionally the ^'removable constriction is moved by a magnetic field.

Optionally the removable constriction is moved by flow of medium.

Optionally the removable constriction is moved by a force on a part of the constriction, such as by pulling on a handle, filament or other tool.

According to a further aspect the invention provides an apparatus for culture of objects comprising a flow channel comprising a constriction as above, the apparatus further comprising: a container which fits within the tube or channel, adapted to contain one or more objects while allowing exchange of medium between the inside and the outside of the container.

In a preferred embodiment more than one container is provided, the containers adapted to stack within the tube or flow channel so as to reduce contact of the object (s) within a first container with the second container.

Optionally the containers are moved within the tube or channel by an external force such as gravity, angular acceleration, magnetic field or force from e.g. a tool introduced into the tube or channel.

In a further aspect the invention provides an apparatus as above, additionally comprising: a first fluidic channel and at least a second fluidic channel branching from the first, along which fluid may be flowed into or out from the first channel, for flow of either the same, or a different medium into or out of the first channel .

In a further aspect the invention provides an apparatus according to the above, additionally comprising: one or more actuators (i.e. actuating means) adapted to move the containers or spacers within the tube or channel, positioning or moving an object within the tube or channel by means of a force from one or more actuators on one or more spacers or containers, or causing it not to move in response to another force.

In a preferred embodiment the spacer or containers are magnetic and move in response to a magnetic field acting through a wall of the flow channel.

In one embodiment the spacers or containers move under force , from flowing medium and are retained at a given location by a magnetic field at that location. The actuators may be static, or may move, and may comprise permanent magnets or electromagnets. The field may be controlled by physically moving a permanent magnet away from the channel.

In a further embodiment, an actuator may move along the channel to draw a spacer or container along the channel.

In a further embodiment, the apparatus may comprise more than one actuator, which may be operated in sequence to cause or control movement of the spacer (s) or container (s) .

According to a further aspect the invention provides an apparatus comprising: a first tube or channel comprising a port at a location between its ends; one or more spacers or containers within the channel acting to space or contain objects; means to locate the spacers or containers in a given location relative to the port, for example magnetic location means as disclosed above; means to introduce the object into or remove it from the tube or channel via the port.

In a further embodiment of this aspect, the apparatus comprises : a cylindrical tube or channel comprising a port at a location between its ends, a substantially cylindrical container for objects, the container comprising an orientation means such as a permanent magnet which allows it to be rotated about its axis, with an opening on one side of the cylinder through which the object is introduced, means to locate the container adjacent to the port, for example magnetic location means as disclosed above, means to rotate the container, so bringing the opening to face the port, for example a rotating magnetic field around the channel, and optional means to introduce the object into or remove it from the tube or channel via the port.

The invention will now be described, by way of example only, with reference to the accompanying schematic drawings, in which :-

Figure 1 shows a first embodiment of an apparatus according to the invention.

Figure 2 shows a second embodiment of an apparatus according to the invention.

Figure 3a shows a first view of a third embodiment of an apparatus according to the invention.

Figure 3b shows a second view of a third embodiment of an apparatus according to the invention.

Figure 4a shows a first view of a fourth embodiment of an apparatus according to the invention.

Figure 4b shows a second view of fourth embodiment of an apparatus according to the invention.

Figure 5a shows a fifth embodiment of an apparatus according to the invention.

Figure 5b shows a sixth embodiment of an apparatus according to the invention.

Figure 6a shows a seventh embodiment of an apparatus according to the invention.

Figure βb shows an eighth embodiment of an apparatus according to the invention.

Figure 7a shows a ninth embodiment of an apparatus according to the invention.

Figure 7b shows a tenth embodiment of an apparatus according to the invention.

Figure 8 shows a three-quarter view of an apparatus according to the invention.

Figure 9 shows an embodiment similar to that in figure 1, adapted for operation in any orientation, and so suitable for operation and transport a part of a transport appliance of the invention.

Figure 10 shows a cross sectional diagram of an assembly which forms part of an appliance of the invention, showing manifold means for a number of devices and means to control their temperature.

Figure 11 shows a cross sectional diagram of an embodiment comprising the features of that in figure 10.

Figure 12 shows a further embodiment of a fluidic and thermal assembly forming part of an appliance according to the invention.

Figure 13 shows a second cross section of an assembly similar to the embodiment shown in figure 12, and

Figure 14 shows a diagram of an appliance according to the invention, adapted for culture and transport of objects, which comprises a device of the invention for retaining the objects, and optionally a fluidic and thermal assembly. Figure 1 shows a first embodiment of a device according to the invention, comprising a tubular flow channel 10 sized to accommodate an object 20, the channel comprising a constriction 12 here shown in the form of a porous plug. The device comprises an inlet port 14 and an outlet port 16, here shown as a connector, which may be a standard fluidic in-line connector, connected to a fluidic line 18 which may lead to a source or drain of fluid, pressurised or not. Inlet port 14 serves to provide a means of pipetting an object into the channel. In the case that the channel is large enough, the inlet port might be omitted. In a one embodiment, inlet port 14 is removable and may be replaced with a fluidic line connector as 16 and 18. In other embodiments, a fluidic connector is provided that plugs directly into 14.

The device can be assembled simply from standardly- available components and materials, and unlike the devices of the prior art that act to provide a small, controlled flow medium environment for cellular objects, does not have to be microfabricated. In some embodiments the channel is formed from a glass capillary or rigid plastic tube, preferably transparent to allow observation using a microscope 22. The constriction is shown here being formed from a porous plug B this might be of any suitable material, such as a filter material, for example porous polyethylene or polypropylene, which may be treated to render it hydrophilic, such as supplied by Porvair Ltd., Wrexham, UK as >VYON= TM. Other forms of constriction are possible, dependent on being able to introduce them into the channel. A precipitated porous silicate plug, such as that formed from sodium silicate solution on heating as known in the art, is suitable in some embodiments .

Figure 2 shows an embodiment as in figure 1, except that the constriction is now formed by a second tube 26 inserted into the first. The second tube itself might form the constriction, if its i.d. is too small for an object to pass through, or the end of 26 might be blocked by a porous plug 12.

Figure 3a shows a further embodiment in which the channel 10 is formed or mounted within a device 30 which acts to enclose it. The inlet port might be part of 30, or might be separable from it, and might comprise fluidic connection means as before. Figure 3a illustrates the method of the invention, in which spacers 24a, 24b are shown between discrete objects 20a, 20b, 20c. The spacers occupy the internal space in the channel so that the objects cannot pass them, so retaining the spatial arrangement of the objects. It is within the scope of the invention to place the objects within the channel without the spacers, but this then means that either the channel is sized close to the size of the objects, or they may be able to pass one another, especially if they are^' deformable, so losing spatial information. The spacers are shown in figure 3a as being approximately cylindrical if the channel 10 has circular cross-section, or rectangular if is has rectangular cross- section, but they may be of any size or shape, for example in a preferred method, spherical beads, preferably inert beads of glass or polymer. In preferred embodiment the spacers are designed so that they allow medium to flow past, so they might have a hollow cylindrical centre, has flutes or passages around their edges, or might be porous. Such spacers, with typical dimensions for a device adapted for embryos or oocytes, around 0.5 -lmm, are readily formed by micromoulding.

Figure 3b makes clear that the apparatus of the invention may be adapted to be operated in any orientation, and may for example be loaded vertically and viewed horizontally.

Figure 4a shows a cross-section of a further apparatus of the invention, adapted for loading with an object by sedimentation under gravity or mild centrifugation. The device comprises a channel 40 with an inlet port 42, which tapers towards the channel to allow ready access of the object to the channel, a constriction 44 and an outlet port 46. An optional viewing area is provided for microscopy. The device may be fabricated by standard means, for example by injection moulding, followed by insertion of a porous material to form the constriction, or by laminating. The channel 40 may be of any appropriate cross section. The device is adapted to minimise interaction between the object and the walls of the channel, and so provides a clear pathway to the constriction. The motion of the object may be assisted or caused by entrainment in flow from the inlet to the outlet; as the object will interact minimally with the walls of the channel, if at all, a rolling or rotating motion as disclosed in US6193647, which has no proven benefit, will tend to be avoided.

Figure 4b shows an array of devices as in figure 4a, with an inlet manifold means in the form of a plug-in component 50, and a similar outlet manifold means 52. Of course, the individual devices within an array of this type can be fed with separate fluidic lines, or the manifolding might be done within the structure of the array.

Figure 5 aims to illustrate alternative embodiments of the apparatus of the invention. Figure 5a shows a device in which the constriction is formed by a moulded or embossed feature, as a narrowing 60 of the channel 10. The inlet port might be closed by a lid 66 or fitted with a flow connection 64. A manifold channel 62- is provided within the device, terminated in one or more connections 68. Figure 5b shows a further device, in which fluid flow over an object can be achieved without a flying fluidic connection. Channel 10 now has a side channel 70 for flow of medium, and in one embodiment lid 66 comprises a plug member 72 which acts to block flow of medium, and/or movement of the object, into the side channel according to whether the lid is in place (or partially in place) or absent. The member 72 might be porous, so allowing flow of medium into the side channel, but retaining the object in place, when it is fitted.

Figure 6a shows a further embodiment of the apparatus adapted for location of the object by sedimentation or flow in vertical orientation, which comprises a housing 30 comprising a body part 80 and a base/substrate 82, and other parts numbered as before. The inlet port 42 leads to a sloping portion 81, adapted for ready movement of the object into the channel 40. The objects rests against the constriction 60, which is adapted to bring the object into contact with the base 82. In a preferred embodiment the base is clear, so allowing good observation through it .

Figure 6b shows a further embodiment similar to that in figure 6a, but now with a viewing window 84 in a solid, for example, moulded, body part 80.

Figure 7 shows an embodiment in which objects 20a, 20b, 20c are housed in containers 90a, 90b, 90c- in the flow channel 40. The containers are held against flow by a constriction 60, which may be sized so as to hold up the containers, while it would be difficult to hold individual objects. The containers might be formed as micromoulded devices, comprising one or several objects, optionally in more than one compartment in each container. They may be porous, or have a mesh base, or have an open cylindrical shape with a small opening at the base. They are adapted to allow medium to reach the contents and to flow through or past them along the channel. They may be held in place by a lid or stopper, or by a holding means for example a moveable stopper 92, which may be a sliding friction fit to the channel, and may comprise a magnetic material and so be moved by magnetic actuation using an external magnet as shown at 94. Alternatively, magnet 94 might control the movement of the stopper driven by flowing medium. Figure 7b shows an alternative embodiment in which the containers are themselves

magnetic and may be moved or retained against flow by one or more magnets 94.

The embodiments in figures 7a and 7b show containers with objects within them - these might be spacers in an alternative embodiment, the spacers moved or controlled magnetically and the objects left free to move between them. In a preferred embodiment, the spacers are adapted to that they stack with clearance for the object between them, for example by means of projections moulded around the circumference at each end.

Figure 8 shows a diagram of the devices of the invention in array form. As they are intended to be inspected from the side, the array are preferably a single row or optionally a double row of channels, which in a preferred embodiment and method are used substantially vertically, and once the objects are in place at the constriction, may be turned to the horizontal for ease of observation on an inverted microscope as shown. The array devices may be adapted to interfit with an appliance, the appliance designed to carry out all or part of the steps of the method, if required, for example to load objects into the channels, make fluidic connections to the device, to supply fluid such as medium to the device, and interact with other features of the 'device as may be present, such a means to control temperature, change the conditions in the medium, or allow access of gas to the channels within the device for C02 / 02 equilibration.

Figure 9 shows an embodiment similar to that in figure 1, adapted for operation in any orientation, and so suitable for operation and transport a part of a transport appliance of the invention. Figure 9 shows a device 100 comprising a tubular flow channel 102 sized to accommodate one or more objects 102, and including a constriction 104 which acts to retain the objects against flow through the channel. The constriction 104 may be formed in any manner as previously described. When fluid flow is required the device 100 may be connected to a fluid flow circuit by means of an upstream connector 108 and flow line 110 and a downstream connector 112 and flow line 114, so enabling flow of liquid media for example from a reservoir to a waste receiver. Change of media within the device may then be achieved by changing the input media flowing through the circuit. In a preferred embodiment the device comprises a retaining cap 116, which includes a second constriction 118 in the flow path through it, again formed in any manner described earlier. When cap 116 is in place, the objects are retained in the channel 102 while fluid is able to flow from line 110 through the device to line 114. The retaining cap 116 allows the objects to be positively retained in the channel 102 in any orientation of the device 100 when this is disconnected from the fluidic circuit. In alternative embodiments the second constriction 118 may be provided as part of the upstream flow connector 108.

The device and connections in figure 9 may form part of a culture and transport appliance of the invention. A single device 100 might be provided, or a plurality of device, with the fluidic circuit connected in series or in parallel through them. The connections, and control of fluid flow through the devices 100, can be made using either conventional fluidic components, such as used for example in flow injection analysis or HPLC, or by means of custom-formed microfluidic manifolds, valves and pumps.

In preferred embodiments the appliance of the invention comprises means to control the temperature of the devices 100.

Figure 10 shows a cross sectional diagram of an assembly which forms part of an appliance of the invention, showing manifold means for a number of devices and means to control their temperature. The assembly 119 comprises a number of devices 100, an inlet manifold component 120 with an inlet manifold channel 122, connected to an input line 110 by means of an inlet connector 124, and an outlet manifold component 126 with an outlet manifold channel 128, connected to an outlet line 114 by means of an outlet connector 130. The devices 100 are reversibly -connectable to the manifolds, in preferred embodiments by means of fluid-tight plug-in connections. In the embodiment in figure 10 the inlet constrictions 118 are shown as part of the inlet manifold component 120, but in preferred embodiments a retaining cap 116 including constriction 118 is provided between channel 102 and inlet manifold component 120.

In preferred embodiments the flow assembly comprising the devices 100 and the inlet and outlet manifolds is housed in a temperature control means, comprising a substantially uniform temperature block 132 which is preferably shaped to retain the assembly in close contact with the block. In a preferred embodiment the block is formed from metal and preferably in two parts, separable to allow the assembly to be mounted between them. The joining faces 132 preferably join into thermal contact to assist temperature uniformity.- In preferred embodiments a heater 138, connected by leads 140 may be provided as part of the block 130. One or more temperature sensors 142 are provided to measure the temperature of the block. In figure 10 these are shown located close to the devices 100; they may be located more remote from the devices, especially if the block 130 is highly conductive.

Figure 11 shows a cross sectional diagram of an embodiment comprising the features of that in figure 10, showing the block 132 comprising two parts 144 and 146. Further heaters 148 and sensors 152 may be provided in the second part of the block. These may be controlled in common with the heater in the first part of the block so as to achieve a substantially equal temperature in the two parts of the block.

Figure 12 shows a further embodiment of a fluidic and thermal assembly forming part of an appliance according to the invention. The assembly 200 comprises a device 202 which comprises one or more subunits as in the embodiment in figure 6a, each comprising a channel 204 sized to ^• accommodate one or more objects, and a constriction 206 which retains the Objects in the channel. The device 202 is adapted to facilitate movement of objects in the channels 204 without a rolling motion, for example by sedimentation under gravity, or entrainment in flow in a substantially vertical orientation in which interaction of the object (s) with the walls of the channel is minimal. In assembly 200, each channel 204 of the device 202 is in fluidic communication with an inlet manifold channel 210 by means of an inlet manifold component 208, and with an outlet manifold channel 214 by means of an outlet manifold component 212. In preferred embodiments the fluidic path to each channel 204 passes via an inlet constriction 216 located in the inlet manifold component 208. The device 202 and manifold components 208, 212 are housed within a thermal control block 220, preferably formed from two subcomponents 222, 224. In preferred embodiments both components are conductive; alternatively, component 224 furthest from the channel (s) in the device 202 may be insulating. A heater 226 is located in the conducting block 222, connected by power leads 228, and a temperature sensor 230 is provided connected by sensor leads 232. The subcomponents 222, 224 are kept in contact by fixing or clamping means (not shown) or by means of close interfitting.

Figure 13 shows a second cross section of an assembly similar to the embodiment shown in figure 12, with common parts having the same numerals. The device 202 is shown as having four channels, though it will be understood that it might have one or any other practical number. The heater 226 and power leads 228 are shows in a different orientation from in the embodiment in figure 12. Inlet fluidic line 240 and outlet line 242 are shown as formed integrally with the manifold components 208 and 212 respectively. In alternative embodiments the manifold components are parts of a common component.

It will be understood that the fluidic and thermal assemblies of figures 10-13 show only certain embodiments of the invention and that other embodiments of assembles incorporating any of the embodiments of figures 1-8 are within the scope of the invention.

Figure 14 shows a diagram of an appliance according to the invention, adapted for culture and transport of objects, which comprises a device of the invention for retaining the objects, and optionally a fluidic and thermal assembly as previously described. The appliance 300 comprises a reversibly openable outer housing 302, preferably capable of being transported and toughened, shock resistant as required. In preferred embodiments and outer insulation layer 304 is provided as part of the outer housing. The appliance comprises an inner payload 305 comprising a reversibly openable inner housing 306, which in preferred embodiments comprises an inner insulating layer 308. The payload housing defines an inner space 310, which in preferred embodiments is adapted to maintain a gas atmosphere independently of the atmosphere elsewhere within the appliance or outside it; in preferred embodiments the inner housing 306 is gas-tight and adapted to retain positive and negative pressure with respect to ambient.

The payload 305 comprises a fluidic and thermal assembly of the invention 312, for example as shown in figures 10-13, and a fluidic circuit comprising such components as are required to supply fluids to the assembly and retain fluids flowed from it. In the preferred embodiment shown in figure 14, the inlet fluid line 110 passes to a pump 314 and then via a first valve 316 to a first media reservoir 322 and also via a second valve 318 to a second media reservoir 324. The outlet fluid line 114 passes to a waste reservoir 326. This arrangement allows a first medium to be flowed through the assembly from the first reservoir, then replaced by a second medium from the second reservoir. Further inlet flow lines, valves and reservoirs may be provided to give further sequences of media flow, and optionally mixing by including further pumps and/or using mixing methods as known in the art.

A gas inlet 330 to the appliance is provided, and a gas flow path from the inlet via first gas valve 332, inlet path 334 to inner space 310, then via outlet path 336 and second gas valve 338 and outlet 340 to atmosphere. When the gas valves are open, the inner space may be filled with' a desired gas composition, for example 5% C02 in air, suitable for culture of the objects in the assembly 312. The valves may then be closed to retain the gas atmosphere.

A control means 350 is provided within the appliance, controlling heaters in the assembly and optionally elsewhere in the payload via power lines 352 and receiving sensor inputs from the assembly and optionally elsewhere in the appliance via input lines 354. A power supply 356 comprising batteries is provided, which may be charged via a line power cable 358.

In preferred embodiments the inlet and outlet manifold components, fluidic lines 110, 114 and the reservoirs form a tubeset that may be fitted together into the assembly 312, pump 314 and valves 316 and 318, used and then disposed of (in some embodiments) after use. The heater block (s) and associated fittings and closure devices in the embodiments in figures 10-13 remain part of the appliance.

In use, objects are first .loaded in media into the one or more fluidic device (s) of the invention and retaining caps fitted where these are required. The fluidic device (s) are then connected to the tubeset and mounted in the thermal assembly. In preferred methods of operating the appliance, the power supply is charged, the thermal assembly is brought to temperature and the fluidic lines are pre-filled with the correct medium and substantially flushed of air before connection to the device (s). Medium is then flowed through the tubeset and device (s) to remove air bubbles so far as is required. The payload housing is then closed, and gas flowed through the space 310. Once the gas valves are closed, the appliance is ready for autonomous action under control of the control means. In preferred embodiments, the control means includes a program that determines the actions of the control means.

Claims

1. A method for culturing embryos and/or other cellular entities comprising: -

providing a medium-filled tube or channel having a constriction, introducing a cellular entity or group of cellular entities into the tube or channel and causing it to move to the constriction avoiding substantial interaction with the walls of the tube or channel, making fluidic connection to at least one end of the tube or channel, and flowing medium along the tube or channel to control or modify the conditions around the object.

2. A method as claimed in claim 1 in which spacers are introduced into the tube or channel between successive cellular entities or groups of cellular entities .

3. A method as claimed in claim 1 in which the cellular entities are introduced into containers being located in the tube or channel.

4. A method as claimed in any preceding claim in which the object is caused to move towards the constriction by sedimentation, or angular acceleration.

5. A method as claimed in any preceding claim in which the object is caused to move towards the constriction by flowing the medium whilst the channel or tube is inclined away from a horizontal • orientation.

6. A method as claimed in any preceding claim in which the tube is a plastic or glass capillary tube.

7. A method as claimed in any preceding claim in which the channel is a channel formed in a microfluidic device .

8. An apparatus for culturing embryos and/or other cellular entities comprising a tube or flow channel adapted to hold one or more cellular entities, the flow channel having an inlet through which a cellular entity may be introduced, and a source of fluid for generating a flow of fluid in the flow channel towards a constriction past which the cellular entity cannot move in use, the apparatus being adapted for use such that the cellular entity moves in the tube or channel avoiding substantial interaction with the wall of the tube or channel.

9. An apparatus as claimed in claim 8 in which the tube or channel is inclined away from horizontal so as to avoid substantial interaction with the walls of the tube or channel.

10. An apparatus as claimed in claim 9 in which- the tube or channel makes an angle of greater than 20 degrees

^■to the horizontal.

11. An apparatus as claimed in claim 10 in which the tube or channel makes an angle of greater than 40 degrees to the horizontal.

12. An apparatus as claimed in claim 10 in which the tube or channel makes an angle of between 30 and 40 degrees to the horizontal.

13. An apparatus as claimed in any one of claims 10 to 12 in which the apparatus is adapted so that the tube or channel can be returned to substantially horizontal once the entity has reached its desired resting place adjacent the constriction.

14. An apparatus as claimed in any one of claims 8 to 13 in which the tube or flow channel comprises a plastic or glass capillary tube.

15. An apparatus as claimed in any one of claims 8 to 13 in which the tube or flow channel comprises a machined or moulded bore in a solid substrate.

16. An apparatus as claimed in any one of claims 8 to 13 in which the flow channel is a channel formed within a microfluidic device.

17. An apparatus as claimed in any one of claims 8 to 16 in which the constriction comprises a narrowing of the flow channel.

18. An apparatus as claimed in any one of claims claim 8 to 16 in which the constriction comprises a porous element which allows a fluid and/or medium to pass therethrough .

19. An apparatus as claimed in any one of claims 8 to 18 in which the constriction is moveable.

20. An apparatus as claimed in claim 19 in which the constriction includes a magnetic material.

21. An apparatus as claimed in any one of claims 8 to 20 in which the tube or flow channel is adapted such that the cellular entity can move from the inlet to the constriction in a substantially straight line.

22. An apparatus as claimed in any one of claims 8 to 21 in which one or more moveable spacer means are provided in the tube or flow channel for location between adjacent cellular entities in use.

23. An apparatus as claimed in any one of claims 8 to 21 in which one or more moveable container means adapted to receive one or more cellular entities are provided in the tube or flow channel to hold one or more cellular entities in use.

24. An apparatus as claimed in claim 22 in which the one or more spacers are adapted to allow a fluid or medium to flow past in the tube or flow channel whilst preventing cellular entities from flowing past them.

25. An apparatus as claimed in claim 22 or 24 in which the spacers comprise a porous material.

26. An apparatus as claimed in claim 22, 24 or 25 in which the spacers have openings through which a fluid or medium can pass in use.

27. An apparatus as claimed in claim 23 in which the one or more container means are adapted to allow a fluid or medium to flow past in the flow channel whilst retaining the one or more cellular entities.

28. An apparatus as claimed in claim 23 or 27 in which the container means comprise a porous material.

29. An apparatus as claimed in any one of claim 23, 27 or 28 in which the container means have openings through which a fluid or medium can pass in use.

30. An apparatus as specified in any one of claims 22, 24, 25 or 26 in which the spacers include a magnetic material .

31. An apparatus as specified in any one of claims 23, 27, 2.8 or 29 in which the container means include a magnetic material.

32. An apparatus as claimed in claim 19 including actuating means for moving the moveable constriction.

33. An apparatus as claimed in claim 20 including magnetic actuator means for moving the moveable constriction which includes a magnetic material.

34. An apparatus as claimed in any one of claims 23,, 27, 28 or 29 including actuating means for moving the one or more moveable container means.

35. An apparatus as claimed in any one of claims 22, 24, 25 or 26 including actuating means for moving the one or. more moveable spacers .

36. An apparatus as claimed in claim 30 including magnetic actuating means for moving the one or more moveable spacer means.

37. An apparatus as claimed in claim 31 including magnetic actuating means for moving the one or more moveable container means .

38. An appliance for transporting cellular entities comprising an apparatus as claimed in any one of claims 8 to 37 and means for controlling the temperature of the one or more cellular entities in use.

39. An appliance as claimed in claim 39 further comprising means for reversibly detaching the source of fluid from the tube or channel before transport.

40. An appliance for transporting cellular entities as claimed in claim 38 further comprising a fluid flow system for flowing a medium through the tube or flow channel in use.