Patent Application
20100196871
This invention relates to apparatus and methods for culturing cells, maturing oocytes and culturing embryos and other cellular structures in vitro, and means of transportation of cells, oocytes, embryos and other cellular structures.
Various apparatus and methods are known for maturing oocytes and culturing embryos in vitro. In standard practice these processes are achieved using conventional tools such as Petri dishes and well-plates with large wells, such as 4-well to 24-well plates, to contain the oocyte or embryo and maturation or culture media. The oocytes 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 oocytes in particular, may be cultured in the presence of other cells, such as cumulus cells. Maturation or culture is often done in microdrops of media in a Petri dish or well plate, the media covered by inert oil, the media being free to exchange gas with the environment in the incubator. Respiration of the embryo(s) or oocyte(s) is sustained by diffusion of oxygen through a relatively shallow depth (a few mm) of media and oil. The media is usually bicarbonate buffered and the pH is kept constant by means of equilibrium with CO2 in the incubator atmosphere. In some conventional maturation or culture procedures the volume of the media environment in which the oocyte or embryo is contained is important—there is evidence that maturation and culture is more successful if several oocytes or embryos are present together in a small volume of media. This auto/paracrine effect is thought to result from trace chemical substances produced by a first oocyte or embryo affecting the development of a second. Therefore small volumes of media per embryo or oocyte are used—typically 10-20 μl per embryo for bovine embryos, though this volume is smaller in some protocols.
Such systems are unsuitable for transport of embryos or oocytes, and in general practice these are transported, in a portable incubator, inside a sealed vial completely filled with media. This has the disadvantage that gas transport to the embryos or oocytes is from the media only, rather than from a gas atmosphere separated from the embryo(s) or oocyte(s) by a thin liquid layer. The increased diffusion limitation and relatively low solubility of oxygen in aqueous media may lead under some circumstances to development of a hypoxic environment around some or all of the embryos. There is a secondary effect of loss of gas from the media to the atmosphere through the walls of the vial if this has significant permeability. Also, a considerable volume of media is typically used—much larger than the typical volume per embryo used in microdrop culture—so negating the beneficial effects of group culture.
A further requirement is the ability to access certain embryos at a given time at the point of use (typically embryo transfer (ET)). An effective embryo transport apparatus should be usable away from conventional laboratory facilities, allowing a subset of the embryos to be removed while maintaining a controlled gas atmosphere for the unused embryos. This means that ideally a transport apparatus should have partitioned gas environments that can be opened selectively, or a means to replenish the common gas environment from a gas source; though the latter course will require a gas cylinder with consequent weight penalty and regulatory complication.
A further requirement is the ability for transport to be done using conventional shipping in which transport might be in any orientation, the transportable incubator in which the transport container is housed might be dropped or shocked, and shipping might take a significant part of the total embryo culture time, for example up to 72 hours in cases where delays are encountered.
The transport container should also be easy to use and of low cost, as it is preferably a single use, disposable item in common with most laboratory culture equipment. To this end, it is advantageous if the transport apparatus is designed to make use in its design or assembly of conventional laboratory components so far as is practical. The material of the container should be inert at incubation temperature (around 37 C) with respect to leaching of component substances into the media, to avoid the possibility of embryotoxicity. There is widespread doubt about the applicability of relatively low melting point polymers such as polyethylene, polypropylene and other materials such as are found in supplies and containers for cryopreservation, when operated at incubation temperatures for long periods (several hours or more).
A number of apparatus and methods have been proposed for transportation of embryos while maintaining improved culture conditions. None of these have addressed satisfactorily the above concerns.
Seidel et al., U.S. Pat. No. 7,094,527, disclose an embryo transportation apparatus and method comprising a 0.1-0.5 ml volume ET straw filled with media, oocytes and sperm (to achieve fertilisation in situ and then culture and transport the resulting embryos), heat sealed and enclosed within a secondary container or ‘incubation element’ that contains a desired gas atmosphere. Seidel et al. give very few details of the apparatus and the method of use. They do not discuss means to provide gas (oxygen, carbon dioxide) access to the media inside the straw, and the requirement for this is not discussed. By implication the source of controlled gas composition is diffusion through the walls of the straw and the media between the wall and embryos. For a single embryo or a sparse group of embryos resting on the sidewall of the straw oxygen diffusion through the wall will be sufficient to maintain the non-diffusion-limited respiration rate of the embryos (for bovine embryos, approximately 1.4E-14 mol.s-1, H. Shiku et al., Anal. Chem. 73(15) (2001) 3751-8). However, if a closely-spaced group of embryos sediments together, as will happen in particular at a bottom corner of the straw if the straw is transported close to vertically, and especially in a 5% O2 atmosphere as is typically used for culturing bovine embryos, oxygen supply by diffusion to the group of embryos will fall below the optimum value and hypoxic conditions may be established within or around the group. The fixed volume capacities of commercially available straws mean that for the ideal culture volume a set number of embryos should be placed in each straw: e.g., for 10 ul per embryo, 10 for a 0.1 ml straw and 50 for a 0.5 ml straw. If fewer embryos are desired to be shipped, then either the volume per embryo will be greater, or the straw can be partly-filled—but this creates the risk that the liquid column will break up through movement of the straw during transport, leaving embryos in unpredictable liquid volumes, or even dry on the side of the straw. Seidel et al. quote a figure of 10-15 embryos per 50 μl for fertilisation, which is proposed to be done inside the straw, so implying 20-30 presumptive zygotes in a 0.1 ml straw and 100-150 in a 0.5 ml straw, which gives and even greater risk of hypoxia if a substantial proportion of the zygotes are viable and they are closely grouped together at the base or at a corner.
An additional problem is that ET or cryopreservation straws are not designed for prolonged (many hours) contact with media at incubation temperatures: the internal surface area/volume ratio of the straw is high, and the material of the straw is not necessarily inert at incubation temperatures and so may leach trace compounds into the media that compromise embryo development. Embryo straws are intended for use in cryopreservation, at which temperatures the material will leach very slowly, if at all, and the embryos are not metabolically active while in contact with media that may have trace leached compounds within it. The need for the straw to be heat-sealable limits the material to a small group of compounds that have relatively low melting point and consequently have greater surface openness to diffusion of mobile contaminants than polymers with higher melting points.
In summary, the embodiment of Seidel et al. does not achieve the desired ends of a known, predictable gas supply, equivalent operation in any orientation during transport and a controlled, small volume for incubation.
Thouas et al., WO02/074900, disclose incubation inside a capillary open at the ends, in which one or two embryos are placed inside a glass capillary and are incubated preferably while the capillary is vertical, the embryo(s) resting on the liquid/gas meniscus. This provides good gas access to the embryos, and a small, controlled volume of media; the high aspect ratio of the media space means that in fact the effective volume experienced by the embryo(s) in terms of accumulation of auto/paracrine factors will probably be smaller than the total volume, which is in any case much smaller (around 1 ul) than is used in standard protocols. Establishing a 10 μl per embryo culture volume in the system of Thouas et al. would mean the capillary would have to have a much larger diameter and so have poorer retention of the media against movement, shock etc. The glass capillary system does not allow groups of embryos to be cultured inside the capillary away from the meniscus as the glass is impermeable and oxygen transport through the media from the ends of the capillary is too limited. Additionally the system is not operable in any orientation—even a single embryo will suffer limited oxygen supply if it is located in a glass capillary far from the gas supply at the open end.
Ranoux et al., US2006/0228794, disclose an embryo culture container for use primarily in human intravaginal embryo culture, comprising a gas-permeable inner vessel with a closure device for ‘selective access’, at least partially surrounded by a shell that defines a buffer chamber for a controlled gas atmosphere. During incubation in a controlled gas environment the buffer chamber is open to the surrounding environment (e.g. the vagina) via a gas-permeable seal region. When the container is removed from the controlled gas environment the gas path between the surrounding environment and the buffer chamber can be closed by a second closure mechanism associated with the shell. The vessel is adapted for culture of one or a few embryos, and comprises a large chamber, of volume not stated but large enough to admit a catheter or pipette, and a ‘microchamber’, a narrow region of the main chamber, at the bottom of the vessel where the embryos can sediment for inspection. The container of Ranoux et al. addresses some of the concerns for effective embryo transport, but does not achieve a small volume of media per embryo and is not suitable for transport of a group of more than two or three embryos, owing to the use of low-permeability plastic for the inner vessel (which needs to be of rigid material, so precluding use of a high-permeability elastomer), the small dimensions of the microchamber, and the possibility that a group of embryos would accumulate there during transport so potentially inducing hypoxic conditions. The design of the container, with a closure means formed as part of the inner vessel, is inherently difficult to adapt to a much smaller total media volume. It is also a complex and relatively costly design, not well suited to a single-use container for transport of non-human embryos for commercial purposes.
Campbell et al., US2002/0068358, have proposed an apparatus for embryo culture which is adapted for transportation, in which the embryo is retained in a well that has a supply of media and flow generating means to allow the media in the well to be replaced under remote or automatic control. The well is considerably larger than the embryo, so giving poor control of the media environment; there is no provision for oxygen supply to the embryo(s) except through flowing media past them, so precluding build up of beneficial auto/paracrine factors around the embryo(s). Additionally, the design is highly asymmetric, with access to the well through a long tube of significant size and internal volume, and so is unsuitable for operation upside down.
It is an object of the present invention to address these and other difficulties in the design and operation of embryo culture and transport devices of the prior art.
In the description that follows reference will be made to culture and transportation 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 and transportation of oocytes and culturing and transportation 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, oocytes and cells. Therefore the terms maturation and culturing, and oocytes 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 oocytes, or to culturing of embryos, this will be noted.
According to a first aspect, the invention provides a container for culturing and transporting embryos, oocytes and other cellular structures as specified in claims 1 to 20.
According to a second aspect of the invention there is provided an insert for a container for culturing and transporting embryos, oocytes and other cellular structures as specified in claim 21.
According to a third aspect of the present invention, there is provided an apparatus for transporting embryos, oocytes and other cellular structures as specified in claim 22.
According to a fourth aspect of the present invention, there is provided a method of culturing embryos or oocytes as specified in claims 23 to 26.
A preferred embodiment of the present invention is provided by a container comprising a housing, having an at least partly gas-permeable insert that is an interference fit within the housing, the housing and the insert defining at least one gas space and at least one liquid media space in gas communication with one another by diffusion through the insert, and a separable, essentially gas-tight closure means that cooperates with the housing to restrict the passage of gas into the container from the outside environment.
In another preferred embodiment, the container comprises a housing, an insert having an inner face oriented towards the inside of the housing and an outer face, the insert being mounted within the housing so defining a gas space between the inner face of the insert and the housing, the insert comprising a recess open to the outer face, the recess adapted to be a space for liquid media. The insert is preferably formed from a gas-permeable polymer and has one or more walls thin enough (either in whole or part) to give good gas exchange (oxygen and carbon dioxide) with the atmosphere in the gas space. The container further comprises a cap that closes and seals both the gas space and the media space.
Optionally the insert when in place in the housing substantially closes the gas space from the external atmosphere so that without the cap fitted gas exchange between the gas space and the atmosphere is primarily by diffusion through the material of the insert.
Optionally the insert comprises a vent channel or one or more regions of open-pore material so that a gas-phase path exists between the gas space and the atmosphere, this path being closed along with the media space when the cap is fitted.
In an alternative embodiment the container comprises a housing, an insert that is mounted within the housing defining below it a media space, the insert and housing defining a gas space that is open to the atmosphere but which can be closed by a cap.
In a further preferred embodiment the container comprises a housing having a number of wells, and an insert having a number of projections that are adapted to fit into the wells, so defining below them a gas space in each well, each projection comprising a recess open to the outer face of the insert, the recess adapted to be a space for liquid media. The insert is preferably formed from a gas-permeable polymer and has one or more walls thin enough (either in whole or part) to give good gas exchange (oxygen and carbon dioxide) with the atmosphere in the gas space. The container further comprises a cap means that seals both the gas spaces and the media spaces.
Optionally the insert substantially closes the gas space from the external atmosphere so that without the cap fitted gas exchange between the gas space and the atmosphere is by diffusion through the material of the insert.
Optionally the insert comprises a vent channel or one or more regions of open-pore material so that a gas-phase path exists between the gas space and the atmosphere, this path being closed along with the media space when the cap is fitted.
Optionally a further secondary lid or container is provided that encloses the container so establishing a controlled gas atmosphere on the top side of the container, the bottom side or both.
In an alternative embodiment the container comprises a housing having a number of wells, and an insert having a number of projections that are adapted to fit into the wells, so defining below them a media space in each well, each projection comprising a recess open to the outer face of the insert, the recess adapted to be a channel for diffusion of gas to the media space. The insert is preferably formed from a gas-permeable polymer and has one or more walls thin enough (either in whole or part) to give good gas exchange (oxygen and carbon dioxide) of the objects in the media with the atmosphere in the gas space. The container further comprises a cap that seals both the gas spaces and the media spaces.
a shows a first view of a first embodiment of an apparatus according to the invention
b shows a second view of a first embodiment of an apparatus according to the invention
c shows a third view of a first embodiment of an apparatus according to the invention
a shows a first view of a third embodiment of an apparatus according to the invention
b shows a second view of third embodiment of an apparatus according to the invention
a shows a seventh embodiment of an apparatus according to the invention
b shows an eighth embodiment of an apparatus according to the invention
a shows an eleventh embodiment of an apparatus according to the invention
b shows a detail of an eleventh embodiment of an apparatus according to the invention
c shows a detail of an eleventh embodiment of an apparatus according to the invention
a shows a fourteenth embodiment of an apparatus according to the invention.
b shows a fifteenth embodiment of an apparatus according to the invention.
b shows the bottom view of the insert in
c show the configuration of the housing 12, cap 14 and insert 16 as the container 10 is assembled. The insert is sized so that it is a friction fit to the container, so on first insertion into the container it is retained with the outer part projecting from the top of the housing. The media space 18 can now be filled with media and embryo(s). To seal the container the cap is pressed down onto the top of the insert and either pushed or screwed (depending on the cap fitting method) down onto the housing. The inner face 34 of the cap bears on the sealing face 36 of the insert, and as the cap moves downwards onto the housing the insert is forced down into the housing against a reaction force from friction between the outer ring 32 and the inner wall of the housing. When the cap is fully screwed into place, the seal face 38 of the cap seals onto the top edge 40 of the housing, so providing a substantially gas-tight seal; the inner face 34 of the cap is pressed hard up against the seal face 36 of the insert, and the cap simultaneously closes both the media space 18 and gas access to and from the gas space 28. In some embodiments a vent channel 42 is provided that acts to vent gas from the gas space 28 when the insert 16 is forced down by the cap as this is fitted. If present, the vent channel is preferably of small cross section to limit the degree of gas exchange between the gas space and the external atmosphere via the channel.
While the invention is not limited by having the features shown in
The housing 12 and cap 14 can be made from a range of rigid materials, such as plastics, glass, metal or the like. Advantageously the housing and cap are a mass-produced pair, such as a standard laboratory vial plus cap. The insert 16 is then designed and made to fit the pre-existing vial. Appropriate vials are for example Bibby Sterilin Bijou 7 ml (Barloworld Scientific Inc.), in polystyrene or glass. The insert is preferably at least partly resilient—for example a moulded elastomeric component, or a component comprising a rigid core with an elastomeric outer component to give a close friction fit against the wall of the housing. In some embodiments the housing material is impermeable to gas, e.g. glass. This allows the gas space to be filled with a chosen gas composition (e.g. 5% oxygen, 5% carbon dioxide, nitrogen) and to retain the composition while in a different atmosphere, e.g. air. In other embodiments the housing may be permeable to gas, allowing the gas space to exchange gas with its surroundings. If the housing is permeable, the container may be used in air provided the permeability is low enough that the gas atmosphere is retained for the desired culture period. Example 1 describes a preferred design for this embodiment.
The insert material is preferably gas-permeable, and in preferred embodiments is sufficiently permeable that, together with the design of the wall and base thickness and the dimensions of the gas channels 26, the impedance to diffusion of oxygen through the insert is not limiting on oxygen supply to embryos within the gas space. In preferred embodiments the insert is moulded from PDMS, for reasons of high gas permeability, ease of moulding and sterilisation, and lack of embryo toxicity. Suitable PDMS compounds are Silastic S and Sylgard 184 (both from Dow Corning), but others will be suitable also (subject to embryo toxicity checks).
In this embodiment, and in all other embodiments of the invention, the material of the insert may be coated with a surface coating, or the surface chemically modified, by any means known in the art in order to modify the surface properties of the insert, in particular in the region contacting the media space. This might be done to modify for example the absorption or adsorption properties of PDMS, the gas permeability or the ease with which embryos, oocytes or cells adhere to the surface.
In use the gas atmosphere may be introduced into the gas space 28 either by actively flushing the gas space with a gas stream before placing the insert in the housing, or the housing with the insert fitted in place (preferably partially in place as shown in
The insert might also be incubated with water or media of the type to be used in place in the media space, in order that any adsorption or absorption processes associated with the properties of the insert material are completed before the culture media is dispensed.
When the gas space has reached equilibrium the container can be removed from the incubator and loaded with media and embryo(s). In preferred embodiments the equilibration time is sufficiently long that this can be done in the laboratory without significant change in the composition of the gas in the gas space. The cap 14 is then fitted, forcing the insert 18 down into the housing and sealing both the media space and gas access (via diffusion through the insert material) to the gas space.
The container 10 is sized to contain a media space of volume chosen according to the preferred media volume per embryo and number of embryos desired to be cultured. Preferred embodiments have media space volumes between 1 ul and 5 ml, more preferred embodiments volumes between 10 μl and 1 ml and most preferred embodiments between 50 μl and 500 μl. The container may house embryos at any preferred ratio of media volume per embryo. Preferred embodiments contain embryos at ratios between 1 embryo per 0.5 ul and 1 embryo per 100 μl, more preferred embodiments ratios between 1 embryo per 1 ul and 1 embryo per 100 μl, and most preferred embodiments ratios between 1 embryo per 2 ul and 1 embryo per 10 μl. It is a particular advantage of the invention over the containers and culture apparatus of the prior art that small volumes of media per embryo can be used in a configuration that is easy to access and can be shipped in a robust manner, with substantially all the oxygen requirement of the embryos met whatever the orientation of the container.
The housing 12 is sized to contain sufficient gas to sustain the metabolism of the embryos in the container and to allow for leakage from the gas space to the surrounding atmosphere where that differs from the gas in the gas space. Such leakage will occur through the walls of the housing, if that is not made from an impermeable material such as glass; through the seal between the cap and the housing, and through the material of the cap. For a housing made from polystyrene preferred volumes of the gas space 28 are between 1 ml and 20 ml. Volumes less than 1 ml will give relatively little time before the composition in the gas space changes as a result of diffusion through the housing wall; volumes greater than 20 ml are unnecessary for preferred numbers of embryos in the media space and typical times in transit (see example 1 below). However, the volume in practice can be set by choice of practically available and/or easily handlable housings and caps and any volume of gas space 28 is within the compass of the invention.
The dimensions of the gas channels 26 are set by the amount of oxygen required by the embryos in the media space, in the condition that the container is oriented other than in the standard base-down way, in which case the embryos will have sedimented onto a wall 22 of the media space or onto the surface 34 of the cap. In this case they will draw oxygen by diffusion through the material of the wall or, if they are on the cap surface 34, by hemispherical diffusion through the media, ultimately from the wall 22. In this latter condition the availability of a gas-phase oxygen source close by avoids limitation of oxygen supply by diffusion, and in preferred embodiments the channels 26 approach close to the sealing face 36 of the insert in order to deliver gas-phase oxygen in close proximity to the cap surface 34. The dimensions of the gas channels 26 are unimportant in terms of the rate of diffusion of oxygen along them—diffusion in the gas phase is so much faster than in the media or insert material that effectively no diffusion limitation will exist along the length of the channels. The dimensions of channels 26 are therefore set effectively by practical moulding considerations—they are formed, for example, by a narrow upstanding feature on the mould and so are made as large as is practical to minimise the effect of wear and damage on the mould tool.
The insert 16 for container 50 is designed in a similar way to that for container 10, but is now designed to be displaced further into the housing when the lid and lid component are forced down by fitting of the lid. As in container 10, fitting the lid seals both the media space 18 and the gas space 28 in one action.
In an alternative embodiment, the lid component has a plug-like form so that it fits inside, and seals against, the upper portion of the wall of the media space 18. In this case the lid component might be separate from the lid, and adapted to be fitted to the insert 16 before the cap 14 is fitted, lid component 52 then acting as a stopper to seal media space 18 and cap 14 when fitted acting to seal both the top of the media space and the gas space 28 from the outside atmosphere.
In preferred embodiments the lid component 52 is moulded in PDMS, and where lid component 52 is adapted to fit inside insert 16 the lid component and insert are preferably of different grades of hardness of PDMS. Example 2 shows the effect of this embodiment on availability of oxygen to embryos resting on the surface 56 of the lid component.
a and
b shows the base of the insert 16, with B-B showing the position of the cross section in
c-e show cross sections of a further embodiment of the invention, with similar parts to the embodiment in
In use the container 90 is pre-gassed by leaving it with the cap removed or only partially closed in a controlled gas atmosphere. Gas diffuses from the atmosphere into the pores of the insert, which act as a gas reservoir when the cap is closed. This embodiment has an operational advantage in certain circumstances in that the porous insert 16 might be gassed separately from the vial, being taken from the gas atmosphere and placed in the vial just before use. In that way the gas atmosphere is carried into the container within the pores of the insert; the insert physically displaces the air from the housing when it is inserted. The pore density and permeability are chosen so that the insert stores sufficient gas (determined by the pore volume) while having a suitable time constant for in-gassing in the incubator and slow enough out-gassing while handling in the environment that the gas atmosphere established inside the container is close enough to that desired.
a shows a further embodiment of the invention. Container 110 comprises a housing 112, a cap 114 and an insert 116. In use the insert 116 is positioned inside the housing to define a media space 118 between the insert and the closed end of the housing and a gas space 128 between the insert and the end of the housing closed by the cap. The insert is a close fit to the housing so substantially preventing media from passing the insert to reach the gas space. In preferred embodiments the insert comprises an open cell porous hydrophobic material 122, which allows gas to pass through it but substantially prevents media from entering the pores. In preferred embodiments the housing 112 is tapered from open end to closed end to allow easy movement of the insert to its desired position. In preferred embodiments the insert comprises compliant material, so dimensioned that in its desired position it fits closely to the wall of the housing. The housing 112 may be formed from a range of materials, the choice being guided by gas permeability as before. As the embryo(s) is/are in direct contact with the material of the tube embryo toxicity also needs to be taken into account. A suitable housing is the Scimart 2.5 ml polystyrene sample vials, product code SAR-55483 and push fit cap SAR-65782. Suitable material for the insert is VYON™, as previously described.
In use, a known volume of media and embryo(s) are pipetted into the base of the housing 112; the insert is then fitted and moved down the housing until it has displaced substantially all the air between its bottom surface and the top surface of the media; in preferred methods of use the insert will be in contact with the media with no, or only small, air bubbles at the interface. The gas space 128 is then flushed with gas of the desired composition and the cap 114 fitted. The dimensions of the housing and insert are chosen so that the insert fits tightly to the housing at the desired position, i.e. when it is in contact with the media. In order to minimise disturbance to the gas concentration in the media the porous material of the insert may be pre-equilibrated with the chosen gas composition prior to fitting.
An optional additional layer 124 may be provided on the surface of the insert adjacent to the media space as described above for the embodiment shown in
b shows a further embodiment in which container 150 comprises similar parts to the embodiment 110 in
In use the container may be provided as a set of a housing 112, insert 116 and cap 114, the insert being placed in the housing before use; or the insert may be pre-fitted into the housing so as to define the desired media space volume. The media space is then filled by means of the needle or pipette. The slit or opening is preferably sized so that air is displaced from the media space around the needle or pipette while media flows in, leaving no, or only small, bubbles in the media space. The gas space 128 is then gassed with the desired composition and the cap fitted.
The container might also be used as described for the embodiment in
In a preferred embodiment the insert is moulded from a gas-permeable elastomer such as PDMS, which can be formed to have good puncture/re-seal properties as exploited in septa. It will be apparent that the housing can be of different shape, for example a screw-top vial as in
While the inserts 116 in
In use the insert 216 is mounted on the housing 212 and the projections 236 forced home into the wells in the housing. The assembly can now be incubated in the desired gas atmosphere for the time needed for the gas to exchange with air in the gas spaces 228. The insert may also be incubated with media in the media spaces to condition the insert well material, for example through adsorption of media components onto/into the walls. This is particularly relevant if the insert 216 is formed from an elastomer such as PDMS, some grades of which have an affinity for water sufficiently high to deplete media content from a small media volume. Fresh media and embryo(s) are then dispensed into the wells, and the cap means 214 fitted. The closed container will exchange gas with the external atmosphere through the material of the housing 12, the insert 216 and cap means 214. These materials may be chosen to give slow enough gas exchange—particularly loss of carbon dioxide—to be acceptable in use. If necessary a secondary container 240 may be provided to assist control of gas exchange.
The apparatus of
a shows a further embodiment adapted to hold embryos either singly or in small groups in a small volume of media in separate media spaces, so allowing their identity to be tracked. Container 300 comprises a housing 212 as before, the housing comprising one or more wells 226, for example in microtitre plate format. In a preferred embodiment housing 212 is a conventional microtitre plate or microtitre strip, or assembly of strips, of appropriate dimensions (number of wells, well size and shape) to hold the desired number of embryos 220 in the chosen volume of media. The container further comprises an insert 216 provided with one or more well-closing projections 236 each adapted to fit closely a well in the housing 212, defining within one or more of the wells of the housing a media space 218. In preferred embodiments each well 226 of the housing 212 is fitted by a projection 236, leading to a multiplicity of closable media spaces. The insert 216 is similar to flexible elastomeric microtitre plate closures as known in the art, but differs in that it is designed to fit more deeply into the wells and to have known and high gas transport capability into the wells. The media spaces are in diffusive communication with the common gas space 228, preferably by means of a gas channel 246 formed in each of the projections. In preferred embodiments the insert 216 is formed from a gas-permeable elastomeric material such as PDMS, with gas permeability characteristics adequate to allow transport of oxygen across the membrane 224 that closes the well, sufficient to sustain the chosen number of embryos in the well. The gas space 228 is closed by a secondary container 240, comprising a cover and optionally a base. The base may be omitted in some embodiments, for example if a substantially gas-tight seal is formed between the cover and the housing 212, and the base of the wells in housing 212 has low gas permeability—for example if housing 212 is a glass-bottomed microtitre plate.
In use, embryos and media are dispensed into the wells 226 and the insert 216 is fitted. The insert 216 may be formed from a flexible elastomeric material so that it can be inserted into each row of wells in the housing in turn. An insertion aid can be used, such as a roller as known in present technology for fitting flexible microtitre plate closures, or a tool with multiple projections that fit into the gas channels 246 can be used to press the projections 236 into the wells. The insert projections fit into the wells so as to exclude the majority or substantially all of the air initially in the well above the media, and to this end the degree of flexibility of the projections is chosen to allow the air to be displaced. Residual air bubbles in the media spaces 218 rapidly equilibrate with gas from the gas space 228 and so in general are not disadvantageous in this application. The gas space 228 is filled with a controlled gas atmosphere in any convenient way, for example being flushed with gas through optional inlet and outlet ports 242, or being provided with a porous gas-containing body (not shown) as analogous in function to component 122 in
b shows a modification to the insert 216 in which the membrane 224 that closes the media space 218 has a slit 244. The slit 244 is normally closed but can be opened, e.g. by air pressure built up in the well by insertion of the projection 236 into the well, or by insertion of a pipette through it from above once the projection has been fitted. The membrane 224 might be shaped to have a recess on the underside where the slit is located to facilitate opening of the slit. By this means air is expelled easily on fitting and/or the contents of the wells can be accessed individually by means of a pipette or needle to allow removal and/or dispensing of media and embryos, without removing the insert from the well or a group of wells. The membrane 224 might alternatively be adapted to allow puncture by a needle or pipette, and optionally re-closure in the manner of a septum, to achieve the same purpose.
c shows a modification to the insert 216 in which part of the base of the projection 236 is formed from a porous component 248, for example formed from a porous hydrophobic material such as VYON™ as already described, which may be a push-fit into the projection. Such a component allows ready venting of air from the well on insertion of the projection, and good gas transport between the media space and the gas space. In some embodiments the component 248 may be adapted to be removable from the projection, so allowing the contents of the well to be accessed.
Typical dimensions of the container in
In certain applications it may be desirable for the media to contact a material with conventional, preferably low adsorption and absorption properties, or which can be coated to control cell adhesion properties. Polystyrene and polycarbonate are examples of materials conventionally used in cell biology and embryology which have well-understood surface properties, and oxygen permeability high enough for them to be used in the invention provided wall thicknesses are kept low.
In use the insert 516 is first fitted part-way into the housing 512 as in previous embodiments, the outer ring being sized to give a friction fit with the housing; when the cap 514 is fitted the inner surface of the cap, or an optional cap insert or seal component 552, bears down onto the seal surface 524, forcing the insert against friction down into the housing and sealing the media space 518. The media and embryos in the media space now contact the material of the inner component and that of the cap or cap seal 552, which can be chosen to be as inert to absorption as is required. In some embodiments the seal region 524 of the inner component may be omitted, the seal then being made against the upper surface of the outer ring. If the seal region 524 is present, then in preferred embodiments it stops short of the inner diameter of the housing, so allowing a portion of the upper surface of the outer ring to be exposed before the cap is fitted. This allows the gas space 528 to be filled with gas of the desired composition by diffusion through the outer ring 532.
In preferred embodiments, the volume of the media space is between 10 μl and 1 ml; in more preferred embodiments between 50 and 500 μl and in most preferred embodiments, between 50 and 200 μl. In an example of the container 510, the housing is formed from a Bibby Sterilin 7 ml bijou vial, the outer ring is moulded from PDMS, e.g. Silastic S (Dow Corning) and the inner component may be formed by for example vacuum forming in polystyrene. The wall thickness of the inner component is preferably between 0.1 mm and 1 mm; more preferably between 0.15 mm and 0.3 mm.
a shows a further embodiment, in which the insert is formed from a moulded thin polymer section and is optionally a separable component, removable from the housing, a separable component that mounts permanently into the housing in use, for example by a snap-fit means, or might be bonded permanently to it. The container 610 comprises a housing 612, cap 614 and insert 616, the insert defining a media space 618 and the insert and housing together defining a gas space 620 as before. In one embodiment the insert 616 is a separate moulded component that rests on the upper rim of the housing at position 624, and when the cap 614 is fitted is brought into firm contact with the rim through force from the cap. Seal component 622 acts to compress the insert evenly around the rim, and is preferably permeable to gas to allow gas supply via the lid as described before. The rim of the housing is preferably smooth and flat in region 624 to allow good sealing. In some embodiments the rim of insert 616 is coated with or enveloped in a region of elastomer, such as PDMS, to assist sealing.
In a preferred embodiment insert 616 is bonded to housing 612 in region 624 with a permanent bond, such as ultrasonic bonding or heat-sealing. In some embodiments the bond is formed so as to be substantially gas-tight.
In preferred embodiments the insert 616 comprises one or more apertures 626, which act to allow a known degree of diffusion of gas between the gas space 620 and an external atmosphere when the cap 614 is absent. This allows ready gassing of the interior of the container when placed in an incubator, and also allows gas communication between the gas space and the seal component 622 in embodiments where this is present, and gas permeable.
The insert may have a stepped cross-section, as in
In a further preferred embodiment the insert 616 is supported within the housing by means of features formed on the housing, the insert or both.
In a further alternative embodiment, the housing and the rim region 644 are so dimensioned that the rim region extends to the base of the housing, so supporting the insert at the correct position; alternatively a further component, such as a cylindrical insert, may be positioned in the base of the housing so as to support the rim region 644 in the same way as shown for the features 642.
In a preferred embodiment the housing may be formed from polystyrene, and the insert 616 may be formed by moulding or vacuum forming, again in polystyrene. Other polymers may be used as appropriate for the required dimensions. The insert wall surrounding the media space is preferably thin to allow adequate gas permeation across it. In preferred embodiments the wall is less than about 0.5 mm thick, in more preferred embodiments less than 0.3 mm and in most preferred embodiments between 0.2 and 0.3 mm thick. The region of the insert against which the seal 622 bears is thick enough to give rigidity to support the sealing pressure, and may be equipped with strengthening ribs (not shown in
In an example of use, the embodiment in
The container in
Alternatively, the insert may be a frictional fit to the housing as before, that reaction force being provided by friction, or features are provided on the housing, insert or both to provide the reaction force in the manner of
The insert 756 in this embodiment may be formed in a number of ways. In a preferred embodiment the region 730 is formed from moulded polystrene, and is supported at a depth within the housing by features moulded in the housing and/or the region 730. In
The present invention has application also in culture of an extended multicellular structure having a high density of cells per unit area, such as a tissue sample, a skin sample, an organ or part thereof, a cellular layer on a support membrane, or a product in the field of regenerative medicine such as replacement tissue, skin, corneal tissue etc., where availability of oxygen is an important requirement while keeping the concentration of CO2 dissolved in the media, and hence the pH, within acceptable limits. Conventional culture systems known in the art, such as petri dishes, well plates and the like rely for oxygen availability mainly on diffusion from a gas/media interface, through a depth of media to the cellular structure, and in particular often only facilitate oxygen availability from one side of the cellular structure: for example, in the case of a culture dish formed from low or moderate gas permeability material, to the side closer to the atmosphere above the media. Just as for embryos, oocytes or other cellular structures as previously disclosed, culture of an extended cell layer or tissue sample can be improved by facilitating supply of oxygen to more than one side of the culture space, and in particular to the two sides parallel to the major surfaces of the extended cellular structure. Transport of such cellular structures is also not facilitated by presently available apparatus, as it is not adapted for transport while providing ready oxygen supply. In the situation where transport is done in a container wholly or mainly filled with media, the low rate of diffusion of oxygen through the media may lead to disadvantageously low oxygen concentration in the vicinity of the cellular structure; if the container is only partially filled with media there is a risk that if the container is turned upside down during transport part of the cellular structure will be left without media coverage.
An apparatus for culture of an extended cellular structure in media with oxygen access from both sides of a body of media is disclosed in application US2008/0092027. However, that apparatus is a simple modification of existing culture plate formats; it is unsuitable for operation outside a conventional incubator and makes no provision for operation except at standard horizontal orientation, and hence is unsuitable for culture during transport. Also the geometry and design of the apparatus disclosed does not control, or aim to reduce, the diffusion distance of oxygen through the media, and hence optimise the access of oxygen to the cellular structure.
a shows a further embodiment 800 of the invention, in which the media space 818 has a flat aspect ratio and in some embodiments is adapted to retain and locate an extended cellular structure such as those listed above. The container comprises a housing 812, a closure 814 that engages with the housing, a media space 818 and a gas space 828. The closure 814 is adapted to form an essentially gas-tight seal at least in the region of areas 838 and 840 when it is fully fitted to the housing, separating the interior of the housing from the external environment, and also closes the media space 818. The housing 812 comprises a region 824 of higher gas permeability per unit area that forms a barrier between the media space 818 and the gas space 828, and a region 830 that is preferably thicker and capable of supporting a gas-tight seal based on pressure on its upper surface. The region 830 will in general be of lower gas permeability per unit area than region 824. The high permeability barrier region 824 preferably forms most or essentially all of the wall of media space 818 facing the gas space 828. In some embodiments the region 824 extends to the sidewalls 820 of the media space. In the embodiment 800 the media space has a flat geometry, smaller parallel to barrier region 824 than perpendicular to it, so allowing improved diffusion of gas from the gas space, through the barrier 824 and media in the media space, to the side of the media space distant from region 824. Optionally a gas port 826 is provided in the region 830 that allows ready gas exchange between the gas space 828 and the external environment when the closure is loosened or removed, so allowing the gas space to be equilibrated rapidly with a desired atmosphere inside the incubator.
The housing 812 has a lower permeability to gas (e.g. O2, CO2) in regions 832 separated by the gas seal region 840 from the region 824, in order to contain the desired gas atmosphere in the gas space 82S. This may be achieved by using the same material but with a thicker profile or by using a different material of lower permeability, or by coating or otherwise reducing the permeability of the wall of the housing. The housing may be formed from bonded subcomponents as would be known to those skilled in the art so as to provide higher and lower gas permeabilities in the desired regions.
In
c shows a further embodiment 804 of the invention with similar components and features to the embodiment in
Optionally, in all embodiments of the invention, a gas closure means may be provided that allows access from the exterior to the gas space, so allowing exchange of gas between a gas environment, for example in a conventional gas incubator, and the gas space while the media space is closed by the closure 814.
The closure 812 may engage with the housing in any manner known in the art, though a fitting that does not substantially pressurise the interior of the container is preferred. Preferred embodiments include a screw fitting with a thread either on the inside or the outside of the housing, i.e. either inside or outside the seal region 838, 840. A snap fitting may also be used. The closure may have a partial closure condition that leaves a gas path open from the exterior to the interior of the housing and a fully closed condition that restricts gas passage between the exterior and the interior. For example in the case that the closure 814 has a screw thread engaging with the housing the gas pathway to the interior might be open when the lid is partially screwed down and closed when it is fully screwed down. One or more location means may be provided on the closure, the housing or both to facilitate location in one or more closure condition. For example, a protrusion and matching recess might be used to provide a ‘click-stop’ action to locate the closure in conditions with the open and the closed gas pathway.
d shows a further embodiment 806 in which the closure 814 is adapted to have a first condition in which it closes the media space 818 while leaving a gas pathway open from the exterior to the interior of the container, and a second condition in which the media space is closed and the gas pathway is also closed. In preferred embodiments the sealing component 822 in the lid is adapted to close the media space while leaving further compression possible as the closure is moved from the first condition towards the second. In
In this and other embodiments the seal component and the closure may be dimensioned so that the seal surface closes the media space 818 in a first condition of the closure, while leaving a gas pathway open between the interior of the container and the exterior. As shown in
One or more ports 856 through the closure may be provided that assist gas access to the interior of the housing in the open condition. A gas pathway might also be provided via the region 858 between the closure and housing, for example via screw thread, a groove in the surface of the closure or the housing or similar means. The container might be approximately cylindrical and the closure 814 might be a screw fit to the housing; alternatively the closure might be a snap-fit to the housing, preferably with two snap-fit positions, a first position as shown in
The embodiments in
Any size or shape of the container is within the scope of the invention. In preferred embodiments the dimensions of the container and its features are chosen in the light of the materials chosen for fabrication, taking into account their permeability to gas and the thickness or other dimensions or features that are needed to render them suitable for use in the design, for example to give them mechanical stability. They are also chosen to suit the type of cellular structure to be cultured and/or transported, taking into account aspects such as its size, any support or backing materials such as support membranes associated with the cellular structure, its oxygen demand and its consumption of nutrients dissolved in the media such as glucose. For certain cellular structures such as embryos or oocytes preferred volumes of media per structure are used for culture and so the media space has preferred volumes which depend on the number of cellular structures to be cultured and/or transported together, as previously described. In preferred embodiments for embryos or oocytes the volume of the media space will be in the range 0.1-100 ul per embryo or oocyte to be transported.
Other cellular structures do not require a preferred volume; rather considerations of oxygen access and easy handling are important. Diffusion through the media is usually the limiting factor on oxygen supply to the cellular structure, and in preferred embodiments the diffusion distance through the media is chosen to provide a concentration at the cellular structure that is estimated to be appropriate for the cellular entity to be cultured. In preferred embodiments for extended cellular structures the maximum distance within the media space from the gas/media barrier to a position where the cellular structure might be located (for example if it is free to sediment)—is preferably between 0.01 and 10 mm, more preferably between 0.01 and 5 mm. In preferred embodiments adapted for extended cellular structures the media space is preferably between 0.1 and 10 mm between its major surfaces, more preferably between 0.5 and 5 mm.
Diffusion limitation through the barrier will also limit the oxygen flux to the cellular structure, or result in a lower oxygen concentration at the cellular structure for a given flux. Dimensions of the one or both barriers depend on the permeability of the barrier structure(s) and are chosen so us not to add excessively to the overall diffusional impedance for oxygen between the gas phase and the media. The barrier may have thicker portions for support and thinner portions for gas diffusion. The following preferred dimensions are for the thinner, gas diffusion portions. Depending on the material used, the barrier is preferably between 0.02 and 10 mm thick and more preferably between 0.05 and 5 mm thick. For example, for a barrier formed mainly from a high permeability elastomer such as PDMS the thickness is preferably in the region 0.2 to 5 mm, for a barrier formed mainly from a high permeability rigid polymer such a TPX the thickness is preferably in the region 0.05 to 1 mm, more preferably in the range 0.05 to 0.4 mm, and for a barrier formed mainly from a lower permeability rigid polymer such a polystyrene the thickness is preferably in the range 0.02 to 0.3 mm, more preferably in the range 0.05 to 0.2 mm. It will be apparent to a skilled person that the thickness of the barrier, or other dimensions of the container, can be chosen appropriately with regard to the permeabilities of the chosen materials.
Typical permeabilities P(O2) for oxygen transport through polymers that might be used for the barrier material are given in units of 10−13 cm3.cm.cm−2.Pa−1 as polystyrene (PS): P(O2)=2; TPX: P(O2)=20; PDMS P(O2)=400. [Goodfellow, Inc., materials supply catalogue (www.goodfellow.com) download 18th April 08. P(O2) is quoted at 25° C. for PS and TPX, at 0° C. for PDMS—for the purpose of order of magnitude estimation in this example the differences between P(O2) at 25° C., 0° C. and the typical operating temperature of 38° C. are not important].
With these parameters, dimensions of the barrier(s) 824 (860) may be chosen based on a known or estimated oxygen flux per unit area to the cellular structure, using Fick's law of diffusion to give the drop in concentration across the barrier(s) as will be apparent to those skilled in the art. For illustration, examples of measurements of oxygen uptake rates (OUR) for different cell types are given in Peng C-A, Paulson B. O., Annals Biomedical Engineering 1996 vol. 24(3) p. 373-381, Cho et al., Biotechnology and Bioengineering 97(1) 2007 p. 188-199, and discussion of oxygen diffusion in media in Mentzen et al. Respiration Physiology 100 (1995) p. 101-106. For example, using OUR figures from these references, for a cellular structure with 106 cells.cm−2, a barrier formed from PS has a preferred thickness range is 0.02-0.1 mm; a TPX barrier has a preferred thickness range of 0.02-0.4 mm and a PDMS barrier has a preferred thickness up to 5 mm. For cellular structures of different OUR per cell and/or different cell densities per unit area, the preferred dimensions may differ from the above.
The container of the invention preferably provides a ready gas diffusion pathway from the gas space to the side of the media space remote from the first barrier. In preferred embodiments this is provided by a gas phase diffusion pathway through a closure gas space 850 and a second barrier 860 (
The gas space 828 is dimensioned to contain enough of the desired atmosphere to supply oxygen to the cellular structure and to compensate for losses to the exterior through gas permeable components or through the gas closure seal. In preferred embodiments the gas space has a volume in the range 0.1-100 ml, in more preferred embodiments in the range 1-40 ml.
It will be evident to the skilled person that based on the above discussion the container according to the invention can be designed and sized to suit a wide range of cellular structures of different oxygen demand.
A container as in
The insert was sized to be an interference fit with the wall of the vial and on filling the media space with 500 μl media, fitting the cap forced the insert down into the vial and provided leak free sealing of the media space.
The total oxygen demand for 50 embryos over 72 hr is 1.8E-7 mol (assuming an individual oxygen demand of 1.4E-14 mol.s-1 per embryo (H. Shiku et al., Anal. Chem. (2001) 73(15) 3751-8), equivalent to 0.87 ml of gas (at 25 C) if the gas is depleted from 5% to 4.5% oxygen. The gas space 28 was approximately 5.3 cm3 and so oxygen depletion in the gas space (neglecting other factors and loss through the lid) is approximately 0.1%.
The change in gas composition owing to diffusion through the walls of the vial is exponential with a time constant that depends on the volume and surface area of the gas space, the wall thickness and gas permeability of the vial. Polystyrene Bibby Sterilin bijou vials have a quoted carbon dioxide permeability of 75E-10 mm.cm3.cm-2.(cm Hg)-1.s-1, and oxygen permeability of 15E-10 mm.cm3.cm-2.(cm Hg)-1.s-1 (www.barloworld.com, Sterilin website, download March 2007). For a gas space of volume 5.3 cm3 and a wall thickness of 1.5 mm (measured) the time constant for loss of gas through the wall (at the same total pressure inside and out) is 275 hr. For an initial internal atmosphere of 5% carbon dioxide and air outside, this leads to a change from 5% to 4% carbon dioxide in approximately 61 hr, and 5% to 3.5% in 98 hr. This is small enough not to compromise embryo development through change in pH of the media. If the internal oxygen content is 5%, oxygen will diffuse inwards from an air atmosphere: the diffusion coefficient is 5 times lower than that of carbon dioxide and the driving force (20% vs. 5%) is 3 times higher—so the increase in percent oxygen will not be significant over 72 hr.
Diffusion of oxygen to the embryos was estimated (i) with the embryos resting on the base 24 of the media space and (ii) with them resting on the lid. Having the embryos resting on the base is the best case and, given the provision of the gas channels 26 and the thin wall in their vicinity, is also a reasonable estimate of the situation when the container is on its side. Having the embryos resting on the lid is the worst case, in that the lid is assumed to be impermeable and O2 diffusion to the embryos is assumed to be wholly through the media.
Oxygen arriving at the embryos through the PDMS is in excess of that needed for respiration of a group of 50 embryos arranged in a disc at an embryo:gap ratio of 1:1 in hexagonal symmetry. The additional contribution from hemispherical diffusion is calculated below. Oxygen solubility in PDMS=0.18 cm3(STP)/cm3.atm and diffusion coefficient 3.4E-5 cm2.s-1 (Merkel et al. (1996) quoted in Zanzotto et al., Biotechnology and Bioengineering 87(2) (2004) 243-254). For 50 embryos in a disc at an embryo:gap ratio of 1:1 in hexagonal symmetry, the disc radius is 0.085 cm, and the oxygen flux for non-diffusion limited respiration (=50×1.4E-14 mol.s-1) creates a concentration at the inner PDMS surface of 3.2E-8 mol.cm-3, equivalent to that in equilibrium with gas containing 3.2% oxygen. The distance in the media from the wall to the embryos is assumed to be negligible (e.g. 10 um) and so contributes negligibly to the overall concentration gradient between the gas atmosphere and the embryos. Oxygen concentration of 3.2% is sufficient for good bovine embryo development (J. G. Thompson et al. J. Reproduction and Fertility 118 (2000) 47-55). If 20% oxygen is used in the gas atmosphere, clearly even less of a diffusion limitation problem will arise.
For embryos resting on the lid, the concentration at the embryos is found using the hemispherical diffusional impedance from the nearest gas phase source, through the media to the embryos. The embryos are assumed to be arranged in hexagonal symmetry with a gap:embryo ratio ranging from 0 (the embryos are in contact in hexagonal close packing) to 3. Obviously the embryos will be arranged in a more random way, though the spacing may be comparable. For comparison in the example dimensions above uniform distribution of the embryos over the lid or the base of the media space is at gap:embryo ratio of only 7 (for media space diameter=7 mm and embryo diameter=100 um), and if the embryos or oocytes tend to group together for example by sedimentation to a confined lowest part of the container if this is tilted, or because the embryos or oocytes are sticky and tend to stick together if they touch, some or all of the embryos could become closely packed in practice. For ease of estimation the properties of PDMS are taken the same as those of media (water). This is justified as (a) the PDMS containment of the media space has relatively thin walls (1.5 mm) compared with the typical diffusion dimension in the media (which is a minimum at approximately the radius of the media space, 3.5 mm, and a maximum at approximately the length of the media space, 13 mm), and (b) in the hemispherical diffusion geometry the outer dimension of the diffusion region and the properties of the region near it have much less impact on the diffusional impedance than does the radius of the inner boundary of the diffusion region and the properties of the region near the inner boundary.
An oxygen concentration at the embryos of 1E-8 mol.cm3, the concentration in the media in equilibrium with 1% oxygen in the gas phase, is considered to be the minimum desirable for bovine embryo culture (Thompson et al. (2000) op. cit.). The flux to the embryos is then calculated using hemispherical diffusion in elements of the cylindrical media space integrated in spherical coordinates over the cylinder.
The critical parameter in the estimation is the inner radius of the diffusion region, which is taken here to be a hemisphere of radius a=(2/pi) r(disc), where r(disc) is the radius of the disc over which the embryos are arranged as described above, by analogy to the result for diffusion-limited current at disc microelectrodes (K. B. Oldham and C. G. Zoski, J. Electroanal. Chem. 256 (1988) 11-19). The results show that for a separation between embryos of less than 3 diameters, respiration will be diffusion limited with local concentration equivalent to equilibrium with 1% O2 or below, even for optimally small diffusion distances from a gas-phase source (that cannot be realised in a practical design). It is unlikely that embryos will group together closely by chance (though if they show tendency to stick together, or to the wall of the media space, such that random movement in the media will lead to aggregation, that assumption might break down). Therefore an embryo gap:space ratio of 3 was used, with resulting r(disc)=0.165 cm and a=0.105 cm.
The flux to the group of 50 embryos in the embodiment in
The largest contribution comes from the shortest diffusion path, radially from the wall of the media space closest to the embryos; this shows the importance of the gas channels 26 in providing ready gas access to this region. Without these channels, the oxygen diffusion rate to the embryos when resting on the lid or the walls 22 would be much lower.
Time to Equilibrate the Gas in the Gas Space with an External Gas Atmosphere
The main route to equilibration is diffusion through the insert, in particular diffusion through the wall 22 and base 24 of the media space, and the composition will change exponentially with a time constant that depends on the volume of the gas space, the permeability and effective area and thickness of the insert and any pressure differential across the insert (which will be negligible in practice as gas tends to leak past the insert as it is being pushed into the housing—it will be zero if the insert has a gas through path 42 as in
The corollary of this is that if the container is removed from the controlled gas atmosphere and left uncapped while embryos are loaded the gas concentration inside the vial will change with the same time constants: the carbon dioxide concentration in the gas space will fall from 5% to 4.5% in around 30 minutes, and the oxygen concentration will rise from 5% to 6% in 11 minutes, which gives adequate time for the media to be added and the cap closed before excessive shift in concentration.
A container as in
The lid component 52 was designed to augment dissolved oxygen availability at a group of embryos resting on the lid when the container is upside down. Availability of sufficient flux of dissolved oxygen at a group at the centre of the lid means that at least this amount will be available at a position towards the edge of the lid, for example when the container is upside down and tilted away from vertical.
The flux contribution through the PDMS lid is modelled by calculating the sum of diffusional impedances (i) through the annulus represented by the PDMS in region 58 in
This shows that a solid PDMS lid component 52 provides sufficient O2 diffusional flux to maintain a group of 50 embryos at 3:1 spacing free of respiratory limitation through O2 diffusion limitation. The lid component 52 might also be provided with a gas space 54, which would serve to increase the diffusion rate if a larger number of embryos (or lower O2 content atmosphere) were to be used.
The gas channels 26 are closed by the rim 64 and so diffusion via this route, while non-zero, will be negligible compared with through a vent channel 66. The channel 66 in
| Number | Date | Country | Kind |
|---|---|---|---|
| 0707776.1 | Apr 2007 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB08/01427 | 4/23/2008 | WO | 00 | 3/17/2010 |
