WO2013001315A1 - Materials and methods for cell culture - Google Patents

Abstract

The invention provides materials and methods for culturing mammalian embryos reliably and reproducibly in vitro from pre-implantation to post-implantation stages. The methods of the invention comprise contacting a blastocyst-stage embryo with culture medium containing cord blood or a fraction thereof such as cord serum, wherein the embryo is located on a polystyrene or gel substrate.

Description

Materials and methods for cell culture

Field of the invention

The invention relates to cell culture, and more particularly provides materials and methods for culturing mammalian embryos reliably and reproducibly from pre-implantation to post- implantation stages.

Background to the invention

Implantation of the mammalian embryo into the uterus is a defining characteristic of the phylum and is critical for successful development. Whereas pre-implantation development can be followed in culture, this is not possible for

implanting embryos, which must be transferred into

pseudopregnant females to permit continued development. This has seriously hindered our understanding of how the major body axes of the embryo first arise, as their development is initiated during implantation.

Experimental procedures to reproduce blastocyst development from pre-implantation to post-implantation stages have previously been described^1-8 but not extensively used. Their neglect has been due to inherent irreproducibility .

For example, in Hsu, Nature 231, 100 (1971), the author explains that all embryos cultured by the method described there appeared to be defective in one tissue or another.

Thus, although correct development of some lineages may have been observed in individual embryos, the development of all lineages was not observed simultaneously. In subsequent studies, although later stage embryos may have been produced, correct development appears to occur at low frequency and can be sporadic. See, for example, Wilson and Jenkinson, J.

Reprod. Fert. 1974, 39, 243-249, discussing the success rates of others' approaches. The reasons for the lack of

reproducibility are unknown. Partially as a result of these inherent difficulties, dynamic, high resolution observations of this crucial stage of

development, during which the first signalling events

specifying the major body plan are initiated, have never been made .

Reliable and reproducible methods for culturing embryos past the implantation stage in vitro would also greatly increase the availability of stem cells.

Summary of the invention

The present inventors set out to develop conditions that would allow reproducible in vitro culture of embryos from pre- implantation to post-implantation stages, with a relatively high frequency of success. An added advantage of the methods developed by the inventors is that they permit optical (e.g. microscopic) analysis of the developing embryo, enabling imaging of the early morphogenetic events that occur during the transition from the pre- to post-implantation stages of development, and accurate lineage tracing through these stages of development by time-lapse microscopy. Such analysis has not previously been possible.

The invention provides an in vitro method of culturing a mammalian embryo, comprising contacting a mammalian blastocyst stage embryo with culture medium comprising cord blood or a fraction thereof, wherein said embryo is located on

(i) a polystyrene substrate; or

(ii) a gel substrate, wherein the surface of said gel

substrate comprises at least one extracellular matrix protein.

The invention further provides a culture system comprising a mammalian blastocyst stage embryo in culture medium comprising cord blood or a fraction thereof, wherein said embryo is located on

(i) a polystyrene substrate; or (ii) a gel substrate, wherein the surface of said gel

substrate comprises at least one extracellular matrix protein.

The inventors have found that the use of cord blood in the medium, at the appropriate stage of development, and in combination with the particular substrates described herein facilitates reproducible culture of the embryo to stages of development which can normally be achieved only post- implantation. In order to achieve optimum results, the embryo may be cultured in medium containing cord blood or a fraction thereof at least from attachment of the blastocyst to the substrate. Typically, when first released from the zona pellucida, the blastocyst is initially free-floating.

Subsequently, the blastocyst attaches to the substrate via the trophoblast cells which spread to form a flat outgrowth.

Appearance of such outgrowths may be taken as an indication of attachment .

As a guide, this may correspond approximately to Theiler stage 6 (for mouse embryos), Carnegie stage 4 for human embryos, and equivalent stages of development for other mammalian species.

For example, the blastocyst may be cultured in medium

containing cord blood or a fraction thereof from no later than 12 hours after attachment, no later than 11 hours after attachment, no later than 10 hours after attachment, no later than 9 hours after attachment, no later than 8 hours after attachment, no later than 7 hours after attachment, no later than 6 hours after attachment, no later than 5 hours after attachment, no later than 4 hours after attachment, no later than 3 hours after attachment, no later than 2 hours after attachment, or no later than 1 hour after attachment.

Blastocysts may be obtained directly from pregnant females between mating and implantation. This may involve sacrifice of the female (for non-human mammals) . For example, it is common laboratory practice to obtain blastocysts from pregnant mice at around day 3.5 of pregnancy (designated "E3.5") by removing the uterus and flushing it with suitable buffer or culture medium.

A blastocyst obtained from a pregnant female may be contained within a zona pellucida. It may be allowed to emerge

naturally (or "hatch") from the zona pellucida in culture without further intervention. Alternatively, the method may comprise the step of removing the blastocyst from the zona pellucida. Suitable methods are well known to the skilled person and an illustrative method involving treatment with acidified Tyrode's solution is provided in the Examples below. Artificial removal of the zona pellucida may increase the rate of attachment of the blastocyst to the substrate and further increase the likelihood that the embryo in question will develop successfully.

The blastocyst-stage embryo may itself have been cultured from an earlier developmental stage. Thus the method may comprise the earlier steps of

(a) providing said embryo at a pre-blastocyst stage of development, and

(b) culturing said embryo to blastocyst stage.

The pre-blastocyst stage of development may be a single cell embryo, e.g. a fertilised egg. Fertilisation may have been performed in vitro; indeed the method of the invention may comprise the step of fertilising the egg in vitro.

Alternatively, the single cell embryo may have been obtained by nuclear transfer, e.g. by transfer of a somatic cell nucleus into an enucleated egg. Thus, the method of the invention may comprise a step of nuclear transfer.

Alternatively, the pre-blastocyst embryo may have been obtained from a pregnant female. When the blastocyst is cultured from a pre-blastocyst stage of development, the method may comprise the step of removing the blastocyst from the zona pellucida at the appropriate time. However the blastocyst stage embryo is obtained, the

blastocyst stage embryo may previously have been present in culture medium which does not comprise cord blood. Thus the method may comprise the steps of: (i) providing a first in vitro culture comprising said blastocyst stage embryo in a first culture medium, wherein said first culture medium does not comprise cord blood or a fraction thereof; and (ii) either

(a) removing said first culture medium from said blastocyst stage embryo and contacting said blastocyst stage embryo with a second culture medium comprising cord blood or a fraction thereof; or

(b) adding cord blood or a fraction thereof to said first culture medium; to provide a second in vitro culture comprising said

blastocyst stage embryo in a second culture medium, wherein said second culture medium comprises cord blood or a fraction thereof . The first culture medium may contain serum from a different source, such as calf serum or foetal calf serum, for example at concentrations of 5-25%, e.g. 5-15%, 10%-10%, 15%-25%, e.g. about 10%, about 15% or about 20%. Such serum may have been inactivated, e.g. it may be heat inactivated serum.

At the time of first contact with medium containing cord blood, the blastocyst stage embryo may be at a developmental stage corresponding approximately to Theiler stage 6 (for mouse embryos), Carnegie stage 4 for human embryos, or equivalent stages for other species.

However it will be appreciated that the embryo may be first contacted with medium containing cord blood at an earlier stage if desired. For example, all earlier culture of the embryo may have been performed in medium containing cord blood. However, it may be that the embryo is first contacted with medium containing cord blood at the blastocyst stage, e.g. at attachment of the blastocyst to the gel substrate, such as no later than 12 hours after attachment, no later than 11 hours after attachment, no later than 10 hours after attachment, no later than 9 hours after attachment, no later than 8 hours after attachment, no later than 7 hours after attachment, no later than 6 hours after attachment, no later than 5 hours after attachment, no later than 4 hours after attachment, no later than 3 hours after attachment, no later than 2 hours after attachment, or no later than 1 hour after attachment .

The methods of the invention find particular use in enabling the embryo to be cultured to a post-implantation stage of development. Thus the methods typically comprise culturing said blastocyst stage embryo to a later stage of development, typically a post-implantation stage of development. The culture medium may contain cord blood or the appropriate fraction thereof throughout these later stages of culture.

The post-implantation stage of development may be the egg cylinder stage in rodents (e.g. in mice) or its equivalent in other mammals, such as the embryonic disc stage in primates (e.g. humans) . These represent Theiler stage 7 in mice, Carnegie stage 5(a) in humans, and their equivalents in other species . This developmental stage may be particularly desirable, at least in part because it contains many more pluripotent cells (which may be referred to as embryonic stem cells) than the earlier blastocyst stage. Such embryos are therefore extremely useful sources of stem cells.

The methods of the invention may therefore comprise the step of removing one or more cells from said embryo, e.g. from the inner cell mass. The cell may be an epiblast cell. Epiblast is derived from the inner cell mass and differentiates to form all three layers of the trilaminar germ disc during

gastrulation . Said cell may be a pluripotent cell, e.g. an embryonic stem cell. Such pluripotent cells may have many uses, e.g. in therapeutic cloning or other therapeutic methods. It will be appreciated that cells may nevertheless be removed from the embryo at blastocyst stage if desired.

However, the methods may comprise culturing the embryo to still-later stages of development if required.

The cell removed from the embryo may be used for genetic testing, e.g. for a genetic abnormality or condition.

In any of the methods described herein, an effective fraction of cord blood may be utilised. That fraction is typically a cell-free fraction of cord blood and may be cord blood serum or plasma. Serum may be preferred. Alternatively the cord blood serum or plasma may be further fractionated. The cord blood or fraction thereof may be included in the culture medium at any effective concentration. Thus the culture medium may contain about 5% to about 50%, about 10% to about 30%, about 10% to about 20%, about 15% to about 25%, e.g. about 10%, about 15% or about 20% of cord blood, serum, or plasma. Serum may be particularly suitable. Where other fractions are used, they may be used at a suitable concentration to provide an equivalent level of any given component as the specified amount of serum would provide.

The cord blood may be from any suitable mammalian species. It may be desirable to use cord blood from the same species as the embryos to be cultured. Alternatively the cord blood and the embryos may be of different species. Human cord blood may be particularly effective and may be used for any species of embryo. Other readily available sources of cord blood may include livestock animals such as cow, sheep, pig or goat.

When a gel substrate is used, the surface of the gel substrate comprises at least one extracellular matrix protein. The extracellular matrix protein (s) may be incorporated into the gel, or the gel may be coated with the extracellular matrix protein ( s) .

The at least one extracellular matrix protein may comprise one or both of collagen and laminin. Either or both may be used in combination with other extracellular matrix proteins such as fibronectin. In certain embodiments, the extracellular matrix protein comprises collagen and optionally laminin and/or fibronectin. The collagen may be Type I collagen.

The gel may be a hydrogel. For example, the gel may be a polyacrylamide gel. Alternatively, the gel may comprise or consist substantially of basement membrane matrix.

The gel substrate may be elastically deformable. The gel substrate may, for example, have a Young's modulus of about 5xl0³ Nrrf² to about lOOxlO³ Nrrf², e.g. about 25xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 50xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 60xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 70xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 75xl0³ Nm^"2 to about lOOxlO³ Nm^"2 , about 80xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 90xl0³ Nm^"2. Additionally or alternatively, the surface of the gel may have a Young's modulus of about 5xl0³ Nm^~2 to about lOOxlO³ Nm^~2, e.g. about 25xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 50xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 60xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 70xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 75xl0³

Nm^"2 to about lOOxlO³ Nm^"2 , about 80xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 90xl0³ Nm^"2. This may be determined by any suitable technique such as atomic force microscopy. Additionally or alternatively, the gel substrate may have characteristics (e.g. elasticity and/or surface compliance) equivalent to those of a polyacrylamide gel as described in the examples, i.e. a gel formed from an aqueous solution of 10% acrylamide and 0.01-0.5% N, N' -methylenebisacrylamide , e.g. 0.1-0.5% Ν,Ν' -methylenebisacrylamide, e.g. 0.2-0.4% Ν,Ν'- methylenebisacrylamide, e.g. 0.25-0.35% Ν,Ν'- methylenebisacrylamide, e.g. 0.3% N, N' -methylenebi sacrylamide when polymerised with ammonium persulphate at a final

concentration of 1/2000 w/v (i.e. ΙΟμΙ of 10% solution per 2ml of polymer solution) and TEMED at a final concentration of

1/2000 v/v (i.e. Ιμΐ per 2ml of polymer solution) .

Throughout this specification, the terms "equivalent to" and "about" should be taken to mean within 10%, more preferably within 5%, of the specified value, unless the context requires otherwise .

The gel substrate may be provided on a solid support such as glass, e.g. borosilicate glass, or plastics material. A glass support may be particularly appropriate for methods requiring imaging of the embryo (e.g. by microscopy) .

The gel substrate may be bonded, e.g. covalently bonded, to the solid support.

When a polystyrene substrate is used, the substrate may be conventional plastic cultureware, which is readily commercially available. It is not necessary that the

polystyrene substrate is coated with extracellular matrix protein(s), although any of the above-described coatings (e.g. collagen and/or laminin, optionally also in combination with fibronectin) may be used.

The substrate may have a surface topography which facilitates retention of one or more embryos and their associated culture medium within a defined region of the substrate.

Thus, for example, the surface of the substrate may comprise one or more receptacles adapted to contain a culture

comprising appropriate culture medium and one or more embryos. The receptacles may, for example, be wells or troughs.

The substrate may be formed in or upon a suitable template or mould in order to achieve the required topography.

The substrate may comprise a single receptacle or a plurality of receptacles.

Each receptacle may have a depth of about 250μιη to about 400μπι, e.g. about 300μπι to about 350μπι. Additionally or alternatively, said plurality of receptacles may have a mean depth of about 250μιτι to about 400μΓη, e.g. about 300μΓη to about 350μιιι.

The substrate may carry one embryo or a plurality of embryos. Where the substrate comprises one or more receptacles, each said receptacle may independently contain one embryo or a plurality of embryos, e.g. 2, 3, 4 or 5 embryos, or more. In some embodiments, each embryo is located in a different respective well. In alternative embodiments, each receptacle comprises a plurality of embryos, e.g. 2, 3, 4 or 5 embryos, or more. Each individual culture may have a volume of 1-20 μΐ, e.

10 μΐ, e.g. 5-10 μΐ, e.g. for a culture of 1 to 5 embryo culture volume of 1-2 μΐ per embryo may be particularly suitable .

The invention also relates to methods of imaging embryos during development. Thus the method may comprise the step of recording one or more images of the embryo. The method may comprise recording a single image, for example at a predetermined stage of development, or a plurality of images at pre-determined stages of development or at pre-determined times. The invention further extends to a method of imaging an embryo during development, the method comprising providing a culture system as described above and imaging apparatus, and recording one or more images of said embryo. The method may comprise recording a plurality of images of the same embryo. The plurality of images may be recorded over a pre-determined period of time. In any such methods, the image may be a two dimensional image or a three dimensional image.

Also provided is an imaging apparatus comprising a culture system as described above.

The imaging apparatus may comprise microscopy apparatus, suitable recording apparatus, and optionally image processing apparatus .

The imaging apparatus may comprise a fluorescence microscope, such as a confocal microcope.

In such cases, the substrate used in the culture system will typically be a gel substrate of the invention, optionally on a glass support.

The methods of the invention may be used to monitor the suitability of embryos for transfer to a recipient female mammal and subsequent development to term. Thus the methods of the invention may be applied to a plurality of embryos, and may comprise a step of selecting one or more embryos from said plurality of embryos based on a pre-determined quality measure (e.g. based on pre-determined morphological and/or genetic criteria) . The methods may subsequently comprise the step of transferring the selected embryo or embryos to a recipient female .

The methods of the invention may also be used to investigate the effects of test agents on blastocyst attachment and other aspects of embryo development.

Thus any of the methods described above may comprise the step of contacting the embryo with a test agent and determining the effect of said test agent on development of said embryo.

The invention further provides a method comprising the steps of:

(a) providing a culture system comprising a mammalian blastocyst stage embryo in culture medium comprising cord blood or a fraction thereof, wherein said embryo is located on

(i) a polystyrene substrate; or

(ii) a gel substrate, wherein the surface of said gel substrate comprises at least one extracellular matrix protein; (b) contacting said culture system with a test agent; and

(c) determining the effect of said test agent on the embryo.

The method may comprise contacting the culture system with th> test agent before attachment of the embryo to the substrate. This may allow the effect of the test agent on attachment of the embryo to the substrate to be determined. Thus, the method may comprise the further step of determining the subsequent effect on attachment of the embryo to the

substrate. This may allow identification or characterisation of agents which prevent or inhibit embryonic attachment to or implantation into the uterine wall . Additionally or

alternatively, the method may also allow determination of the effect of the test agent on later developmental stages of the embryo .

Alternatively, the method may comprise contacting the embryo with the test agent after attachment of the embryo to the substrate. This may also allow determination of the effect of the test agent on the ability of the embryo to remain attached to the substrate. Thus, the method may comprise the further step of determining the subsequent effect on attachment of the embryo to the substrate. This may allow identification or characterisation of agents which induce release of an embryo from the uterine wall once attached or implanted thereto.

Additionally or alternatively, the method may also allow determination of the effect of the test agent on later developmental stages of the embryo.

Thus the method may be used to characterise or screen for contraceptive agents, mutagens, teratogens, etc.. Any such methods may involve comparing the results obtained with the test agent with control results. The control results may be, for example, obtained in the absence of a test agent, or in the presence of an agent having known properties, e.g. contraceptive, mutagenic or teratogenic properties.

The methods may be performed in a high throughput format.

Thus a plurality of cultures containing embryos may be contacted with different test agents. Additionally or alternatively, a plurality of cultures containing embryos may be contacted with the same test agent at different times or stages of development. Each said culture may be contained in a separate respective receptacle on the same substrate.

Other features of the method are as described above in relation to other methods of the invention. The effects of the test and other agents on attachment and development may be monitored using imaging techniques as described elsewhere in this specification. Thus the methods may comprise the steps of recording one or more images of the embryo. Such images may be recorded before and/or after contact with the test agent.

The invention will now be described in more detail, by way of example and not limitation, by reference to the accompanying drawings and examples.

Description of the Drawings

Figure 1. Time-course of in vitro cultured mouse blastocysts on standard tissue culture dishes. (A) E3.5 blastocysts were seeded in culture drops containing FCS (Day 0 of in vitro culture) . After one day of culture blastocyst expansion was evident. Trophoblast outgrowths (white arrows) were observed after 2 days of culture indicating attachment to the

substrate. At this point FCS was replaced by HCS and kept for the remaining of the culture period. Embryonic development into an early egg cylinder is observed between days 4 and 5 of culture. (B) Early egg cylinders recovered following in vitro culture of HistoneH2B-GFP expressing embryos, highlighting the formation of distinct epiblast (EPI), extra-embryonic ectoderm (ExE) and visceral endoderm (VE) domains. (C) Immunostaining of egg cylinders (carried out as described previously¹⁹ showing correct spatial expression of 0ct4, Cdx2 , and Gata4. Nuclei are counterstained with DAPI . Figure 2. Optimisation of polyacrylamide gels for embryo culture and live imaging. (A) Manufacturing process for the polyacrylamide gels used in this study. For details see

Material and Methods. (B) End point for 5 day in vitro culture of E3.5 blastocysts on acrylamide : bis-acrylamide matrices of different stiffness and coating. Acrylamide : bisacrylamide ratio (as an index of stiffness) and protein coating are indicated. Detailed Description of the Invention

Embryo

The term "embryo" is used in this specification to refer to a mammalian organism from the single cell stage. The single cell may be a fertilised egg, or any other totipotent cell which is capable of developing into an adult organism under appropriate conditions. The single totipotent cell may have been derived by artificial means such as nuclear transfer, in which a nucleus from a somatic cell is transferred into an enucleated egg .

While mammalian embryogenesis has some common features across all species, it will be appreciated that different mammalian species develop in different ways and at different rates, which may make comparison difficult. In general, though, the fertilized egg undergoes a number of cleavage (passing through two cell, four cell and eight cell stages) before undergoing compaction to form a solid ball of cells called a morula, in which the cells continue to divide. Ultimately the internal cells of the morula give rise to the inner cell mass and the outer cells to the trophectoderm. The morula in turn develops into the blastocyst, which is surrounded by trophectoderm and contains a fluid-filled vesicle, with the inner cell mass at one end.

Theiler has established numbered stages of murine development. The earliest stages, as applied to (C57BLxCBA) l mice, are described in the "emouse digital atlas"

(http://www.emouseatlas.org) as follows:

Theiler dpc* Cell number (C57BLxCBA)Fl mice

Stage

(range)

1 0-0.9 1 One-cell egg

(0 -2.5)

2 1 2-4 Dividing egg

(1 -2.5)

3 2 4-16 Morula (1-3.5)

4 3 16-40 Blastocyst, Inner cell mass

(2-4) apparent

5 4 Blastocyst (zona-free)

(3-5.5)

6 4.5 Attachment of blastocyst,

(4-5.5) primary

endoderm covers

blastocoelic surface of inner cell mass

7 5 Implantation and formation

(4.5-6) of egg

cylinder Ectoplacental cone appears, enlarged epiblast, primary endoderm lines mural trophectoderm

8 6 Differentiation of egg

(5-6.5) cylinder .

Implantation sites 2x3mm. Ectoplacental cone region invaded by maternal blood,

Reichert ' s membrane and proamniotic cavity form

*: "dpc" indicates days post conception, with the morning after the vaginal plug is found being designated 0.5 dpc or E0.5.

Similarly, so-called "Carnegie stages" have been established to describe stages of human development. Each stage is defined by the development of specific structures, and can be used to define equivalent stages in development of other species. The earliest Carnegie stages are as follows:

Carnegie stage Days since ovulation Characteristic

(approx. ) events/structures

1 1 fertilization; polar bodies

2 2-3 cleavage; morula;

compaction

3 4-5 blastocyst and

blastocoele; trophoblast and

embryoblast

4 6 syncytiotrophoblast;

cytotrophoblast; anchoring to endometrium

5(a) 7-8 implantation;

embryonic disc; bilaminar germ disc; primary yolk sac;

5 (b) 9-10 formation of

trophoblast lacunae; complete penetration into endometrium; amniotic cavity; primary umbilical vesicle

5(c) 11-16 pre-chordal plate;

extra-embryonic mesoblast; secondary yolk sac

6 17 primitive streak, primitive node, primitive groove; secondary umbilical vesicle; primordial germ cells; body stalk

The methods of the invention enable reliable culture through to post-implantation stages, corresponding to Theiler stage 7 and beyond, Carnegie stage 5 (b) and beyond, and corresponding stages in other species.

Certain defects are only detectable after the implantation stage once the body plan develops. Amongst other benefits, the methods of the invention allow embryos to be observed and screened in vitro to a later stage of development than has previously been possible, beyond the implantation stage, enabling such defects to be detected. Thus, where the embryos are to be transferred to a recipient female mammal, they may be screened so that only embryos lacking such defects are transferred. The screening may be based on genetic tests (which may be performed on a cell removed from the embryo) and/or on non-invasive morphological analysis (e.g. using imaging techniques such as those described herein) .

The methods of the invention may be applied to embryos from any suitable mammalian species, such as:

primates, including humans, great apes (e.g. gorillas, chimpanzees, orang utans), old world monkeys, new world monkeys; rodents (e.g. mice, rats, guinea pigs, hamsters); cats; dogs; lagomorphs (including rabbits); cows; sheep;

goats; horses; pigs; and any other livestock, agricultural, laboratory or domestic mammals.

In some embodiments, the methods are applied to any non-human mammal, including but not limited to those described above.

Cord blood

The term "cord blood" is used throughout this specification to refer to umbilical cord blood, i.e. the blood which remains in the umbilical cord and/or placenta post partum. Collection of such blood is routine and is often considered desirable because cord blood is known to contain stem cells, including haematopoietic stem cells, which have many applications.

The methods of the present invention do not rely on the presence of stem cells per se in the cord blood itself, since a cell-free fraction of cord blood is typically used. The cell-free fraction may be plasma or serum, although further fractionation or concentration of either may be performed before addition to the relevant culture medium.

Typically the culture medium to be used does not contain other serum products, such as foetal calf serum, which are commonly used in cell culture. The cord blood used may be from any mammalian species. It may be from the same species as the embryo to be cultured, or it may be from a different species. Human cord blood has been found to be particularly effective and may be used for any species of embryo but need not be used in all circumstances. Livestock animals may provide other convenient sources of cord blood. Thus the cord blood may, for example, be cow, pig, sheep or goat cord blood.

The cord blood or fraction thereof may have been

"inactivated", i.e. treated to inactivate components such as complement which could damage cells or exert other undesirable effects in cell culture. For example, serum is often inactivated by heat treatment (e.g. at 56^°C for 30 minutes) and is then referred to as heat inactivated serum.

The embryo is preferably contacted with the cord blood or fraction thereof at the time of attachment to the substrate, at the very least within 12 hours, preferably within 11. 10,

9, 8, 7, 6, 5, 4, 3, 2 or 1 hour of such attachment. Previous methods of embryo culture have used cord blood, but it is believed that the methods of the present invention employ the cord blood at an earlier stage of embryonic development than these earlier methods. Without being bound by any particular theory it is believed that this earlier contact with cord blood is significant in the reproducibility of the present methods, and the high proportion of embryos which develop through to egg cylinder stage and beyond.

Substrate

According to the invention, embryos are cultured on a polystyrene substrate or a gel substrate.

A gel is commonly recognised to be a substance with properties intermediate between the solid and liquid states. Gels are essentially colloidal, with a disperse solid phase and a continuous liquid phase. The solid phase is typically an extended three-dimensional network or matrix, often of polymeric material, which may be cross-linked. The liquid phase is commonly water (or an aqueous solution) and such gels are often referred to as hydrogels . Hydrogels are

particularly suitable for use in the present invention.

The gel may be a polyacrylamide gel, e.g. a gel comprising a cross-linked polymer matrix formed by polymerisation of acrylamide and bis-acrylamide (e.g. Ν,Ν'- methylenebisacrylamide) . Polymerisation may be initiated using ammonium persulphate and NNN' -M-tetramethylethyldiamine (TEMED) .

Alternatively, the gel may comprise or consist substantially of basement membrane matrix. The basement membrane matrix may comprise one or more of laminin, collagen, heparan sulphate proteoglycan and entactin, amongst other components. The gel may be formed from basement membrane extract, which may be isolated from a suitable basement membrane-secreting cell type, such as Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. Basement membrane extracts produced from EHS cells are commercially available under the trade names Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) and Cultrex (Trevigen Inc., Gaithersburg, MD, USA) . Their ma or component is laminin, followed by collagen IV, heparan sulphate

proteoglycan and entactin. The skilled person is well aware of how to form gels using such products, e.g. by following the manufacturer's instructions.

Other suitable gel types may include alginate gels and polyethylene glycol (PEG) based gels

The gel may be elastically deformable. It is appreciated that certain gels, including polyacrylamide gels, are not

rigorously elastic. Rather, they are more properly described as visco-elastic . However, for practical purposes of the present invention, they can be treated as elastic. In such cases, the gel may be therefore be characterised in terms of its Young's modulus, which is a measure of elasticity or stiffness. Young's modulus E for any particular gel can be determined very simply by applying a known force (e.g. a weight) to a gel web of known length and cross-sectional area, and determining the extent of stretching which results. E is calculated using the formula: E = FL₀/A₀AL where F is the force applied, L₀ is the original length of the web, A₀ is the original cros s- sectional area through which the force is applied, and L₀ is the original length of the web.

The gel substrate may, for example, have a Young's modulus of about 5xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 25xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 50xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 60xl0³ Nrrf² to about lOOxlO³ Nrrf², e.g. about 70xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 75xl0³ Nm^"2 to about lOOxlO³ Nm^"2 , about 80xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 90xl0³ Nm^"2. Measurement of Young's modulus for polyacrylamide gels is described, for example, in Pelham & Wang, PNAS USA, 94, 13661-13665, 1997, as corrected (PNAS USA 1998, 95(20) : 12070) .

Surface stiffness, or compliance, may also be used to

characterise the gel substrate. Surface compliance may be determined, for example, by atomic force microscopy, and may also be expressed in terms of Young's modulus. Thus the the surface of the gel may have a Young's modulus of about 5xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 25xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 50xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 60xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 70xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 75xl0³ Nm^"2 to about lOOxlO³ Nm^"2 , about 80xl0³ Nm^"2 to about lOOxlO³ Nm^"2, e.g. about 90xl0³ Nm^" . Additionally or alternatively, the gel substrate may have characteristics (e.g. elasticity and/or surface compliance) equivalent to those of a polyacrylamide gel formed from an aqueous solution of 10% acrylamide and 0.01-0.5% Ν,Ν'- methylenebisacrylamide, e.g. 0.1-0.5% Ν,Ν'- methylenebisacrylamide, e.g. 0.2-0.4% Ν,Ν'- methylenebisacrylamide, e.g. 0.25-0.35% Ν,Ν'- methylenebisacrylamide, e.g. 0.3% N, N' -methylenebi sacrylamide when polymerised with ammonium persulphate at a final concentration of 1/2000 w/v (i.e. ΙΟμΙ of 10% solution per 2ml of polymer solution) and TEMED at a final concentration of 1/2000 v/v (i.e. Ιμΐ per 2ml of polymer solution) .

The surface of the gel substrate comprises at least one extracellular matrix protein. The extracellular matrix protein (s) may be incorporated into the gel, or the gel may be coated with the extracellular matrix protein (s) after polymerisation of the gel.

The at least one extracellular matrix protein may comprises one or both of collagen and laminin. Collagen may be particularly preferred. Either or both may be used in combination with fibronectin or other extracellular matrix proteins. In certain embodiments, the extracellular matrix protein comprises collagen and optionally laminin and/or fibronectin. The collagen may be Type I collagen, e.g. from rat tail, although other types may be used.

Methods for coating gels with such proteins are well known in the art and illustrative methods are described in the examples below. Typically the extracellular matrix protein (s) are chemically cross-linked to the gel surface using a suitable bifunctional linker molecule which is capable of reacting with both the protein and the gel. Reaction between the respective functional groups of the linker and the protein and gel may be controlled by any suitable means. For example, the functional groups may be photo-activatable, i.e. activatable by irradiation, e.g. by UV irradiation. The surface of the gel may carry any suitable functional group for reaction with the linker, but amine groups may be particularly suitable. It will be apparent that some gels (for example, those comprising or consisting of basement membrane matrix) will already contain suitable matrix proteins. For others

(polyacrylamide, alginate, etc.) it may be necessary to incorporate suitable proteins into the gel components before gelatini sation or coat suitable proteins onto the gel surface after gelatinisation .

The gel substrate may be provided on a solid support. The solid support may be glass, e.g. borosilicate glass. A glass support may be particularly appropriate for methods requiring imaging of the embryo (e.g. by microscopy) . The gel substrate may be bonded, e.g. covalently bonded, to the solid support. The skilled person will be aware of suitable methods and chemistries which may be applied, and illustrative methods are described in the examples below.

As already described, a polystyrene substrate may be provided by conventional commercially available polystyrene

cultureware. If desired, the surface may be coated with one or more extracellular matrix proteins as described above.

Whatever the nature of the substrate, it may have a surface topography which facilitates retention of one or more embryos and their associated culture medium within a defined region of the substrate.

Thus, for example, the surface of the substrate may comprise one or more receptacles adapted to contain a culture

comprising appropriate culture medium and one or more embryos. Each receptacle may comprise or consist of a concave or recessed portion of the substrate surface. Where a gel is used, the gel substrate will typically form the bottom surface and optionally also any walls (substantially upright surfaces) of such receptacles.

The receptacles may have any suitable shape as viewed from above. They may have approximately equal dimensions along notional orthogonal X-Y axes, e.g. they may be substantially round, square, hexagonal, or any other suitable shape. Such receptacles may conveniently be referred to as "wells".

Alternatively the receptacles may be elongate, i.e.

substantially longer along one of said axes than the other.

Such receptacles may conveniently be referred to as "troughs".

Whether wells or troughs, the cross-sectional shape of the receptacle may be tailored as desired. For example, the bottom surface of the receptacle may be substantially flat, or it may be more or less curved providing a U-shaped cross section. In either case, the internal sides of the receptacle may be substantially upright, or may be sloped such that the top of the receptacle is broader than the bottom.

The substrate may be formed in or upon a suitable template or mould in order to achieve the required topography.

Alternatively the substrate may be substantially planar, with walls provided to partition one or more regions of the substrate surface into suitable receptacles. In such

embodiments, the planar substrate will form the bottom surface of the receptacles. The walls may be formed from, or covered with, a gel similar or identical to that of the substrate, or they may be formed from different material. substrate may comprise a single receptacle or a plurality receptacles .

Each receptacle may have a depth of about 250μπι to about 400μιιι, e.g. about 300μιτι to about 350 m. Additionally or alternatively, said plurality of receptacles may have a mean depth of about 250μιτι to about 400μιτι, e.g. about 300μιη to about 350μπι.

Especially when the receptacles are wells, they may be ordered on the substrate in an array, i.e. in a grid pattern having regular spacing in substantially orthogonal directions.

Whatever the topography of the substrate, the substrate may carry one or more embryos. Where the substrate comprises one or more receptacles, each said receptacle may independently contain one or more embryos, e.g. 2, 3, 4 or 5 embryos, or more. In some embodiments, each embryo is located in a different respective well. In alternative embodiments, each receptacle comprises a plurality of embryos, e.g. 2, 3, 4 or 5 embryos, or more.

The methods of the invention may be applied in culture volumes of any appropriate size. However, without wishing to be bound by any particular theory, it is presently believed that embryo development is facilitated when a plurality of embryos is cultured together. This effect may be mediated by factors secreted by the embryos themselves. Thus it may be desirable to minimise the culture volume (as far as practically possible) to maximise the concentration of such development- promoting factors. For example, a culture volume of 5-10 μΐ has been found to be particularly effective, especially for cultures of 1 to 5 embryos, e.g. 1, 2, 3, 4 or 5 embryos. A culture volume of 1-2μ1 per embryo may be particularly suitable .

Imaging methods and apparatus

The invention also extends to methods of imaging an embryo during development, the method comprising providing a culture system as described above and imaging apparatus, and recording an image of said embryo. The method may comprise recording a plurality of images of the same embryo. The plurality of images may be recorded over a pre-determined period of time, thus giving illustrating the development of the embryo over time.

The imaging apparatus may comprise microscopy apparatus, suitable recording apparatus, and optionally image processing apparatus .

Typically, fluorescent markers, such as fluorescent dyes or fluorescent marker proteins, are used in the imaging of embryonic development. Such markers may be added to the culture system. For example, fluorescent dyes may be added to visualise particular molecules or cellular structures. For example, DAPI is used to stain DNA. Additionally or

alternatively, the embryo may produce such fluorescent markers endogenously, e.g. it may contain one or more cells which express a fluorescent marker protein. Such cells may have been genetically modified in order to confer the ability to express such a marker protein.

Thus, fluorescence imaging apparatus may be particularly suitable for the methods described. The imaging apparatus may thus comprise a fluorescence microscope, such as a confocal microcope .

Confocal microscopes image a single point of a specimen at any given time but allow generation of two dimensional or three dimensional images by scanning different points in a specimen in a regular raster to provide image data which can be assembled into a two or three dimensional image. For example, scanning a specimen in a single plane enables generation of a two dimensional image of a slice through the specimen. A plurality or "stack" of such two dimensional images can be combined to yield a three dimensional image. Fluorescence microscopy typically requires the specimen to be illuminated with UV light. Many plastics themselves fluoresce when illuminated with UV light and so are not suitable for use in such imaging techniques . Much commercially available plastic cultureware is also too thick to be useful in such techniques. Thus, imaging methods involving fluorescence will typically be carried out using the gel substrate of the invention, optionally on a glass support. Examples

The establishment of the anterior-posterior (AP) axis in the mouse embryo is clearly visible after embryo implantation at embryonic day 5.5 (E5.5), when a distinct group of visceral endoderm cells, the AVE, forms at the distal tip of the egg cylinder ⁹. The subsequent unilateral movement of these cells towards the proximal region of the embryo establishes the future anterior pole ^10-12. Gene expression within the AVE and AVE migration are regulated by signals, such as BMP4, that are derived from the extra-embryonic ectoderm ^13-15. Nodal

signalling within the epiblast (EPI) is also essential for AVE formation ^16-17. Although these events around E5.5 are essential for AP axis development, the expression of the AVE markers, Leftyl ¹¹'¹⁸ _r Hex ¹¹ and Cerl ¹⁹ appears already initiated at the blastocyst, opening up the possibility that these earlier cells could be the true AVE progenitors. However, the test of this hypothesis would require direct observation of these early expressing cells from pre- to post-implantation stages and this was previously impossible. Here we validate our new in vitro culture system by both recapitulating these recent findings and extending them to identify AVE progenitors within implanting embryos. By the use of a transgenic mouse reporter line in which GFP monitors expression of the AVE marker

Cerberus , Cerl ²⁰ , and by time-lapse tracking of cells, we can now follow how the descendents of individual cells of the implanting blastocyst develop into the AVE of the egg

cylinder . In vitro culture from blastocyst to egg cylinder

Mouse embryos can be recovered, cultured, and filmed at pre- implantation stages. However, the development of the mouse embryo in the period between the blastocyst and post- implantation egg cylinder stages has remained a "black box" due to the lack of a reproducible method of in vitro culture that enables live time-lapse observations of this process. Yet this is the developmental time when the first signalling centres specifying the major body axes form and start to function, leading to a period of extensive morphogenetic and epigenetic transformations ^21-22.

To determine the origins and development of precursor cells for the AVE, however, we required a system that would allow us to identify these cells and then follow them during this hidden implantation period. We therefore re-assessed

techniques for culturing embryos through this stage using conditions described in the 1970s ^1-2,5 as our starting point for establishing a reproducible method. We began by culturing freshly collected zona-free E3.5 mouse blastocysts for 5 days in standard plastic tissue culture dishes in the presence of foetal calf serum (FCS) . We found that once the blastocysts were observed to be attached, the replacement of FCS with human cord serum (HCS) was key to obtaining successful development of the embryos into early egg cylinders. Within 2 days of in vitro culture, the blastocysts expanded and attached to the plastic substrate whereupon trophoblast outgrowths began to form (Fig. 1) . Egg cylinders grew out from the surface in approximately 40% of the attached embryos (Fig. 1) . Such embryos were morphologically indistinguishable from freshly collected E6.5 embryos, and showed the same spatial patterns of expression of extra-embryonic ectoderm (ExE) , visceral endoderm (VE) and EPI marker genes (Fig. 1) . This culture system was, however, not suitable for our purposes, since the plastic dishes used were not amenable to live imaging. We therefore attempted to use commercially-available brands of optically friendly culture dishes with a variety of coatings, but found that none of these permitted correct embryo development. As glass coverslips coated with protein- coated polyacrylamide gels have been successful in the culture and differentiation of mesenchymal stem cells (²³, Fig. 2A) , we considered whether they might also be compatible with both embryonic development and continuous imaging from pre- to post-implantation stages. We found that two variables were of importance: the surface stiffness of the polyacrylamide hydrogel, and the protein coating chemically bonded to that surface, which constitutes the contact region of the substrate for the embryo. Appropriate N' N' -methylenebisacrylamide and acrylamide concentration ratios were determined, and a surface coating comprising type I rat-tail collagen was found to be the most effective (data not shown) . In addition, we formed 300 to 350 μιιι deep wells that allowed attachment of the embryos without constraining expansion. Using these

conditions, almost 90% of embryos attached to the matrix and of these 40% formed egg cylinders.

Gels formed from solubilised basement membrane extract from

EHS cells (Matrigel; BD Biosciences, Franklin Lakes, NJ, USA) were also found to provide good results when used as an alternative to polyacrylamide (data not shown) .

To study the cellular dynamics of embryo development during implantation, we removed E3.5 blastocysts from the zona pellucida and seeded these onto such matrices in media containing 20% FCS . This allowed us to perform time-lapse observations, which revealed the dynamic nature of the events that take place during implantation. In accordance with previous snapshot observations, during the first 24 hours of in vitro culture we observed expansions and contractions of the blastocysts. During Day 2 of culture, thickening of the mural trophectoderm surrounding the blastocyst cavity became evident. Towards the end of day 2, the embryo contracted around its attachment site and subsequently its spreading was evident on Day 3 of culture. Once all blastocysts had attached, the medium was removed and replaced by medium containing 20% HCS . We observed that trophoblast cells, with typical large nuclei and vacuolated cytoplasms, appeared to undergo proliferation and migrated across the collagen surface. The embryo became flattened and the classical blastocyst structure was lost since the EPI and the primitive endoderm (PE) no longer morphologically resembled those found in pre-implantation stages . During Day 4 the EPI and the VE (derived from PE) acquired the expected morphological organisation found in freshly collected egg cylinders at the corresponding stage. In addition, the ExE became recognisable close to the site of attachment highlighting the nascent proximo-distal axis of the growing early egg cylinder. The embryonic VE containing the EPI started to detach from the surface, while the extraembryonic components remained firmly implanted onto the matrix. Complete egg cylinders were consistently found from Day 5 onwards and the embryonic and extraembryonic components were indistinguishable from those observed in embryos that had developed in utero (Fig. 1) . At this point, cultured embryos could either be left to develop further under these conditions or sliced from the gels and cultured under the same conditions as embryos freshly collected from the uterus.

Origins of Cerl expression in the mouse embryo

We have previously shown how the asymmetric development of cell clones in the E3.5 mouse blastocyst relates to the asymmetry of the post-implantation E6.5 egg cylinder ¹⁰'²⁰. we and others have also shown that the expression of AVE markers starts before implantation^18-19. However, due to the necessity of transferring pre-implantation embryos to foster mothers to allow their further development, the continuity of these processes could not be addressed. The method we have now developed for the culture and imaging of Cerl-GFP reporter embryos on collagen-coated polyacrylamide gels provides the first opportunity to definitively demonstrate the pre- implantation origins of the AVE (and thus of the AP axis) by continuous time-lapse observations. We collected E3.5 blastocysts from female mice mated with Cerl-GFP male mice and seeded them onto our collagen-coated polyacrylamide gels. This allowed us to observe the behaviour of Cerl-GFP expressing cells as blastocysts grew and developed into egg cylinders. Early on day 2 of in vitro culture, time-lapse observations revealed the onset of Cerl-GFP expression in a single cell of the PE in the implanting blastocyst. As development continued, this single cell divided to give two Cerl-GFP expressing cells and new neighbouring cells acquired de novo expression of Cerl-GFP. Interestingly, the more intensively expressing cells then seemed to consolidate into a single AVE domain. On the fourth day of culture, as the egg cylinder grew to extend out of the culture plane, this cluster of cells could be seen to occupy its distal tip. This pattern of cells expressing Cerl- GFP and their dynamics is in agreement with observations on the expression of Cerl-GFP in freshly collected embryos ¹⁹. Together these data indicate that development of embryos on collagen-coated polyacrylamide matrices leads to the formation of egg cylinders that are equivalent to freshly collected embryos from the uteri of pregnant females both in morphology and pattern of AVE gene expression. Moreover, these

observations directly demonstrate that the AVE has its roots in the pre-implantation blastocyst.

DISCUSSION

The technical advances we describe here greatly extend the value of the mouse embryo as the model of choice for studying embryonic development in mammals. We present the design, test, and proof of efficiency of a synthetic support, which, for the first time, enables high-resolution live-imaging of mouse embryo implantation and development from the free-floating blastocyst stage into the early egg cylinder. In order to validate this approach as a means of monitoring embryo development during this period, we have followed the origin of the AVE. These studies confirmed that AVE-marker gene expression is initiated in pre-implantation embryos and that these progenitors are able to seed the future AVE, in agreement with earlier indications suggested by snapshot observations of embryos recovered from the uterus ^18-19.

We found that the most important factors enabling mouse embryos to be cultured beyond the blastocyst stage were the use of serum from human umbilical cord blood within the culture medium and the substrate used for the culture. We also confirmed that attachment of embryos to a substrate is a prerequisite for subsequent development. In fact, we found conventional tissue culture plastic to be effective but not suitable for fluorescent microscopy of the living embryos. The most successful alternative that we found for this purpose was a collagen-coated polyacrylamide hydrogel covalently bonded to a glass coverslip with 300 to 350 μιτι deep wells to harbour the embryo. This provides a powerful culture system with which we can continuously examine the stages of early mouse embryo development that are otherwise inaccessible for study.

As a proof of its suitability, we show that this system recapitulates events that we are able to examine either in blastocysts, cultured up until the point at which they would normally implant, or in embryos recovered from the uterus at the egg cylinder stage. The ability to perform time-lapse imaging in implanting embryos allowed us to identify the single cells that begin to express an AVE marker in the PE at the blastocyst stage and follow subsequent development of the AVE per se . We also identify that the cell that expresses Cerl at a higher level than its neighbours in the distal VE of the egg cylinder is able to pioneer AVE migration. The integrity of this cell and its surrounding community is required for the migratory process, since ablation of this single cell prevents both AVE migration and its expansion. Ablation of its immediate Cerl-expressing neighbour also prevents migration but permits some expansion of the Cerl domain. These findings are in accord with a recent study that reported the

maintenance of the integrity of tight and adherens junctions during AVE migration²⁴. This therefore suggests that a group of AVE cells undergo coordinated migration and that some form of cell-cell communication within the AVE is necessary for these cells to migrate. The nature of this particular "community" effect will be an interesting future topic.

In conclusion, we present a powerful new method for mouse embryo culture with huge potential for both developmental and reproductive biologists. This platform opens up the

possibility of studying implantation from an embryonic perspective. For instance, using chemically defined culture medium it will now be possible to precisely define which growth factors are essential for embryo implantation and successful development. Furthermore, when extended to human embryos, this system could give insight into these important developmental stages. In a clinical context such an approach could be used, for example, to identify substances or culture conditions that either improve embryo implantation and development, or those that could prevent implantation and hence be used as contraceptive agents.

MATERIALS AND METHODS.

Embryo collection and culture.

Blastocysts were collected in M2 medium with 4mg/ml of BSA from Fl (C57BL/ 6XCBA/H) females mated with Cerl-GFP males ²⁰. Zona pellucidas were removed by treatment with acidified Tyrode's solution and embryos were washed in KSOM medium as described before (Torres-Padilla et al . , 2008) . After a short recovery, embryos were transferred to 5μ1, culture drops of in vitro culture media 1 (IVC1: CMRL-1066 (Invitrogen)

supplemented with 200 mM L-glutamine (Invitrogen), lOOmM Sodium pyruvate (Invitrogen) , Penicilin and Streptomycin (Invitrogen), and 20% Fetal Calf Serum (FCS, Invitrogen) either in plastic tissue culture dishes or on polyacrylamide matrices (see below) . On the evening of Day 2, the medium was replaced by mouth pipeting with in vitro culture media 2 (IVC2) , which is identical to the above one except for the serum component: we used 20% Human Cord Serum (HCS;

Addenbrokes Hospital, Cambridge, UK) . IVC2 media was replaced on a daily basis for the remainder of the culture. For postimplantantion studies, E5.5 embryos were collected from Fl females mated with Cer-l-GFP

males and cultured in IVC2. Fluorescence intensity was measured using Image-J software. All embryo cultures were performed at 37oC in a 5% C02 atmosphere.

Polyacrylamide gels suitable for embryo culture .

Polyacrylamide gels of characterised surface stiffness covalently bonded to glass substrates have been used to study the effect of surface stiffness on mesenchymal stem cell differentiation (Engler et al . , 2006) . We present a modified surface topography on the polyacrylamide gels which allows mouse embryo culture in well plates and petri-dishes and simultaneous time-lapse confocal imaging of the cultures.

Circular grade 1 borosilicate glass coverslips (Menzel-Glaser, Thermo Scientific) were cleaned in a 0.1M sodium hydroxide solution (>98%, Sigma-Aldrich) . Successive dip-coating in APTMS (97%, Sigma-Aldrich), and 0.5% glutaraldehyde solutions (-50% solution, Fluka/Biochemika, Sigma-Aldrich) were then carried out, with wash-steps in distilled water after each APTMS and glutaraldehyde treatment. Polyacrylamide gel solutions were made up containing varying concentrations of N, N' -methylenebisacrylamide solution (2% in water,

Fluka/Biochemika, Sigma-Aldrich) - from 0.02% to 0.5%; and 10% acrylamide solution (40% in water), Fluka/Biochemika, Sigma- Aldrich) . Appropriate volumes of gel solution were made up using distilled water. Polyacrylamide gel was polymerised using 10% ammonium persulphate solution (98%, Sigma-Aldrich) in distilled water (1:167 volume), and NNN'-M- Tetramethylethyldiamine (TEMED) (Sigma-Aldrich) (1:250 volume) . Initial screening of embryo attachment was carried out using gels varying in N' N' -methylenebi sacrylamide concentration, but all data reported was from embryos cultured on hydrogels of 0.3% N' N' -methylenebisacrylamide

concentration.

Polyacrylamide surfaces were coated with the extracellular matrix component, collagen. A 0.2 mg/ml water solution of type

I rat-tail collagen (9.92 mg/ml, BD Bioscience) was attached to the polyacrylamide gels using a 0.5 mg/ml in water solution of U.V. light activated heterobi functional molecule, sulfo- SANPAH, (N-Sulfosuccinimidyl- 6- [4 ' -azido-2 ' -nitrophenylamino ] hexanoate; Pierce Biotechnology) . The stainless steel mould

(Fig. 2) for moulding gels into the correct shape was manufactured by the Precision Manufacturing Centre, University of Nottingham. The mould was fabricated from 6mm thick stainless steel. Embryos were cultured in one large

cylindrical well in the centre of the gel (1130 μπι diameter,

300 ]im height) , or placed one embryo in one well of five (each well 480 ]im wide and 350 ]im in length and height) .

Time-lapse confocal imaging.

Time-lapse imaging during in vitro culture was performed using a confocal spinning disk microscopy system from Intelligent Imaging Solutions. Image stacks with 5 μιτι z planes were captured every 15 - 20 minutes, scanning up to 100 μπι of the developing embryo. Imaging was interrupted for 2 - 4 hours every day for changing medium and adjusting the focus of the microscope. Image analysis was performed with Slidebook 5.0.

Laser ablation.

Elimination of the selected cell by laser irradiation was performed in an Olympus inverted FV1000 microscope with the

405 nm laser irradiating for 500 ms the selected area.

Confocal images of embryos were captured prior to and after ablation, and following 16 hours of culture using the same system. References

1. Hsu, Y.C. Differentiation in vitro of mouse embryos beyond the implantation stage. Nature 239, 200-202 (1972) . 2. Hsu, Y.C. Differentiation in vitro of mouse embryos to the stage of early somite. Dev Biol 33, 403-411 (1973) .

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8. Huang, F.J., Wu, T.C. & Tsai, M.Y. Effect of retinoic on implantation and postimplantation development of mouse embryos in vitro. Hum Reprod 16, 2171-2176 (2001) .

9. Beddington, R.S. & Robertson, E.J. Axis development and early asymmetry in mammals. Cell 96, 195-209 (1999). 10. Weber, R.J., Pedersen, R.A., Wianny, F . , Evans, M.J. & Zernicka-Goetz , M. Polarity of the mouse embryo is anticipated before implantation. Development 126, 5591-5598 (1999) . 11. Thomas, P.Q., Brown, A. & Beddington, R.S. Hex: a homeobox gene revealing periimplantation asymmetry in the mouse embryo and an early transient marker of endothelial cell precursors. Development 125, 85-94 (1998) . 12. Srinivas, S., Rodriguez, T., Clements, M., Smith, J.C. &

Beddington, R.S. Active cell migration drives the unilateral movements of the anterior visceral endoderm. Development 131, 1157-1164 (2004) . 13. Richardson, L., Torres-Padilla, M.E. & Zernicka-Goetz, M.

Regionalised signalling within the extraembryonic ectoderm regulates anterior visceral endoderm positioning in the mouse embryo. Mech Dev 123, 288-296 (2006) . 14. Rodriguez, T.A., Srinivas, S., Clements, M.P., Smith, J.C.

& Beddington, R.S. Induction and migration of the anterior visceral endoderm is regulated by the extra-embryonic

ectoderm. Development 132, 2513-2520 (2005) .

15. Soares, M.L., et al . Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi. BMC Dev Biol 5, 28 (2005) .

16. Brennan, J., et al . Nodal signalling in the epiblast patterns the early mouse embryo. Nature 411, 965-969 (2001)

17. Robertson, E.J., Norris, D.P., Brennan, J. & Bikoff, E.K. Control of early anterior-posterior patterning in the mouse embryo by TGF-beta signalling. Philos Trans R Soc Lond B Biol Sci 358, 1351-1357; discussion 1357 (2003) . 18. Takaoka, K., et al . The mouse embryo autonomously acquires anterior-posterior polarity at implantation. Dev Cell 10, 451- 459 (2006) . 19. Torres-Padilla, M.E., et al. The anterior visceral endoderm of the mouse embryo is established from both preimplantation precursor cells and by de novo gene expression after implantation. Dev Biol 309, 97-112 (2007) . 20. Mesnard, D., Filipe, M . , Belo, J. A. & Zernicka-Goetz , M.

The anterior-posterior axis emerges respecting the morphology of the mouse embryo that changes and aligns with the uterus before gastrulation . Curr Biol 14, 184-196 (2004) . 21. Perea-Gomez, A., et al . Regionalization of the mouse visceral endoderm as the blastocyst transforms into the egg cylinder. BMC Dev Biol 7 , 96 (2007) .

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While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth are

considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention. All documents cited herein are expressly incorporated by reference .

Claims

Claims

1. An in vitro method of culturing a mammalian embryo, comprising contacting a mammalian blastocyst stage embryo with culture medium comprising cord blood or a fraction thereof, wherein said embryo is located on

(i) a polystyrene substrate; or

(ii) a gel substrate, wherein the surface of said gel

substrate comprises at least one extracellular matrix protein.

2. A method according to claim 1 wherein the embryo is cultured in medium containing cord blood or a fraction thereof from attachment of the blastocyst to the substrate.

3. A method according to claim 1 or claim 2 wherein the embryo is cultured in medium containing cord blood or a fraction thereof from no later than 12 hours after attachment, no later than 11 hours after attachment, no later than 10 hours after attachment, no later than 9 hours after

attachment, no later than 8 hours after attachment, no later than 7 hours after attachment, no later than 6 hours after attachment, no later than 5 hours after attachment, no later than 4 hours after attachment, no later than 3 hours after attachment, no later than 2 hours after attachment, or no later than 1 hour after attachment.

4. A method according to any one of claims 1 to 3 wherein the blastocyst-stage embryo has been obtained from a pregnant female .

5. A method according to claim 4 comprising the step of removing the blastocyst from the zona pellucida.

6. A method according to any one of claims 1 to 5 comprising the earlier steps of (a) providing said embryo at a pre-blastocyst stage of development, and

(b) culturing said embryo to blastocyst stage.

A method according to claim 6 wherein the pre-blastocyst of development is a single cell embryo.

8. A method according to claim 7 wherein the single cell embryo is a fertilised egg.

9. A method according to claim 7 wherein the single cell embryo has been obtained by nuclear transfer.

10. A method according to any one of claims 1 to 9 comprising the steps of:

(i) providing a first in vitro culture comprising said blastocyst stage embryo in a first culture medium, wherein said first culture medium does not comprise cord blood or a fraction thereof; and

(ii) either (a) removing said first culture medium from said blastocyst stage embryo and contacting said blastocyst stage embryo with a second culture medium comprising cord blood or a fraction thereof; or (b) adding cord blood or a fraction thereof to said first culture medium; to provide a second in vitro culture comprising said

blastocyst stage embryo in a second culture medium, wherein said second culture medium comprises cord blood or a fraction thereof .

11. A method according to any one of claims 1 to 10

comprising culturing said blastocyst stage embryo to a post- implantation stage of development.

12. A method according to claim 11 wherein the post- implantation stage is egg cylinder stage or embryonic disc stage .

13. A method according to any one of claims 1 to 12

comprising the step of removing one or more cells from said embryo .

14. A method according to claim 13 wherein said one or more cells is taken from the inner cell mass.

15. A method according to claim 14 wherein said cell is an epiblast cell.

16. A method according to any one of claims 13 to 15 wherein said cell is a pluripotent cell.

17. A method according to any one of claims 1 to 16 wherein the cord blood or fraction thereof is included in the culture medium at about 5% to about 50%, about 10% to about 30%, about 10% to about 20%, or about 15% to about 25%, e.g. about 10%, about 15% or about 20%.

18. A method according to any one of claims 1 to 17 wherein the cord blood or fraction thereof is from the same species as the embryo .

19. A method according to any one of claims 1 to 17 wherein the cord blood or fraction thereof and the embryo are from different species.

20. A method according to any one of claims 1 to 19 wherein the cord blood or fraction thereof is human.

21. A method according to any one of claims 1 to 20 wherein the fraction of cord blood is serum.

22. A method according to any one of claims 1 to 21 wherein the substrate is a gel substrate and the extracellular matrix protein comprises collagen and/or laminin.

23. A method according to claim 23 wherein the extracellular matrix protein further comprises fibronectin.

24. A method according to claim 22 or claim 23 wherein the collagen is Type I collagen.

25. A method according to any one of claims 1 to 24 wherein the gel is a hydrogel .

26. A method according to claim 25 wherein the gel is a polyacrylamide gel .

27. A method according to any one of claims 1 to 25 wherein the gel comprises or consists essentially of basement membrane matrix .

28. A method according to any one of claims 1 to 27 wherein the gel substrate is provided on a solid support.

29. A method according to claim 28 wherein the solid support is glass.

30. A method according to any one of claims 1 to 21 wherein the substrate is polystyrene and wherein the surface of the polystyrene substrate is coated with extracellular matrix protein .

31. A method according to claim 30 wherein the extracellular membrane protein comprises collagen and/or laminin.

32. A method according to claim 31 wherein the extracellular matrix protein further comprises fibronectin.

33. A method according to claim 31 or claim 32 wherein the collagen is Type I collagen.

34. A method according to any one of claims 1 to 33 wherein the surface of the substrate comprises one or more receptacles adapted to contain a culture comprising appropriate culture medium and one or more embryos.

35. A method according to claim 34 wherein the or each said culture comprises a plurality of embryos.

36. A method according to claim 34 or claim 35 wherein the or each said culture has a volume of 1-2 μΐ per embryo.

37. A method according to any one of claims 1 to 36

comprising recording one or more images of the embryo.

38. A method according to any one of claims 1 to 37

comprising contacting said embryo with a test agent and determining the effect of said test agent on development of said embryo.

39. A culture system comprising a mammalian blastocyst stage embryo in culture medium comprising cord blood or a fraction thereof, wherein said embryo is located on

(i) a polystyrene substrate; or

(ii) a gel substrate, wherein the surface of said gel

substrate comprises at least one extracellular matrix protein.

40. A culture system according to claim 39 wherein the embryo is attached to the substrate.

41. A culture system according to claim 39 or claim 40 wherein the embryo has been cultured in medium containing cord blood or a fraction thereof from no later than 12 hours after attachment, no later than 11 hours after attachment, no later than 10 hours after attachment, no later than 9 hours after attachment, no later than 8 hours after attachment, no later than 7 hours after attachment, no later than 6 hours after attachment, no later than 5 hours after attachment, no later than 4 hours after attachment, no later than 3 hours after attachment, no later than 2 hours after attachment, or no later than 1 hour after attachment.

42. A culture system according to any one of claims 39 to 41 wherein the cord blood or fraction thereof is included in the culture medium at about 5% to about 50%, about 10% to about

30%, about 10% to about 20%, or about 15% to about 25%, e.g. about 10%, about 15% or about 20%.

43. A culture system according to any one of claims 39 to 42 wherein the cord blood or fraction thereof is from the same species as the embryo.

44. A culture system according to any one of claims 39 to 42 wherein the cord blood or fraction thereof and the embryo are from different species.

45. A culture system according to any one of claims 39 to 44 wherein the cord blood or fraction thereof is human.

46. A culture system according to any one of claims 39 to 45 wherein the fraction of cord blood is serum.

47. A culture system according to any one of claims 39 to 46 wherein the substrate is a gel substrate and the extracellular matrix protein comprises collagen and/or laminin.

48. A culture system according to claim 47 wherein the extracellular matrix protein further comprises fibronectin.

49. A culture system according to claim 47 or claim 48 wherein the collagen is Type I collagen.

50. A culture system according to any one of claims 39 to 50 wherein the gel is a hydrogel .

51. A culture system according to claim 50 wherein the gel is a polyacrylamide gel.

52. A culture system according to any one of claims 39 to 50 wherein the gel comprises or consists essentially of basement membrane matrix.

53. A culture system according to any one of claims 39 to 51 wherein the gel substrate is provided on a solid support.

54. A culture system according to claim 53 wherein the solid support is glass.

55. A culture system according to any one of claims 39 to 46 wherein the substrate is polystyrene and wherein the surface of the polystyrene substrate is coated with extracellular matrix protein.

56. A culture system according to claim 55 wherein the extracellular membrane protein comprises collagen and/or laminin .

57. A culture system according to claim 56 wherein the extracellular matrix protein further comprises fibronectin.

58. A culture system according to claim 56 or claim 57 wherein the collagen is Type I collagen.

59. A culture system according to any one of claims 39 to 58 wherein the surface of the substrate comprises one or more receptacles containing a culture comprising appropriate culture medium and one or more embryos.

60. A culture system according to claim 59 wherein the or each said culture comprises a plurality of embryos.

61. A culture system according to claim 59 or claim 60 wherein the or each said culture has a volume of 1-2 μΐ per embryo .

62. A method of imaging an embryo during development, the method comprising providing a culture system according to any one of claims 39 to 61 and imaging apparatus, and recording an image of said embryo.

63. A method according to claim 62 wherein said image is a two dimensional or three dimensional image.

64. A method according to claim 62 or claim 63 comprising recording a plurality of images of the same embryo.

65. An imaging apparatus comprising a culture system according to any one of claims 39 to 61, microscopy apparatus and suitable recording apparatus .

66. An imaging apparatus according to claim 65 further comprising image processing apparatus.

67. A method or imaging apparatus according to any one of claims 62 to 66 comprising a fluorescence microscope.

68. A method or imaging apparatus according to any one of claims 62 to 66 comprising a confocal microcope.

69. A method or imaging apparatus according to any one of claims 62 to 68 wherein said culture system comprises a gel substrate, optionally on a glass support.

70. A method for investigating the effect of a test agent on embryo development comprising the steps of:

(a) providing a culture system according to any one of claims 39 to 61;

(b) contacting said culture system with a test agent; and (c) determining the effect of said test agent on the embryo.

71. A method according to claim 70 comprising contacting the culture system with the test agent before attachment of the embryo to the substrate.

72. A method according to claim 70 comprising contacting the embryo with the test agent after attachment of the embryo to the substrate.

73. A method according to claim 71 or claim 72 further comprising determining the subsequent effect on attachment of the embryo to the substrate.

74. A method according to any one of claims 70 to 73 further comprising recording one or more images of the embryo.

75. A method, culture system or imaging apparatus according to any of the preceding claims wherein the embryo is non- human .