WO2015022541A1

WO2015022541A1 - Media and methods for culturing embryos and stem cells

Info

Publication number: WO2015022541A1
Application number: PCT/GB2014/052500
Authority: WO
Inventors: Magdalena Zernicka-Goetz; Ivan BEDZHOV
Original assignee: Cambridge Enterprise Limited
Priority date: 2013-08-15
Filing date: 2014-08-14
Publication date: 2015-02-19

Abstract

Media, kits and methods for culturing embryos and stem cells in vitro are provided. The media comprise an insulin receptor agonist, an oestrogen receptor agonist, and a progesterone receptor agonist. The media enable reliable and reproducible in vitro culture of embryos from a pre-implantation stage of development to a post- implantation stage of development.

Description

MEDIA AND METHODS FOR CULTURING EMBRYOS AND

STEM CELLS

FIELD OF THE INVENTION

[1] The invention relates to cell culture, in particular media, kits and methods for culturing embryos and stem cells.

BACKGROUND TO THE INVENTION

[2] Implantation of the mammalian embryo into the uterus is a defining characteristic of the phylum and is critical for successful development. 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 ^{1 2}. However, until recently, the experimental procedures to reproduce blastocyst development from pre-implantation to post-implantation that have been described^3"10 have not been extensively used due to inherent irreproducibility. However, in 2012 Morris and co-workers described an in-vitro culture system that allowed the dynamics of the anterior-posterior axis formation to be followed in the developing mouse embryo. This system used an in-vitro culture medium containing human cord serum¹¹.

[3] It is an object of the present invention to provide further and improved in-vitro culture media and methods for culturing embryos from a pre-implantation stage of development to a post-implantation stage of development.

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

[5] It is also an object of the present invention to provide in-vitro culture media and methods for culturing stem cells. [6] 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. In particular, the defined media described herein provide culturing environments which reduce variability and provide consistency, particularly during certain critical stages of embryonic development. 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.

SUMMARY OF THE INVENTION

[7] The invention provides an in vitro culture medium comprising: an insulin receptor agonist, an oestrogen receptor agonist, and progesterone receptor agonist. Preferably, the medium is capable of supporting development of a mammalian embryo on a substrate from a pre-implantation stage of development to a post-implantation stage of development. The medium may also be free, or substantially free, of human serum.

[8] The medium may comprise insulin, or an analogue thereof. The medium may comprise oestrogen, or an analogue thereof. The medium may comprise progesterone, or an analogue thereof. [9] The medium may comprise a basal medium. The basal medium may comprise water, salts, amino acids, a carbon source, vitamins, lipids and a buffer.

[10] The medium may comprise an albumin. The medium may comprise a non- human mammalian serum, e.g. fetal calf serum (FCS), which may be included in the culture medium at a concentration of 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%. Alternatively, the medium may be serum-free, or substantially free of serum. [11] The pre-implantation stage may be the blastocyst stage, for example prior to the attachment of the blastocyst to the substrate. The post-implantation stage may be upon or any stage subsequent to the outgrowth of trophoblastic cells. In particular the post-implantation stage may be the emergence of the egg cylinder at the egg cylinder stage in rodents (e.g. in mice) or its equivalent in other mammals, such as at the emergence of the embryonic disc at the embryonic disc stage in primates (e.g. humans). Other pre-implantation and post-implantation stages are described below. [12] The insulin receptor agonist may be one or more of insulin, IGF -I, and/or IGF-II, and/or an analogue thereof. The concentration of the insulin receptor agonist (e.g. insulin) in the culture medium may be about 0.1 mg/1 to about 200 mg/1, about 0.5 mg/1 to about 100 mg/1, about 1 mg/1 to about 50 mg/1 , about 2 mg/1 to about 25 mg/1 , or about 5 mg/1 to about 12.5 mg/1 e.g. about 10 mg/1. The concentration of the insulin receptor agonist (e.g. IGF-1 or IGF-2) in the culture medium may be about 0.05 ng/ml to about 300 ng/ml, about 0.25 ng/ml to about 200 ng/ml, about 1 ng/ml to about 150 ng/ml, about 5 ng/ml to about 100 ng/ml, or about 25 ng/ml to about 75 ng/ml e.g. about 50 ng/ml. [13] The oestrogen receptor agonist may be one or more steroidal oestrogens, for example β-estradiol and/or a metabolite thereof (for example, 2-hydroxyestradiol and 4-hydroxyestradiol), estrone, estriol, and/or estetrol, and/or an analogue thereof. Additionally or alternatively, the oestrogen receptor agonist may be one or more nonsteroidal oestrogens, for example a xenoestrogen, a phytoestrogen and/or a mycoestrogen, and/or an analogue thereof. The concentration of the oestrogen receptor agonist in the culture medium may be about 1 nM to about 100 nM, about 2 nM to about 50 nM, about 3 nM to about 25 nM, about 4 nM to about 12.5 nM, about 5 nM to about 12 nM, about 6 nM to about 11 nM, about 7 nM to about 10 nM, or about 7.5 nM to about 9 nM e.g. about 8 nM.

[14] The progesterone receptor agonist may be progesterone and/or an analogue thereof. The concentration of the progesterone receptor agonist, or an analogue thereof, in the culture medium may be about 1 ng/ml to about 2 ug/ml, about 5 ng/ml to about 1.5 μg/ml, about 10 ng/ml to about 1 μg/ml, about 20 ng/ml to about 750 ng/ml, about 50 ng/ml to about 500 ng/ml, or about 100 ng/ml to about 300 ng/ml e.g. about 200 ng/ml.

[15] The in vitro culture medium may be free of serum, substantially free of serum or essentially free of serum and may further comprise a serum replacement. The serum replacement may be included in the culture medium at about 5% to about 60%, about 10% to about 50%, about 15% to about 45%, or about 20% to about 40%. Preferably the in vitro culture medium is free of serum or substantially free of serum and comprises 30% serum replacement.

[16] The culture medium may further comprise one or more of transferrin, selenium (for example sodium selenite, in this case provided as a salt), and/or ethanolamine, and/or an analogue thereof. Preferably, the culture medium comprises transferrin, selenium (for example sodium selenite, in this case provided as a salt) and ethanolamine. For example, the culture medium may comprise ITS-X (Invitrogen, 51500-056). The concentration of transferrin, or an analogue thereof, in the culture medium may be about 0.01 mg/1 to about 500 mg/1, about 0.05 mg/1 to about 250 mg/1, about 0.1 mg/1 to about 100 mg/1, about 0.5 mg/1 to about 25 mg/1, about 1 mg/1 to about 10 mg/1, or about 2.5 mg/1 to about 7.5 mg/1 e.g. about 5.5 mg/1. The concentration of selenium (for example sodium selenite), or an analogue thereof, in the culture medium may be about 0.0001 mg/1 to about 0.1 mg/1, about 0.0002 mg/1 to about 0.05 mg/1, about 0.0005 mg/1 to about 0.02 mg/1, about 0.001 mg/1 to about 0.01 mg/1, or about 0.005 mg/1 to about 0.0075 mg/1 e.g. about 0.0067 mg/1 . The concentration of ethanolamine, or an analogue thereof, in the culture medium may be about 0.01 mg/1 to about 500 mg/1, about 0.025 mg/1 to about 250 mg/1, about 0.05 mg/1 to about 100 mg/1, about 0.1 mg/1 to about 50 mg/1, about 0.25 mg/1 to about 25 mg/1, about 0.5 mg/1 to about 10 mg/1, or about 1 mg/1 to about 5 mg/1 e.g. about 2 mg/1. [17] The culture medium may comprise L-glutamine at a concentration as further defined herein. Preferably L-glutamine is used at a concentration of about 2 mM. [18] The culture medium may comprise sodium pyruvate at a concentration as further defined herein. Preferably sodium pyruvate is used at a concentration of at about 1 mM. [19] The culture medium may comprise one, more than one or all components selected from the group consisting of L-glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine, each at a concentration as further defined herein. Preferably, the culture medium may further comprise L-glycine at a concentration of about 7.5 mg/1, L-alanine at a concentration of about 9 mg/1, L- asparagine at a concentration of about 13 mg/1, L-aspartic acid at a concentration of about 13 mg/1, L-glutamic acid at a concentration of about 14.5 mg/1, L-proline at a concentration of about 11.5 mg/1 and L-serine at a concentration of about 10.5 mg/1.

[20] The culture medium may further comprise an agonist of the activin type 1 or type 2 receptors, for example, activin and/or nodal, and/or an analogue thereof. The concentration of the agonist (e.g. activin) in the culture medium may be about 1 ng/ml to about 200 ng/ml, about 5 ng/ml to about 100 ng/ml, about 10 ng/ml to about 50 ng/ml, about 15 ng/ml to about 25 ng/ml e.g. about 20 ng/ml. [21] The culture medium may comprise a reducing agent, for example N-acetyl-L- cysteine, glutathione, dithiothreitol (DTT) or 2-mercaptoethanol (β-mercaptoethanol), and/or an analogue or substitute thereof. The concentration of the reducing agent in the culture medium may be about 0.5 μΜ to about 250 μΜ, about 5 μΜ to about 200 μΜ, about 7.5 μΜ to about 150 μΜ, about 10 μΜ to about 100 μΜ, about 15 μΜ to about 50 μΜ, about 17.5 μΜ to about 40 μΜ, or about 20 μΜ to about 30 μΜ e.g. about 25 μΜ. Preferably the culture medium further comprises N-acetyl-L-cysteine at a concentration of at about 25 μΜ.

[22] The culture medium is capable of supporting development of a mammalian embryo on a substrate. The substrate may comprise a solid support, preferably comprising a plastics material or glass. Alternatively, the substrate may be in contact with a solid support, wherein the solid support preferably comprises a plastics material or glass. Where the substrate is in contact with a solid support, the substrate may comprise a matrix, preferably comprising at least one extracellular matrix protein or analogue thereof. The extracellular matrix protein may be one or more of collagen, laminin, fibronectin, vitronectin and/or gelatin. Preferably, the extracellular matrix protein is collagen and/or laminin. The matrix may activate signalling through β- integrin receptors. The substrate may comprise cells or a tissue, or an extract thereof . Alternatively, the substrate does not comprise cells or a tissue or a feeder-cell layer. Preferably the substrate does not comprise uterine epithelial cells or uterine endometrium. Preferably, the medium does not comprise a conditioned medium.

[23] The invention also provides a culture medium supplement for producing the in vitro culture medium of the invention comprising an insulin receptor agonist, an oestrogen receptor agonist, and a progesterone receptor agonist.

[24] The culture medium supplement may comprise insulin, or analogue thereof. The medium may comprise oestrogen, or an analogue thereof. The medium may comprise progesterone, or an analogue thereof.

[25] The culture medium supplement can be constituted such that when converted to the final medium for use in the in vitro culturing of embryos, any of the in vitro culture media embodiments defined herein are produced. In all cases, upon conversion, the final medium thereby produced is capable of supporting development of a mammalian embryo on a substrate from a pre-implantation stage of development to a post-implantation stage of development.

[26] Any of the optional additional components, such as defined herein, may be included in the culture medium supplement or may be provided as separate supplements. Components of the supplement may be provided in amounts such that when reconstituted any of the working amounts defined herein are produced, provided that the medium is capable of supporting development of a mammalian embryo on a substrate from a pre-implantation stage of development to a post-implantation stage of development.

[27] For example, the culture medium supplement may comprise one or more components, or analogues thereof, selected from transferrin; sodium selenite; ethanolamine; sodium pyruvate; L-glutamine; L-glycine; L-alanine; L-asparagine; L- aspartic acid; L-glutamic acid; L-proline; L-serine; and N-acetyl-L-cysteine.

[28] The culture medium supplement may be constituted such that the individual components are concentrated relative to the final in vitro culture medium by between about x5 to about x500, about x25 to about x250, about x50 to about x200, or about x75 to about xl50 e.g. about xlOO.

[29] The invention also provides a kit for culturing a mammalian embryo.

[30] The kit may comprise (1) any of the in vitro culture media defined herein comprising: insulin, an insulin analogue, or an insulin receptor agonist, oestrogen, an oestrogen analogue, or an oestrogen receptor agonist, and progesterone, a progesterone analogue, or a progesterone receptor agonist and wherein the medium is capable of supporting development of a mammalian embryo on a substrate from a pre- implantation stage of development to a post-implantation stage of development and wherein the medium may also be free, or substantially free, of human serum; and (2) any substrate as defined herein. [31] The kit may also comprise (1) any of the culture medium supplements, as defined herein, for producing the in vitro culture medium of the invention; and (2) (I) a basal medium, as defined herein; and/or (II) one or more separate supplements comprising one or more of the components as defined herein. For example, separate supplements may comprise one or more components, or analogues thereof, selected from transferrin; sodium selenite; ethanolamine; sodium pyruvate; L-glutamine; L- glycine; L-alanine; L-asparagine; L-aspartic acid; L-glutamic acid; L-proline; L- serine; and N-acetyl-L-cysteine.

[32] In all cases the kit may further comprise a substrate for culturing the mammalian embryo. The substrate may comprise a solid support. Preferably, the solid support is of a plastics material or glass. The substrate may not be pre-coated. Optionally, the substrate may comprise a matrix. The matrix may comprise at least one extracellular matrix protein. The extracellular matrix protein may be one or more of collagen, laminin, fibronectin, vitronectin and/or gelatin. Preferably, the extracellular matrix protein is collagen and/or laminin. The matrix may activate signalling through β-integrin receptors. The substrate may comprise cells or a tissue, or an extract thereof. Alternatively, the substrate may not comprise a cell, tissue or feeder-cell layer, preferably the substrate does not comprise uterine epithelial cells or uterine endometrium. 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. Preferably, the substrate is suitable for imaging, for example time-lapse imaging as discussed below. [33] The invention also provides an in vitro method of culturing a mammalian embryo, comprising contacting a mammalian embryo with a culture medium of the invention, wherein the embryo is cultured on or in a substrate from a pre-implantation stage of development to a post-implantation stage of development. [34] The pre-implantation stage may be the blastocyst stage, for example prior to the attachment of the blastocyst to the substrate. The post-implantation stage 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). Other pre-implantation and post-implantation stages are described below.

[35] The method may comprise the step of removing the blastocyst from the zona pellucida.

[36] The method may further comprise the earlier steps of providing said embryo at a pre-blastocyst stage of development, and culturing said embryo to blastocyst stage. The culturing may be performed using the culture medium of the invention. The pre-blastocyst stage of development may be a single cell embryo, for example a fertilised egg. The single cell embryo may be obtained by nuclear transfer. [37] The methods may involve the culture of an embryo on a substrate from a pre- implantation stage of development to a post-implantation stage of development using only serum-free culture medium, or culture medium substantially free of serum. [38] The methods may comprise the steps of providing a first in vitro culture comprising an embryo in a first culture medium, wherein the first culture medium comprises fetal calf serum; and removing the first culture medium from the embryo and contacting the embryo with a second culture medium that is serum-free, or substantially free of serum, to provide a second in vitro culture comprising the embryo in a serum-free culture medium (or a medium substantially free of serum).

[39] Fetal calf serum may be 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%.

[40] The step of removing the first culture medium from the embryo and contacting the embryo with a second culture medium that is serum-free, or substantially free of serum, may be performed at a post-implantation stage, preferably the egg cylinder stage or embryonic disc stage, or, alternatively, at a pre-implantation stage. Pre-implantation and post-implantation stages are described below.

[41] The methods may comprise the step of removing one or more cells from said embryo. The one or more cells may be taken from the inner cell mass, for example an epiblast cell. The cell(s) may be pluripotent cell(s). The one or more cells may be taken from extraembryonic lineages e.g. the trophectoderm and/or the primitive endoderm, and/or derivatives of these lineages. The cell(s) may be unipotent or multipotent. For example, such cells may be useful for genotyping an embryo without the destruction of the embryo.

[42] The substrate used in the methods may comprise at least one extracellular matrix protein. The extracellular matrix protein may be one or more of collagen, laminin, fibronectin and/or gelatin. Preferably, the extracellular matrix protein is collagen and/or laminin. The matrix may activate signalling through β-integrin receptors. The substrate may comprise cells or a tissue, or an extract thereof. Alternatively, the substrate does not comprise cells or a tissue. The substrate may comprise a solid support. Preferably, the solid support is a plastics material or glass. 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 or each culture may comprise a plurality of embryos. The or each culture may have a volume of 15 μΐ to about 20 μΐ per embryo. Preferably, the substrate is suitable for imaging, for example time-lapse imaging as discussed below. [43] The methods may further comprise the step of recording one or more images of the embryo. Additionally, or alternatively, the methods may further comprise the steps of contacting the embryo with a test agent and determining the effect of the test agent on development of said embryo. [44] The invention also provides an in vitro method of culturing a mammalian embryo comprising contacting a mammalian embryo with a culture medium of the invention, wherein said embryo is cultured on a substrate from a pre-implantation stage of development to produce a rosette of polarized cells. The rosette may be formed at a stage corresponding to Carnegie stage 4 or 5a (Theiler stage 6 or 7 or 8).

[45] The substrate used in the methods may comprise at least one extracellular matrix protein. The extracellular matrix protein may be one or more of collagen, laminin, fibronectin and/or gelatin. Preferably, the extracellular matrix protein is collagen and/or laminin. The matrix may activate signalling through β-integrin receptors. The substrate may comprise cells or a tissue, or an extract thereof. Alternatively, the substrate does not comprise cells or a tissue. The substrate may comprise a solid support. Preferably, the solid support is a plastics material or glass. 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 or each culture may comprise a plurality of embryos. The or each culture may have a volume of 15 μΐ to about 20 μΐ per embryo. Preferably, the substrate is suitable for imaging, for example time-lapse imaging as discussed below.

[46] The in vitro method of of culturing a mammalian embryo may further comprise the steps of removing one or more cells from the rosette of polarized cells and culturing the cells to produce differentiated cells. The methods may further comprise producing a rosette of polarized cells as described above, culturing the rosette of polarized cells using the culture conditions of the invention as described herein, subjecting said rosette of polarized cells to culture conditions which propote differentiation, and thereby producing differentiated cells. The differentiated cells may thereafter be isolated from said rosette and further cultured. The differentiated cells may be selected from the group consisting of exocrine secretory epithelial cells, hormone secreting cells, cells of the integumentary system, cells of the nervous system, metabolism and storage cells, barrier function cells, extracellular matrix cells, contractile cells, blood and immune system cells, germ cells, nurse cells and interstitial kidney cells. Further types of differentiated cells that may be produced using the methods of the invention are described below. Cell types that may be produced from the rosette of polarized cells as described herein include endoderm, ectoderm and mesoderm as well as cells of the germ line.

[47] The invention also provides differentiated cells obtainable by the methods of the invention. The differentiated cells may be selected from the group consisting of exocrine secretory epithelial cells, hormone secreting cells, cells of the integumentary system, cells of the nervous system, metabolism and storage cells, barrier function cells, extracellular matrix cells, contractile cells, blood and immune system cells, germ cells, nurse cells and interstitial kidney cells. Further types of differentiated cells that may be produced using the methods of the invention are described below. [48] Differentiated cells may be produced from the one or more cells removed from the rosette of polarized cells by the methods of the invention with an efficiency of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100%. [49] The invention also provides an in vitro method of culturing stem cells, comprising contacting the stem cells with a culture medium of the invention, wherein the stem cells are cultured on or in a substrate.

[50] The substrate may be a matrix and the method may comprise the step of suspending the stem cells in the matrix. The substrate may comprise at least one extracellular matrix protein. The extracellular matrix protein may be one or more of collagen, laminin, fibronectin and/or gelatin. Preferably, the extracellular matrix protein is collagen and/or laminin. The matrix may activate signalling through β- integrin receptors. The substrate may comprise cells or a tissue, or an extract thereof. Alternatively, the substrate does not comprise cells or a tissue. The substrate may comprise a solid support. Preferably, the solid support is a plastics material or glass. The surface of the substrate may comprise one or more receptacles adapted to contain a culture comprising appropriate culture medium and one or more stem cells. The or each culture may comprise a plurality of stem cells. The or each culture may have a volume of 1-2 μΐ. Preferably, the substrate is suitable for imaging, for example time- lapse imaging as discussed below.

[51] The stem cells may be embryonic stem cells. Alternatively, the stem cells are induced pluripotent stem cells. The stem cells may be non-human mammalian stem cells. The stem cells may be human stem cells. Further types of stem cells that may be used in the methods of the invention are described below.

[52] The methods may enable the culture of stem cells to form typical embryonic structures with a central cavity that correspond to the morphology of the embryonic lineage found in E4.75 - E5.5 mouse embryos in vivo.

[53] The methods may further enable the culture of stem cells to form an embryo corresponding to one of the pre-implantation or post-implantation stages described below.

[54] The methods may enable the culture of stem cells to produce a sphere of cells having a lumen and apical-basal polarity. The sphere of cells may correspond to the morphogenetic structure formed at a stage corresponding to Carnegie stage 4 or 5a (Theiler stage 6 or 7 or 8). The methods may further comprise the steps of removing one or more cells from the sphere of cells and culturing the cells to produce differentiated cells. The methods may further comprise producing a sphere of cells as described above, culturing the sphere of cells using the culture conditions of the invention as described herein, subjecting said sphere of cells to culture conditions which propote differentiation, and thereby producing differentiated cells. The differentiated cells may thereafter be isolated from said sphere and further cultured. The differentiated cells may be selected from the group consisting of exocrine secretory epithelial cells, hormone secreting cells, cells of the integumentary system, cells of the nervous system, metabolism and storage cells, barrier function cells, extracellular matrix cells, contractile cells, blood and immune system cells, germ cells, nurse cells and interstitial kidney cells. Further types of differentiated cells that may be produced using the methods of the invention are described below. [55] The methods may further comprise producing a sphere of cells using the culture conditions of the invention as described herein and wherein the culture conditions further comprise activin and bFGF, subjecting said sphere of cells to culture conditions comprising BMP4, SCF, EGF and LIF and thereby producing primordial germ cells. The primordial germ cells may thereafter be isolated from said sphere and further cultured. In such methods the further culture conditions comprising BMP4, SCF, EGF and LIF may be imposed for 3 days or 4 days. In such methods, the stem cells from which the sphere of cells are produced may be embedded in matrigel during the step of culturing in conditions which further comprise activin and bFGF. [56] The invention also provides differentiated cells obtainable by the methods of the invention. The differentiated cells may be selected from the group consisting of exocrine secretory epithelial cells, hormone secreting cells, cells of the integumentary system, cells of the nervous system, metabolism and storage cells, barrier function cells, extracellular matrix cells, contractile cells, blood and immune system cells, germ cells, nurse cells and interstitial kidney cells. Further types of differentiated cells that may be produced using the methods of the invention are described below.

[57] Differentiated cells may be produced from the one or more cells removed from the sphere of cells by the methods of the invention with an efficiency of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100%.

[58] The invention also provides a method for investigating the effect of a test agent on embryo development comprising the steps of: culturing a mammalian embryo using a culture medium of the invention; contacting the embryo with a test agent; and determining the effect of the test agent on the embryo. [59] The step of determining the effect of the test agent on the embryo may comprise comparing a phenotype or a genotype in the presence of said test agent with the phenotype or genotype in the absence of said test agent. [60] The method may comprise contacting the embryo with the test agent before attachment of the embryo to the substrate. Alternatively, the method may comprise contacting the embryo with the test agent after attachment of the embryo to the substrate. The method may further comprise the step of determining the subsequent effect on attachment of the embryo to the substrate.

[61] The method may comprise recording one or more images of the embryo.

[62] The invention provides the use of an in vitro culture medium of the invention for culturing a mammalian embryo on a substrate from a pre-implantation stage of development to a post-implantation stage of development.

[63] The invention also provides the use of an in vitro culture medium of the invention for culturing stem cells on a substrate. [64] Examples of substrates that may be used are provided below.

[65] The invention provides a method of imaging an embryo during development comprising culturing a mammalian embryo using a culture medium of the invention and imaging apparatus, and recording an image of said embryo. The image may be a two dimensional or three dimensional image. A plurality of images may be recorded of the same embryo.

[66] The invention also provides an imaging apparatus comprising a kit of the invention, microscopy apparatus and suitable recording apparatus. An imaging apparatus may further comprise image processing apparatus. Additionally, an imaging apparatus may further comprise a fluorescent microscope. Additionally, or alternatively, an imaging apparatus may further comprise a confocal microscope. BRIEF DESCRIPTION OF THE DRAWINGS

[67] Fig.l Schematic representation of the early mouse development. During the first four days, starting from the fertilization, the embryo is free floating in the maternal reproductive tract. The fertilized egg is undergoing a series of cleavage divisions, without changing the overall size of the embryo. After the early lineage specifications are accomplished, the mature blastocyst implants into the uterine wall (E4.5). During peri-implantation stages (black box) the embryo becomes hidden from view and hardly accessible for experimental manipulations. At this time the embryo undergoes dramatic morphogenic transformation leading to the formation of the egg cylinder at E5.5. After setting up the body axes, the tissues of the egg cylinder give rise to the fetus and contribute to the placenta and the yolk sac.

[68] Fig.2 Development of mouse blastocyst beyond implantation outside the body of the mother using defined (serum-free) medium in accordance with Example 1, and schematic representation of the main steps of the in vitro culture process. [69] Fig.3 Similar organization of the mouse embryonic lineage in in vivo recovered at E6.0 and in vitro cultured embryos (in accordance with Example 1) at day 4.

[70] Fig.4 Development of mouse blastocyst beyond implantation outside the body of the mother using FCS-containing medium and defined (serum-free) medium in accordance with Example 2, and schematic representation of the main steps of the in vitro culture process.

[71] Fig.5 Development of human blastocyst beyond implantation outside the body of the mother using FCS-containing medium and defined (serum-free) medium in accordance with Example 3.

[72] Fig.6 Culture of mouse ES cells in 3D extracellular matrix using defined (serum-free) medium in accordance with Example 4. A) Schematic representation of the ES cell culture method in 3D extracellular matrix. B) Confocal images of CAG- GFP ES cells. C) Comparison between epiblast morphology of E4.75-E5.0 embryo versus mouse ES cells grown for 48 hours in matrigel. DETAILED DESCRIPTION

[73] The invention provides an in vitro culture medium comprising: an insulin receptor agonist, an oestrogen receptor agonist, and progesterone receptor agonist. [74] In all embodiments described herein, the medium is capable of supporting development of a mammalian embryo on a substrate from a pre-implantation stage of development to a post-implantation stage of development. The medium may be capable of supporting development of a non-human mammalian embryo on a substrate from a pre-implantation stage of development to a post-implantation stage of development. The medium may be capable of supporting development of a human embryo on a substrate from a pre-implantation stage of development to a post- implantation stage of development.

[75] The medium may comprise insulin, or an insulin analogue. The medium may comprise oestrogen, or an oestrogen analogue. The medium may comprise progesterone, or a progesterone analogue.

[76] As described above, the culture medium may contain other components, or analogues thereof. The term "analogue" is used in this specification to refer to a biologically active analogue of any of the components of the culture medium. Such an analogue may be natural or synthetic.

[77] The specific biologically active ligands and compounds used in the media defined herein, such as insulin, progesterone, activin etc. are used for illustrative purposes. However, one of skill in the art will readily recognise that analogues of such ligands and compounds may equally be used as alternatives, provided that they retain the relevant biological activity. One of skill in the art will be able to identify, in a routine manner, other biologically active compounds that are suitable for use as substitutes. For instance, these may be naturally occurring compounds or compounds which can be made by synthetic or semi-synthetic methods.

[78] The term "analogue" may refer to a compound which may be structurally related to the relevant molecule. The term "agonist" may refer to a compound which might not be structurally related to the relevant molecule. For example, an agonist may activate the relevant receptor by altering the conformation of the receptor. Nevertheless, in both cases the terms are used in this specification to refer to compounds or molecules which can mimic, reproduce or otherwise generally substitute for the specific biological activity of the relevant molecule.

[79] In addition, the culture medium may contain a basal medium. The basal medium may comprise water, salts, amino acids, a carbon source, vitamins, lipids and a buffer. Suitable carbon sources may be assessed by one of skill in the art from compounds such as glucose, sucrose, sorbitol, galactose, mannose, fructose, mannitol, maltodextrin, trehalose dihydrate, and cyclodextrin. Basal media are commercially available, for example, under the trade names Advanced DMEM/F12 (Gibco, 12634- 010) and CMRL-1066 (Invitrogen or Sigma). [80] The components of Advanced DMEM/F12 are set out in Table 1 below.

Table 1

Folic Acid 441 2.65 0.00601

Zinc sulfate (ZnS04-7H20) 288 0.864 0.003

Proteins

AlbuMAX® II 400

Human Transferrin (Holo) 7.5

Insulin Recombinant Full

10

Chain

Reducing Agents

Glutathione, monosodium 307 1 0.00326

Trace Elements

Ammonium Metavanadate 116.98 0.0003 0.0000026

Manganous Chloride 198 0.00005 0.0000003

Sodium Selenite 173 0.005 0.0000289

Other Components

D-Glucose (Dextrose) 180 3151 17.51

Ethanolamine 97.54 1.9 0.0195

Hypoxanthine Na 159 2.39 0.015

Linoleic Acid 280 0.042 0.00015

Lipoic Acid 206 0.105 0.00051

Phenol Red 376.4 8.1 0.0215

Putre seine 2HC1 161 0.081 0.000503

Sodium Pyruvate 110 110 1 Thymidine 242 0.365 0.00151

The components of CMRL-1066 are set out in Table 2 below.

Table 2

Other Components 2'Deoxyadenosine 251 10 0.0398

2'Deoxycytidine 227 10 0.0441

2'Deoxyguanosine 267 10 0.0375

5 -Methyl-deoxycytidine 225 0.1 0.000444

Co-carboxylase 461 1 0.00217

Coenzyme A 768 2.5 0.00326

D-Glucose (Dextrose) 180 1000 5.56

Diphosphopyridine

663 7 0.0106 nucleotide (NAD)

FAD (flavin adenine

786 1 0.00127 dinucleotide)

Glutathione (reduced) 307 10 0.0326

Phenol Red 376.4 20 0.0531

Sodium acetate-3H20 136 83 0.61

Sodium glucuronate-H20 236 4.2 0.0178

Thymidine 242 10 0.0413

Triphosphopyridine

743 1 0.00135 Nucleotide (NADP)

Tween 80® 5

Uridine 5'- triphosphate 484 1 0.00207

[82] The in vitro culture medium or the basal medium may be supplemented with one or more components such as L-glutamine, sodium pyruvate, non-essential amino acids (for example MEM NEAA, Gibco, 15070-063), penicillin and/or streptomycin.

[83] L-glutamine may be included in the culture medium at a concentration of about 0.1 mM to about 40 mM, about 0.2 mM to about 20 mM, about 0.5 mM to about 10 mM, about 1 mM to about 5 mM or about 1.5 mM to about 2.5 mM e.g. about 2 mM. Preferably, L-glutamine is included in the culture medium at a concentration of about 2mM.

[84] Sodium pyruvate may be included in the culture medium at a concentration of about 0.05 mM to about 20 mM, about 0.1 mM to about 10 mM, about 0.25 mM to about 5 mM, or about 0.5 mM to about 2.5 mM e.g. about 1 mM. Preferably, sodium pyruvate is included in the culture medium at a concentration of about ImM.

[85] Non-essential amino acids may be included in the culture medium, for example, comprising glycine (about 1 mg/1 to about 25 mg/1 or about 5 mg/1 to about

10 mg/1 e.g. about 7.5 mg/1), L-alanine (about 1 mg/1 to about 25 mg/1 or about 5 mg/1 to about 10 mg/1 e.g. about 9 mg/1), L-asparagine (about 5 mg/1 to about 30 mg/1 or about 10 mg/1 to about 15 mg/1 e.g. about 13.2 mg/1), L-aspartic acid (about 5 mg/1 to about 30 mg/1 or about 10 mg/1 to about 15 mg/1 e.g. about 13 mg/1), L-glutamic acid (about 5 mg/1 to about 50 mg/1 or about 10 mg/1 to about 20 mg/1 e.g. about 15 mg/1), L-proline (about 5 mg/1 to about 30 mg/1 or about 10 mg/1 to about 15 mg/1 e.g. about

11 mg/1) and/or L-serine (about 5 mg/1 to about 30 mg/1 or about 10 mg/1 to about 15 mg/1 e.g. about 11 mg/1). Preferably, the culture medium may comprise L-glycine at a concentration of about 7.5 mg/1, L-alanine at a concentration of about 9 mg/1, L- asparagine at a concentration of about 13 mg/1, L-aspartic acid at a concentration of about 13 mg/1, L-glutamic acid at a concentration of about 14.5 mg/1, L-proline at a concentration of about 11.5 mg/1 and L-serine at a concentration of about 10.5 mg/1.

[86] Penicillin may be included in the culture medium at a concentration of about 1 unit/ml to about 500 units/ml, about 2 units/ml to about 250 units/ml, about 5 units/ml to about 100 units/ml, about 10 units/ml to about 50 units/ml, or about 20 units/ml to about 30 units/ml e.g. about 25 units/ml. Streptomycin may be included in the culture medium at a concentration of about 1 μg/ml to about 500 μg/ml, about 2 μg/ml to about 250 μg/ml, about 5 μg/ml to about 100 μg/ml, about 10 μg/ml to about 50 μg/ml, or about 20 μg/ml to about 30 μg/ml e.g. about 25 μg/ml. Preferably, the culture medium may comprise penicillin at a concentration of about 25 units/ml and/or streptomycin at a concentration of about 25 μg/ml.

[87] The culture medium may be free of serum or substantially free of serum or essentially free of serum. The culture medium may comprise a serum replacement medium. Such serum replacement media are commercially available under the trade names KSR (KnockOut™ Serum Replacement, Invitrogen, 10828-010) and N2B27 (e.g. Invitrogen, ME100137L1). Alternatively, the culture medium may comprise a serum replacement medium as described in WO 98/30679 (in particular, Tables 1 to 3), the contents of which is expressly incorporated by reference. The serum replacement medium may be included in the culture medium at about 5% to about 60%, about 10% to about 50%, about 15% to about 45%, or about 20% to about 40%, e.g. about 30%. Preferably the in vitro culture medium is free of serum or substantially free of serum and comprises 30% serum replacement.

[88] The culture medium may comprise a basal medium, as defined above, (e.g. Advanced DMEM/F12) supplemented with , an insulin receptor agonist e.g. Insulin (e.g. about 2 mg/1 to about 25 mg/1), Transferrin (e.g. about 1 mg/1 to about 10 mg/1), Selenium e.g. sodium selenite (e.g. about 0.001 mg/1 to about 0.01 mg/1), Ethanolamine (e.g. about 0.5 mg/1 to about 10 mg/1), an oestrogen receptor agonist e.g. β-estradiol (e.g. about 5 nM to about 10 nM), a progesterone receptor agonist e.g. Progesterone (e.g. about 50 ng/ml to about 500 ng/ml) and a reducing agent e.g. N- acetyl-L-cysteine (e.g. about 17.5 μΜ ΐο about 40 μΜ).

[89] The culture medium may further comprise one or more of L-glutamine (e.g. about 1 mM to about 5 mM), Sodium pyruvate (e.g about 0.25 mM to about 5 mM), non-essential amino acids (e.g. comprising glycine (e.g. about 5 mg/1 to about 10 mg/1), L-alanine (e.g. about 5 mg/1 to about 10 mg/1), L-asparagine (e.g. about 10 mg/1 to about 15 mg/1), L-aspartic acid (e.g. about 10 mg/1 to about 15 mg/1), L-glutamic acid (e.g. about 10 mg/1 to about 20 mg/1), L-proline (e.g. about 10 mg/1 to about 15 mg/1) and/or L-serine (e.g. about 10 mg/1 to about 15 mg/1) , Penicillin (e.g. about 10 units/ml to about 50 units/ml) and/or Streptomycin (e.g. about 10 μg/ml to about 50 Mg/ml).

[90] The culture medium may comprise 15 to 45% KSR (KnockOut Serum Replacement). The culture medium may be serum-free, or substantially serum free. Alternatively, the culture medium may comprise KSR and FCS, optionally about 5 to 15% KSR and 5-15% FCS. The culture medium may comprise about 10% to about 30% FCS.

[91] The culture medium may consist of, or consist essentially of, a basal medium, an insulin receptor agonist, an oestrogen receptor agonist, a progesterone receptor agonist, a reducing agent, transferrin, selenium, ethanolamine and an albumin. The albumin may be provided by a non-human mammalian serum (e.g. FCS) and/or serum-replacement. The culture medium may be serum-free, or substantially serum free. Each component of the culture medium may be present in an amount such that the culture medium is suitable for supporting the development of a mammalian embryo on a substrate from a pre-implantation stage of development to a post- implantation stage of development.

[92] In one embodiment there is provided a defined in vitro culture medium that is free or substantially free of serum comprising a basal medium comprising water, salts, amino acids, a carbon source, vitamins, lipids and a buffer; and further comprising the components insulin, an insulin analogue, or an insulin receptor agonist; oestrogen, an oestrogen analogue, or an oestrogen receptor agonist; progesterone, a progesterone analogue, or a progesterone receptor agonist; transferrin, or analogue thereof; sodium selenite, or analogue or substitute thereof; ethanolamine, or analogue thereof; sodium pyruvate; L-glutamine; L-glycine; L-alanine; L-asparagine; L-aspartic acid; L- glutamic acid; L-proline; L-serine; N-acetyl-L-cysteine; a serum substitute, optionally wherein the defined in vitro culture medium comprises 30% serum substitute; and wherein said components are provided in amounts such that the medium is capable of supporting development of a mammalian embryo on a substrate from a pre- implantation stage of development to a post-implantation stage of development; and wherein the medium further comprises penicillin and streptomycin. Components of the medium may be provided in any of the amounts defined herein, provided that the medium is capable of supporting development of a mammalian embryo on a substrate from a pre-implantation stage of development to a post-implantation stage of development.

[93] In a further embodiment there is provided a defined in vitro culture medium that is free or substantially free of serum that is capable of supporting development of a mammalian embryo on a substrate from a pre-implantation stage of development to a post-implantation stage of development comprising the components listed at Table 3. It will be appreciated that the formulation as listed at Table 3 is intended for illustrative purposes and is not intended to limit the scope of the invention. Table 3 - complete formulation of IVC defined culture medium (final/working concentrations)

Thiamine hydrochloride 1.519

Vitamin B 12 0.476 i-Inositol 8.82

Inorganic Salts

Calcium Chloride (CaC12) (anhyd.) 81.62

Cupric sulfate (CuS04-5H20) 0.00091

Ferric Nitrate (Fe(N03)3"9H20) 0.035

Ferric sulfate (FeS04-7H20) 0.2919

Magnesium Chloride (anhydrous) 20.048

Magnesium Sulfate (MgS04) (anhyd.) 34.188

Potassium Chloride (KC1) 218.26

Sodium Bicarbonate (NaHC03) 1706.6

Sodium Chloride (NaCl) 4896.85

Sodium Phosphate dibasic (Na2HP04)

49.714 anhydrous

Sodium Phosphate monobasic (NaH2P04-H20) 43.75

Zinc sulfate (ZnS04-7H20) 0.6048

Proteins

AlbuMAX® II 280

Human Transferrin (Holo) 10.75

Insulin Recombinant Full Chain 17 β-estradiol 8nM

Progesterone 200 ng/ml

Reducing Agents

Glutathione, monosodium 0.7

Trace Elements

Ammonium Metavanadate 0.00021

Manganous Chloride 0.000035

Sodium Selenite 0.0102

Other Components

D-Glucose (Dextrose) 2205.7

Ethanolamine 3.33

Hypoxanthine Na 1.673

Linoleic Acid 0.0294

Lipoic Acid 0.0735

Phenol Red 5.67

Putre seine 2HC1 0.0567

Sodium Pyruvate 187 Thymidine 0.2555

N-acetyl-L-cysteine 2μΜ

Penicillin 25 units/ml

Streptomycin 25 μg/ml

Serum replacement

30%

(KSR KnockOut Serum Replacement)

[94] In all of the in vitro culture medium embodiments defined herein the culture medium further may be free, substantially free or essentially free of one or more of an EGF receptor agonist or an analogue thereof, such as EGF or an EGF substitute; an FGF receptor agonist or an analogue thereof, such as FGF or an FGF substitute; a LIF receptor agonist or an analogue thereof, such as LIF or a LIF substitute; a BMP receptor agonist or an analogue thereof, such as a BMP, or a BMP substitute; a WNT receptor agonist or an analogue thereof, such as WNT or a WNT substitute. The culture medium further may be free, substantially free or essentially free of a TGF beta receptor agonist or an analogue thereof. Unless otherwise indicated, the culture medium further may be free, substantially free or essentially free of nodal, activin, stem cell factor or members of the hedgehog family of proteins.

[95] Terms such as "about" should be taken to mean within 10%, more preferably within 5%, of the specified value, unless the context requires otherwise.

[96] The term "embryo" is used in this specification to refer to a mammalian organism from the single cell stage. The embryo may have been obtained from a pregnant female. 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. Alternatively, the embryo may be produced from oocytes and sperm derived from induced pluripotent stem cells.

[97] 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 steps (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.

[98] Theiler has established numbered stages of murine development. The earliest stages, as applied to (C57BLxCBA)Fl mice, are described in the "emouse digital atlas" (http://www.emouseatlas.org) as follows:

Ectoplacental cone region invaded by maternal blood, Reichert's membrane and proamniotic cavity form

9a 6.5 (6.25- Pre-streak(PS), advanced

7.25) endometrial reaction, ectoplacental cone invaded by blood, extraembryonic ectoderm, embryonic axis visible

9b Early streak(ES), gastrulation starts, first evidence of mesoderm

10a 7 (6.5-7.75) Mid streak (MS), amniotic fold starts to form

10b Late streak, no bud (LSOB), exocoelom

10c Late streak, early bud (LSEB), allantoic bud first appears, node, amnion closing

11a 7.5 (7.25-8) Neural plate (NP), head process developing, amnion complete l ib Late neural plate (LNP),

elongated allantoic bud

11c Early head fold (EHF)

l id Late head fold (LHF), foregut invagination

12a 8 (7.5-8.75) 1-4 somites, allantois extends, 1st branchial arch, heart starts to form, foregut pocket visible, preotic sulcus at 2-3 somite stage)

12b 5-7 somites, allantois contacts chorion at the end of TS 12 Absent 2nd arch, >7 somites

13 8.5 (8-9.25) Turning of the embryo, 1st branchial arch has maxillary and mandibular components, 2nd arch present

Absent 3rd branchial arch

14 9 (8.5-9.75) Formation & closure of ant.

neuropore, otic pit indented but not closed, 3rd branchial arch visible

Absent forelimb bud

15 9.5 (9-10.5) Formation of post, neuropore,

forelimb bud, forebrain vesicle subdivides

Absent hindlimb bud, Rathke's pouch

16 10 (9.5-10.75) Posterior neuropore closes,

Formation of hindlimb & tail buds, lens plate, Rathke's pouch; the indented nasal processes start to form Absent thin & long tail

17 10.5 (10- Deep lens indentation, adv.

11.25) devel. of brain tube, tail elongates and thins, umbilical hernia starts to form

Absent nasal pits

18 11 (10.5- Closure of lens vesicle, nasal pits,

11.25) cervical somites no longer visible

Absent auditory hillocks, anterior footplate

19 11.5 (11- Lens vesicle completely

12.25) separated from the surface epithelium, Anterior, but no posterior, footplate. Auditory hillocks first visible

Absent retinal pigmentation and sign of fingers

20 12 (11.5-13) Earliest sign of fingers, (splayed- out), posterior footplate apparent, retina pigmentation apparent, tongue well-defined, brain vesicles clear Absent 5 rows of whiskers, indented

21 13 (12.5-14) Anterior footplate indented,

elbow and wrist identifiable, 5 rows of whiskers, umbilical hernia now clearly apparent Absent hair follicles, fingers separate distally

22 14 (13.5-15) Fingers separate distally, only indentations between digits of the posterior footplate, long bones of limbs present, hair follicles in pectoral, pelvic and trunk regions Absent open eyelids, hair follicles in cephalic region

23 15 Fingers & Toes separate, hair follicles also in cephalic region but not at periphery of vibrissae, eyelids open Absent nail primordia, fingers 2-5 parallel

24 16 Reposition of umbilical hernia,

eyelids closing, fingers 2-5 are parallel, nail primordia visible on toes Absent wrinkled skin, fingers & toes joined together

25 17 Skin is wrinkled, eyelids are closed, umbilical hernia is gone Absent ear extending over auditory meatus, long whiskers

26 18 Long whiskers, eyes barely visible through closed eyelids, ear covers auditory meatus

27 19 Newborn Mouse

* : "dpc" indicates days post conception, with the morning after the vaginal plug is found being designated 0.5 dpc or E0.5. [99] 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:

5(b) 9-10 formation of trophoblast lacunae; complete penetration into endometrium; amniotic cavity; primary umbilical vesicle

5(c) 11-16 pre-chordal plate; extraembryonic mesoblast; secondary yolk sac

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

[100] The term "pre-implantation stage" is used in this specification to refer to a stage of development earlier than the stage corresponding to Theiler stage 7, Carnegie stage 5(a), and corresponding stages in other species.

[101] The term "post-implantation stage" is used in this specification to refer to a stage of development later than the stage corresponding to Theiler stage 7, Carnegie stage 5(a), and corresponding stages in other species. A "post-implantation stage" may be determined by detecting the up-regulation of one or more genes by the embryo. For example, such a stage may be determined by detecting one or more of the following changes: the epiblast up-regulates Fgf5; the primitive endoderm differentiates into visceral endoderm that up-regulates Cerl in a subpopulation of cells (the anterior visceral endoderm); the visceral endoderm up-regulates Eomes; and the trophectoderm up-regulate Handl .

[102] The invention enables reliable culture up to or through to post-implantation stages corresponding to: Theiler stage 7, 8, 9(a), 9(b), 10(a), 10(b), 10(c), 11(a), 11(b), 11(c), 11(d), 12(a), 12(b) and beyond (as described above); Carnegie stage 5(a), 5(b) or 5(c) and beyond (as described above); and corresponding stages in other species. [103] 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 stage of development beyond the implantation stage enabling such defects to be detected. Thus, if 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). [104] Screening may comprise determining the effect of a test agent on an embryo. For example, such a method may comprise culturing a mammalian embryo using any of the culture media as defined herein, contacting the embryo with a test agent and determining the effect of said test agent on the embryo. Said determining may, for example, comprise comparing a phenotype or a genotype in the presence of said test agent with the phenotype or genotype in the absence of said test agent.

[105] The invention may be applied to selecting embryos. For example, such a method may comprise culturing a mammalian embryo using any of the culture media as defined herein and selecting an embryo based on a cellular parameter, phenotypic marker or genotypic marker. Such a method may comprise culturing a mammalian embryo using any of the culture media as defined herein, time lapse imaging the embryo for a time period and measuring at least one cellular parameter. The time period may be the duration of at least one cytokinesis event or cell cycle. [106] 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.

[107] The invention may be applied to an embryo from any non-human mammal, including but not limited to those described above. Thus, any of the culture media embodiments defined herein may be capable of supporting development of a non- human mammalian embryo on a substrate from a pre-implantation stage of development to a post-implantation stage of development

[108] Stem cells may be cultured using the media, kits and methods of the invention.

[109] The term "stem cell" is used in this specification to refer a cell capable of retaining a constant potential for differentiation even after cell division. [110] Examples of stem cells include: embryonic (ES) stem cells with pluripotency derived from a fertilized egg or clone embryo; epiblast stem cells; trophoblast stem cells; extraembryonic endoderm (XEN) stem cells; somatic stem cells and pluripotent stem cells that are present in tissues in a living organism e.g. hepatic stem cells, dermal stem cells, and reproductive stem cells that serve as the bases for respective tissues; pluripotent stem cells derived from reproductive stem cells; pluripotent stem cells obtained by nuclear reprogrammed somatic cells; totipotent stem cells and non- totipotent stem cells and the like. Also, partially committed stem cells e.g. progenitor cells may be cultured using the media and according to the methods described herein. [HI] In particular, "a pluripotent stem cell" refers to a stem cell permitting in vitro culture, and having the potential for differentiating into all cells, but the placenta, constituting the body [tissues derived from the three primary germ layers of the embryo (ectoderm, mesoderm, endoderm)] (pluripotency); embryonic stem cells are also included. "A pluripotent stem cell" may be obtained from a fertilized egg, clone embryo, reproductive stem cell, or stem cell in tissue. Also included are cells having differentiation pluripotency similar to that of embryonic stem cells, conferred artificially by transferring several different genes to a somatic cell (also referred to as induced pluripotent stem cells or iPS cells). Induced pluripotent stem cells may be derived from any suitable source (e.g. hair follicles, skin cells, fibroblasts etc.). Pluripotent stem cells can be prepared by known methods.

[112] Any of the stem cells as defined herein may be derived from diseased or non- diseased tissue. [113] The invention may be applied to stem cells 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.

[114] The invention may be applied to stem cells from any non-human mammal, including but not limited to those described above. [115] Examples of stem cells useful in the methods of the invention include embryonic stem cells of a mammal or the like established by culturing a pre- implantation early embryo, embryonic stem cells established by culturing an early embryo prepared by nuclear-transplanting the nucleus of a somatic cell, induced pluripotent stem cells (iPS cells) established by transferring several different transcriptional factors to a somatic cell, and pluripotent stem cells prepared by modifying a gene on a chromosome of embryonic stem cells or iPS cells using a gene engineering technique.

[116] More specifically, embryonic stem cells include embryonic stem cells established from an inner cell mass that constitutes an early embryo, ES cells established from a primordial germ cell, cells isolated from a cell population possessing the pluripotency of pre-implantation early embryos (for example, primordial ectoderm), and cells obtained by culturing these cells. [117] Differentiated cells may be produced using the media, kits and methods of the invention.

[118] Examples of differentiated cells include cells that are derived primarily from the endoderm, cells that are derived primarily from the ectoderm and cells that are derived primarily from the mesoderm and cells that are derived primarily from the germ line.

[119] Cells that are derived primarily from endoderm include exocrine secretory epithelial cells and hormone secretory cells. Exocrine secretory epithelial cells include salivary gland cell, von Ebner's gland cell in tongue, mammary gland cell, lacrimal gland cell, ceruminous gland cell in ear, eccrine sweat gland dark cell, eccrine sweat gland clear cell, apocrine sweat gland cell, gland of Moll cell in eyelid, sebaceous gland cell, Bowman's gland cell in nose, Brunner's gland cell in duodenum, seminal vesicle cell, prostate gland cell, bulbourethral gland cell, Bartholin's gland cell, gland of Littre cell, uterus endometrium cell, isolated goblet cell of respiratory and digestive tracts, stomach lining mucous cell, gastric gland zymogenic cell, gastric gland oxyntic cell, pancreatic acinar cell, paneth cell of small intestine, type II pneumocyte of lung and Clara cell of lung. Hormone secreting cells include anterior pituitary cells, intermediate pituitary cell, magnocellular neurosecretory cells, gut and respiratory tract cells, thyroid gland cells, parathyroid gland cells, adrenal gland cells, Leydig cell of testes, Theca interna cell of ovarian follicle, corpus luteum cell, juxtaglomerular cell, macula densa cell of kidney, peripolar cell of kidney and Mesangial cell of kidney.

[120] Cells that are derived primarily from ectoderm include cells of the integumentary system and nervous system. Cells of the integumentary system include keratinizing epithelial cells (such as epidermal keratinocyte, epidermal basal cell, keratinocyte of fingernails and toenails, nail bed basal cell, medullary hair shaft cell, cortical hair shaft cell, cuticular hair shaft cell, cuticular hair root sheath cell, hair root sheath cell of Huxley's layer, hair root sheath cell of Henle's layer, external hair root sheath cell, hair matrix cell), wet stratified barrier epithelial cells (such as surface epithelial cell of stratified squamous epithelium of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, basal cell of epithelia of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina and urinary epithelium cell). Cells of the nervous system include sensory transducer cells (such as auditory inner hair cell of organ of Corti, auditory outer hair cell of organ of Corti, basal cell of olfactory epithelium, cold-sensitive primary sensory neurons, heat- sensitive primary sensory neurons, Merkel cell of epidermis, olfactory receptor neuron, pain-sensitive primary sensory neurons, photoreceptor cells of retina in eye, proprioceptive primary sensory neurons, touch-sensitive primary sensory neurons, type I carotid body cell, type II carotid body cell, type I hair cell of vestibular system of ear, type II hair cell of vestibular system of ear and type I taste bud cell), autonomic neuron cells (such as cholinergic neural cell, adrenergic neural cell and peptidergic neural cell), sense organ and peripheral neuron supporting cells (such as inner pillar cell of organ of Corti, outer pillar cell of organ of Corti, inner phalangeal cell of organ of Corti, outer phalangeal cell of organ of Corti, border cell of organ of Corti, Hensen cell of organ of Corti, vestibular apparatus supporting cell, taste bud supporting cell, olfactory epithelium supporting cell, Schwann cell, satellite glial cell and enteric glial cell), central nervous system neurons and glial cells (such as astrocyte, neuron cells, oligodendrocyte and spindle neuron) and lens cells (such as anterior lens epithelial cell and crystallin-containing lens fiber cell). [121] Cells that are derived primarily from mesoderm include metabolism and storage cells, barrier function cells, extracellular matrix cells, contractile cells, blood and immune system cells, germ cells, nurse cells and interstitial cells. Metabolism and storage cells include hepatocyte, adipocytes and liver lipocyte. Barrier function cells (lung, gut, exocrine glands and urogenital tract) include kidney cells (such as kidney parietal cell, kidney glomerulus podocyte, kidney proximal tubule brush border cell, loop of Henle thin segment cell, kidney distal tubule cell, kidney collecting duct cell, type I pneumocyte, pancreatic duct cell, nonstriated duct cell, duct cell, intestinal brush border cell, exocrine gland striated duct cell, gall bladder epithelial cell, ductulus efferens nonciliated cell, epididymal principal cell and epididymal basal cell). Extracellular matrix cells include ameloblast epithelial cell, planum semilunatum epithelial cell of vestibular system of ear, organ of Corti interdental epithelial cell, loose connective tissue fibroblasts, corneal fibroblasts, tendon fibroblasts, bone marrow reticular tissue fibroblasts, other nonepithelial fibroblasts, pericyte, nucleus pulposus cell of intervertebral disc, cementoblast/cementocyte, Odontoblast/odontocyte, hyaline cartilage chondrocyte, fibrocartilage chondrocyte, elastic cartilage chondrocyte, osteoblast/osteocyte, osteoprogenitor cell, hyalocyte of vitreous body of eye, stellate cell of perilymphatic space of ear, hepatic stellate cell and pancreatic stelle cell. Contractile cells include skeletal muscle cells including red skeletal muscle cell, white skeletal muscle cell, intermediate skeletal muscle cell, nuclear bag cell of muscle spindle and nuclear chain cell of muscle spindle, satellite cells, heart muscle cells including ordinary heart muscle cell, nodal heart muscle cell and Purkinje fiber cell, smooth muscle cell, myoepithelial cell of iris and myoepithelial cell of exocrine glands. Blood and immune system cells include erythrocyte, megakaryocyte, monocyte, connective tissue macrophage, epidermal Langerhans cell, osteoclast, dendritic cell, microglial cell, neutrophil granulocyte, eosinophil granulocyte, basophil granulocyte, hybridoma cell, mast cell, helper T cell, suppressor T cell, cytotoxic T cell, natural Killer T cell, B cell, natural killer cell, reticulocyte and committed progenitors for the blood and immune system. Germ cells include oogonium/Oocyte, spermatid, spermatocyte, spermatogonium cell and spermatozoon. Nurse cells include ovarian follicle cell, Sertoli cell and thymus epithelial cell, and interstitial cells include interstitial kidney cells.

[122] The invention may be used to produce differntiated cells 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.

[123] The invention may be used to produce differentiated cells from any non- human mammal, including but not limited to those described above.

[124] According to the invention, embryos or stem cells are typically cultured on or suspended in a substrate. The substrate may be a matrix and/or a gel. The substrate may comprise a solid support. Alternatively, the substrate may be a solid support. Preferably, the solid support is a plastics material or glass. 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.

[125] The substrate may comprise a matrix. The matrix may comprise at least one extracellular matrix protein. The at least one extracellular matrix protein may comprise one or more of collagen, laminin, fibronectin and/or gelatin. Any combination of these proteins may be used, for example collagen, fibronectin and gelatin may be used together. For example, this combination may be used in the proportion 1 :2: 13 (collagen : fibronectin : gelatin). In addition, any of these proteins may be used in combination with other extracellular matrix proteins. Collagen and/or laminin may be particularly preferred. The collagen may be Type I collagen, e.g. from rat tail, although other types may be used. The substrate may comprise cells or a tissue, or an extract thereof. Alternatively, the substrate does not comprise cells or a tissue.

[126] The substrate may be 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.

[127] 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. Preferably, the matrix comprises collagen and/or laminin. 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), Cultrex (Trevigen Inc., Gaithersburg, MD, USA) and Geltrex (Invitrogen). Their major 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. [128] Alternatively, 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 N '-M-tetramethylethyldiamine (TEMED). [129] Other suitable gel types may include alginate gels, polyethylene glycol (PEG) based gels and agarose gels.

[130] 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 = FL_o/A_oAL where F is the force applied, L₀ is the original length of the web, A₀ is the original cross-sectional area through which the force is applied, and L₀ is the original length of the web. [131] 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 60x10³ 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. 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).

[132] 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 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.

[133] 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% Ν,Ν'-methylenebisacrylamide when polymerised with ammonium persulphate at a final concentration of 1/2000 w/v (i.e. 10 μΐ of 10% solution per 2 ml of polymer solution) and TEMED at a final concentration of 1/2000 v/v (i.e. 1 μΐ per 2 ml of polymer solution).

[134] The surface of the gel substrate may comprise at least one extracellular matrix protein as described above. 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.

[135] 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.

[136] 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 gelatinisation or coat suitable proteins onto the gel surface after gelatinisation. [137] 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.

[138] The gel substrate may be provided on a plastics material substrate, preferably conventional commercially available polystyrene cultureware, for example ibiTreat microscopy plastic μ-plates. If desired, the surface may be coated with one or more extracellular matrix proteins as described above.

[139] The substrate may be coated with at least one extracellular matrix protein by incubation of a solution of the one of more extracellular matrix proteins on the substrate, e.g. for 10 minutes, followed by washing the excess protein off the substrate, for example using PBS. Chemical crosslinking may not be required. For example, a plastics material substrate may be coated using this method. [140] Whatever the nature of the substrate, it may have a surface topography which facilitates retention of one or more embryos, or stem cells, and their associated culture medium within a defined region of the substrate.

[141] 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, or stem cells. 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.

[142] 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".

[143] 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. [144] The substrate may be formed in or upon a suitable template or mould in order to achieve the required topography.

[145] 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.

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

[147] Each receptacle may have a depth of about 250 μιη to about 400 μιη, e.g. about 300 μιη to about 350 μιη. Additionally or alternatively, said plurality of receptacles may have a mean depth of about 250 μιη to about 400 μιη, e.g. about 300 μιη to about 350 μιη.

[148] 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. [149] 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.

[150] 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 in certain instances it may be desirable to minimise the culture volume (as far as practically possible) to maximise the concentration of such development-promoting factors. Alternatively, it may be desirable to culture embryos in isolation.

[151] For example, the culture volume per embryo may be about 1 μΐ to about 50 μΐ, optionally about 2 μΐ to about 40 μΐ, optionally about 5 μΐ to about 30 μΐ, optionally about 10 μΐ to about 25 μΐ, optionally about 12.5 μΐ to about 22.5 μΐ or about 15 μΐ to about 20 μΐ. The culture volume per embryo may be about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 μΐ or more.

[152] The invention also extends to methods of imaging an embryo during development, the method comprising contacting an embryo with a culture medium as described above and recording an image of said embryo using imaging apparatus.

[153] 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.

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

[155] 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 may be used to stain

DNA or MitoTracker (Invitrogen) may be used to stain the mitochondria.

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. [156] 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 microscope, that can include but is not limited to wide field, scanning and spinning disc confocal, and light sheet microscope.

[157] 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. Spinning disc confocal microscopy provides added advantages over confocal laser scanning microscopy. Additionally, light sheet microscopy can also provide good imaging of embryonic development.

[158] We present powerful new media, kits and methods for embryo and stem cell culture with huge potential for both developmental and reproductive biologists. This platform opens up the possibility of studying implantation from an embryonic perspective. 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. In addition, embryos cultured using the media, kits and methods of the invention are a potential source of pluripotent stem cells and multipotent progenitors that can be used in regenerative medicine.

[159] Culture of stem cells (e.g. embryonic stem cells) using the media, kits and methods of the invention may be used to model the development of the body in vitro, including the transitions of fates and structures, and for the building of organs and systems.

[160] Such culturing can be useful as a source of stem cells for specific tissues and organs, for example skin stem cells or central nervous system stem cells, as a source of stem cells that can be used as an alternative to induced pluripotent stem cells (iPSC) in order to reconstitute organs and/or be used in regenerative medicine, and as a source of stem cells obtained by embryo biopsy to provide an individual's embryonic cells of identical genotype to be stored for future use in regenerative medicine therapies for the benefit of that individual. [161] Culture of embryonic stem cells using the media, kits and methods of the invention may also be used for screening drug candidates in drug discovery applications.

[162] The media, kits and methods of the invention may be used to evaluate the effectiveness of equipment used in the preparation and storage of embryos for IVF.

[163] The media, kits and methods of the invention may be used in assays for optimisation or for the investigation of the effectiveness of any embryo handling procedures prior to implantation, including but not limited to manipulating, culturing, storage, transport etc.

[164] The media, kits and methods of the invention may be used in a method of diagnosis for identifying factors important for successful implantation of embryos and potentially also successful early development beyond the implantation stage.

[165] The media, kits and methods of the invention may be used in a method of culturing induced pluripotent stem cells (iPSCs) and further differentiating said iPSCs into desired cells and tissues. iPSCs, as well as cells and tissues differentiated therefrom, can be used for pharmacological screens and eventually for patient-specific replacement therapy.

EXAMPLES

Example 1

In vitro Culture (IVC) system using defined (serum-free) medium - Mouse embryos [166] Wild-type Fl (C57BL/6 x CBA) female mice (8-12 weeks old) were injected intraperitoneally with 7.5 IU of PMSG (Pregnant Mare's Serum Gonadotropin) followed by 7.5 IU injection of hCG (human Chorionic Gonadotropin) after 48 hours, to induce superovulation. After hCG administration the females were mated with Fl or appropriate transgenic reporter stud males. At 3.5 days post coitum (d.p.c.) the females were humanely killed by cervical dislocation and blastocyst staged embryos were recovered in M2 medium. Zona pellucida was removed by brief exposure to acidic Tyrode's solution (Sigma, T1788). The zona-free embryos were seeded on glass bottom plates coated with Matrigel (BD, 356230) or on ibiTreat microscopy plastic μ-plates (Ibidi) coated with Collagen (4 mg/ml, Sigma C38671VL) / Fibronectin (1 mg/ml, Sigma, Fl 141) / Gelatin (0.1%) in proportion 1 :2: 13. The plates were filled with pre-warmed defined (serum-free) IVC-medium (Advanced DMEM/F12 (Gibco, 12634-010) supplemented with 2 mM L-glutamine (Gibco, 25030-024), 1 mM Sodium pyruvate (Gibco, 11360-039), 1 x MEM NEAA (7.5 mg/1 glycine, 8.9 mg/1 L-alanine, 13.2 mg/1 L-asparagine), 13.3 mg/ml L-aspartic acid, 14.7 mg/1 L-glutamic acid, 11.5 mg/1 L-proline and 10.5 mg/1 L-serine) (Gibco, 11 MO- OSS), Penicillin (25 units/ml) / Streptomycin (25 Mg/ml) (Gibco, 15070-063), 1 x ITS- X (10 mg/1 Insulin, 5.5 mg/1 Transferrin, 0.0067 mg/1 sodium selenite, 2 mg/1 Ethanolamine) (Invitrogen 51500-056), 8 nM β-estradiol (Sigma, E8875), 200 ng/ml Progesterone (Sigma, P0130) and 25 μΜ N-acetyl-L-cysteine (Sigma, A7250), containing 30% KSR (KnockOut Serum Replacement (Invitrogen, 10828-010)). During the next 36-48 hours the embryos attached to the surface and the trophectoderm started to differentiate into giant cells that spread out. After embryo attachment, the culture medium was exchanged with fresh defined (serum-free) IVC- medium. At that time point the egg cylinders had already emerged. For the next 48 hours the embryos were cultured on defined (serum-free) IVC-medium, supporting the egg-cylinder growth. The whole embryo culture was performed on 37°C and 5% CO2. [167] Figure 2 shows the successful development of mouse blastocyst beyond implantation outside the body of the mother using the defined (serum-free) medium in accordance with the method described above. The same observations were seen using a matrigel substrate on a glass support. The same observations are expected when culturing human embryos. The figure shows A) Zona pellucida free blastocyst seeded on ibiTreat microscopy plastic μ-plates (ibidi); B) Embryo attached to the surface with trophectodermal giant cells spreading out (GC); C) Early egg cylinder emerges consisting of epiblast (EPI) surrounding proamniotic cavity (PC) and two extraembryonic lineages - visceral endoderm (VE) and extraembryonic ectoderm (EXE); D) The embryo continues proliferating and the egg cylinder elongates; E) Schematic representation of the main steps of the in vitro culture process with the main lineages annotated; Abbreviations - EPI (epiblast), PrE (primitive endoderm), VE (visceral endoderm), TE (trophectoderm), EXE (extraembryonic ectoderm), GC (giant cells), PC (proamniotic cavity). [168] Figure 3 shows the similar organization of the mouse embryonic lineage in in vivo recovered at E6.0 and in vitro cultured embryos (in accordance with the method described above) at day 4. Confocal images of embryos stained for the apical polarity marker Par6 (gray) and the epiblast cell fate marker Oct4 (white).

Example 2

In vitro Culture (IVC) system using FCS-containing medium and defined (serum- free) medium - Mouse embryos

[169] Wild-type Fl (C57BL/6 x CBA) female mice (8-12 weeks old) were injected intraperitoneally with 7.5 IU of PMSG (Pregnant Mare's Serum Gonadotropin) followed by 7.5 IU injection of hCG (human Chorionic Gonadotropin) after 48 hours, to induce superovulation. After hCG administration the females were mated with Fl or appropriate transgenic reporter stud males. At 3.5 days post coitum (d.p.c.) the females were humanely killed by cervical dislocation and blastocyst staged embryos were recovered in M2 medium. Zona pellucida was removed by brief exposure to acidic Tyrode's solution (Sigma, T1788). The zona-free embryos were seeded on glass bottom plates coated with Matrigel (BD, 356230), or on ibiTreat microscopy plastic μ- plates (Ibidi) with no matrix coating, filled with pre-warmed IVC-medium (Advanced DMEM/F12 (Gibco, 12634-010) supplemented with 2 mM L-glutamine (Gibco, 25030-024), 1 mM Sodium pyruvate (Gibco, 11360-039), 1 x MEM NEAA (7.5 mg/1 glycine, 8.9 mg/1 L-alanine, 13.2 mg/1 L-asparagine), 13.3 mg/ml L-aspartic acid, 14.7 mg/1 L-glutamic acid, 11.5 mg/1 L-proline and 10.5 mg/1 L-serine) (Gibco, 11 MO- OSS), Penicillin (25 units/ml) / Streptomycin (25 μg/ml) (Gibco, 15070-063), ITS-X (10 mg/1 Insulin, 5.5 mg/1 Transferrin, 0.0067 mg/1 Sodium selenite, 2 mg/1 Ethanolamine) (Invitrogen 51500-056), 8 nM β-estradiol (Sigma, E8875), 200 ng/ml Progesterone (Sigma, P0130) and 25 μΜ N-acetyl-L-cysteine (Sigma, A7250), containing 20% FCS (Fetal Calf Serum). During the next 36-48 hours the embryos attached to the surface and the trophectoderm started to differentiate into giant cells that spread out. After embryo attachment the culture medium was exchanged with IVC-medium supplemented with 10% FCS and 10% KSR (KnockOut Serum Replacement (Invitrogen, 10828-010). At that time point the egg cylinders already emerged. For the next 48h the embryos were cultured on defined (serum-free) IVC- medium (as defined in Example 1), supporting the egg-cylinder growth. The whole embryo culture was performed on 37°C and 5% CO2.

[170] Figure 4 shows the successful development of mouse blastocyst beyond implantation outside the body of the mother using FCS-containing medium and defined (serum-free) medium in accordance with the method described above. The figure shows A) Zona pellucida free blastocyst seeded on ibiTreat microscopy plastic μ-plates (ibidi); B) Embryo attached to the surface with trophectodermal giant cells (GC) spreading out; C) Early egg cylinder emerges consisting of epiblast (EPI) surrounding proamniotic cavity (PC) and two extraembryonic lineages - visceral endoderm (VE) and extraembryonic ectoderm (EXE); D) The embryo continues proliferating and the egg cylinder elongates; E) Schematic representation of the main steps of the in vitro culture process with the main lineages annotated; Abbreviations EPI (epiblast), PrE (primitive endoderm), VE (visceral endoderm), TE (trophectoderm), EXE (extraembryonic ectoderm), GC (giant cells), PC (proamniotic cavity).

Example 3

In vitro Culture (IVC) system using FCS-containing medium and defined (serum- free) medium - Human embryos [171] Human zygotes were cultured for 6 days to blastocyst stage using standard protocols. Zona pellucida was removed by brief exposure to acidic Tyrode's solution (Sigma, T1788). The zona-free embryos were seeded on ibiTreat microscopy plastic μ-plates (Ibidi), with no matrix coating, filled with pre-warmed IVC-medium (Advanced DMEM/F12 (Gibco, 12634-010) supplemented with 2 mM L-glutamine (Gibco, 25030-024), 1 mM Sodium pyruvate (Gibco, 11360-039), 1 x MEM NEAA (7.5 mg/1 glycine, 8.9 mg/1 L-alanine, 13.2 mg/1 L-asparagine), 13.3 mg/ml L-aspartic acid, 14.7 mg/1 L-glutamic acid, 11.5 mg/1 L-proline and 10.5 mg/1 L-serine) (Gibco, 11140-035), Penicillin (25 units/ml) / Streptomycin (25 ug/ml) (Gibco, 15070-063), ITS-X (10 mg/1 Insulin, 5.5 mg/1 Transferrin, 0.0067 mg/1 Sodium selenite, 2 mg/1 Ethanolamine) (Invitrogen 51500-056), 8 nM β-estradiol (Sigma, E8875), 200 ng/ml Progesterone (Sigma, P0130) and 25 μΜ N-acetyl-L-cysteine (Sigma, A7250), containing 20% FCS (Fetal Calf Serum). During the following days (from day 6 to day 9) the embryos attached to the surface and the trophectoderm started to differentiate. After embryo attachment (day 9) the culture medium was exchanged with IVC-medium supplemented with 10% FCS and 10% KSR (KnockOut Serum Replacement (Invitrogen, 10828-010)). At that time point different cell layers were already visible. From day 10 to day 13 the human embryos were cultured in defined (serum-free) IVC-medium (as defined in Example 1), supporting the embryo growth. The whole embryo culture was performed on 37°C and 5% CO2.

[172] Figure 5 shows the successful development of human blastocyst beyond implantation outside the body of the mother using FCS-containing medium and defined (serum-free) medium in accordance with the method described above. The figure shows that between day 6 and day 9 the embryo attached to the surface. The embryo is cultured in vitro up to day 13. The epiblast is marked with a dashed line. Lineage annotations: EPI (epiblast), TB (trophoblast).

Example 4

In vitro culture of mouse ES cells in 3D extracellular matrix

[173] Mouse embryonic stem (ES) cells were maintained using standard protocols. On the day of the experiment, the cells were washed once with PBS and incubated for 10 min / 37°C in 0.05% Trypsin-EDTA (Invitrogen, 25300-054). Equal volume of standard ES cell medium is added to stop the reaction. The cells were pelleted by centrifuging for 5 min / 1000 rpm and the medium was sucked out. The cell pellet was resuspended in Matrigel (BD, 356230). The single cells suspension was then plated on ibiTreat microscopy plastic μ-plates (Ibidi) and incubated for 5-10 min until the matrigel solidified and formed a three dimensional gel. After that the plate was filled with pre-warmed defined (serum-free) IVC medium (as defined in Example 1) or pre-warmed IVC medium containing 20% FCS (as defined in Example 2). The medium was exchanged every second day. The ES cells grew and formed clumps in the matrigel. After 48-72 hours, the ES cell clumps formed typical structures with central cavity that correspond to the morphology of the embryonic lineage found in E4.75 - E5.5 mouse embryos in vivo.

[174] Figure 6 shows successful culture of mouse ES cells in 3D extracellular matrix using defined (serum-free) medium in accordance with the method described above. The figure shows A) Schematic representation of the ES cell culture method in 3D extracellular matrix. B) Confocal images of CAG-GFP ES cells grown in matrigel. After 48 hours the first constricted structures with small cavities in the center are formed. C) Comparison between epiblast morphology of E4.75-E5.0 embryo versus mouse ES cells grown for 48 hours in matrigel.

[175] 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.

REFERENCES

1. 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).

2. Bielinska, M., Narita, N. & Wilson, D.B. Distinct roles for visceral endoderm during embryonic mouse development. IntJ Dev Biol 43, 183-205 (1999).

3. Hsu, Y.C. Differentiation in vitro of mouse embryos beyond the implantation stage. Nature 239, 200-202 (1972).

4. Hsu, Y.C. Differentiation in vitro of mouse embryos to the stage of early somite. Dev Biol 33, 403-411 (1973). 5. Pienkowski, M., Solter, D. & Koprowski, H. Early mouse embryos: growth and differentiation in vitro. Exp Cell Res 85, 424-428 (1974).

6. Konwinski, M., Solter, D. & Koprowski, H. Effect of removal of the zona pellucida on subsequent development of mouse blastocysts in vitro. J Reprod Fertil 54, 137-143 (1978).

7. Hsu, Y.C. In vitro development of individually cultured whole mouse embryos from blastocyst to early somite stage. Dev Biol 68, 453-461 (1979). 8. Libbus, B.L. & Hsu, Y.C. Sequential development and tissue organization in whole mouse embryos cultured from blastocyst to early somite stage. Anat Rec 197, 317-329 (1980).

9. Tournaye, H., Van der Linden, M., Van den Abbeel, E., Devroey, P. & Van Steirteghem, A. Effect of pentoxifylline on implantation and post-implantation development of mouse embryos in vitro. Hum Reprod 8, 1948-1954 (1993). 10. Huang, F.J., Wu, T.C. & Tsai, M.Y. Effect of retinoic acid on implantation and postimplantation development of mouse embryos in vitro. Hum Reprod 16, 2171-2176 (2001). 11. Morris, S.A., et al. Dynamics of anterior - posterior axis formation in the developing mouse embryo. Nat Commun. 14; 3: 673 (2012).

Claims

An in vitro culture medium that is free or substantially free of serum comprising: a. insulin, an insulin analogue, or an insulin receptor agonist,

b. oestrogen, an oestrogen analogue, or an oestrogen receptor agonist, and c. progesterone, a progesterone analogue, or a progesterone receptor agonist; wherein the medium is capable of supporting development of a mammalian embryo on a substrate from a pre-implantation stage of development to a post- implantation stage of development.

The in vitro culture medium according to claim 1, wherein the medium comprises an albumin.

The in vitro culture medium according to claim 1 or claim 2, wherein the medium comprises a serum replacement.

The in vitro culture medium according to claim 3, wherein the medium comprises 30% serum replacement.

The in vitro culture medium according to any of claims 1 to 4, wherein the post- implantation stage is the egg cylinder stage or embryonic disc stage.

The in vitro culture medium according to any of claims 1 to 5, wherein the insulin receptor agonist is selected from the group consisting of IGF-I or an analogue thereof and IGF -II or an analogue thereof.

The in vitro culture medium according to any of claims 1 to 6, wherein the oestrogen receptor agonist is selected from the group consisting of β-estradiol or an analogue thereof, estrone or an analogue thereof, estriol or an analogue thereof and estetrol or an analogue thereof.

8. The in vitro culture medium according to any of claims 1 to 7, wherein the medium comprises transferrin or an analogue thereof, sodium selenium and/or ethanolamine or an analogue thereof.

9. The in vitro culture medium according to any of claims 1 to 8, wherein the medium comprises L-glutamine.

10. The in vitro culture medium according to any of claims 1 to 9, wherein the medium comprises sodium pyruvate.

11. The in vitro culture medium according to any of claims 1 to 10, wherein the medium comprises one, more than one or all components selected from the group consisting of L-glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine.

12. The in vitro culture medium according to any of claims 1 to 11, wherein the medium further comprises an agonist of the activin type 1 or type 2 receptors.

13. The in vitro culture medium according to any of claims 1 to 12, wherein the medium comprises a reducing agent such as N-acetyl-L-cysteine, dithiothreitol (DTT) or β-mercaptoethanol.

14. The in vitro culture medium according to any of claims 1 to 13, wherein the medium does not comprise a conditioned medium.

15. The in vitro culture medium according to any of claims 1 to 14, wherein the substrate is a solid support, preferably comprising a plastics material or glass.

16. The in vitro culture medium according to any of claims 1 to 14, wherein the substrate is in contact with a solid support, wherein the solid support preferably comprises a plastics material or glass.

17. The in vitro culture medium according to claim 16, wherein the substrate comprises a matrix.

18. The in vitro culture medium according to claim 17, wherein the matrix comprises at least one extracellular matrix protein, or analogue thereof.

19. The in vitro culture medium according to claim 18, wherein the extracellular matrix protein is collagen or analogue thereof, laminin or analogue thereof, fibronectin or analogue thereof and/or gelatin.

20. The in vitro culture medium according to claim 19, wherein the extracellular matrix protein is collagen or analogue thereof, and/or laminin or analogue thereof.

21. The in vitro culture medium according to claim 20, wherein the extracellular matrix protein is collagen and/or laminin.

22. The in vitro culture medium according to any one of claims 17 to 21, wherein the matrix activates signalling through β-integrin receptors.

23. The in vitro culture medium according to any of claims 1 to 22, wherein the substrate does not comprise a feeder-cell layer, preferably wherein the substrate does not comprise uterine epithelial cells or uterine endometrium.

24. A culture medium supplement for producing the culture medium of any one of claims 1 to 23 comprising:

a. insulin, an insulin analogue, or an insulin receptor agonist,

b. oestrogen, an oestrogen analogue, or an oestrogen receptor agonist, and c. progesterone, a progesterone analogue, or a progesterone receptor agonist; wherein the culture medium thereby produced is capable of supporting development of a mammalian embryo on a substrate from a pre-implantation stage of development to a post-implantation stage of development.

25. The culture medium supplement according to claim 24 comprising one or more components, or analogues thereof, selected from transferrin; sodium selenite; ethanolamine; sodium pyruvate; L-glutamine; L-glycine; L-alanine; L-asparagine; L-aspartic acid; L-glutamic acid; L-proline; L-serine; and N-acetyl-L-cysteine.

26. A kit for culturing a mammalian embryo comprising:

a. an in vitro culture medium of any one of claims 1 to 23; and

b. a substrate as defined in any one of claims 15 to 23.

27. A kit for culturing a mammalian embryo comprising:

a. a culture medium supplement according to claim 24, and

b. (I) a basal medium and/or (II) one or more separate supplements comprising one or more additional components, or analogues thereof, selected from transferrin; sodium selenite; ethanolamine; sodium pyruvate; L-glutamine; L-glycine; L-alanine; L-asparagine; L-aspartic acid; L-glutamic acid; L-proline; L-serine; and N-acetyl-L-cysteine.

28. The kit according to claim 26 or 27, further comprising an additional separate supplement which is a serum replacement medium.

29. The kit according to claim 27 or claim 28, further comprising a substrate as defined in any one of claims 15 to 23.

30. The kit according to claim 29, 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.

31. An in vitro method of culturing a mammalian embryo comprising contacting a mammalian embryo with a culture medium according to any one of claims 1 to 23, wherein said embryo is cultured on a substrate from a pre-implantation stage of development to a post-implantation stage of development.

32. The method of claim 31, wherein the pre-implantation stage is the blastocyst stage.

33. The method of claim 31, wherein the pre-implantation stage is prior to attachment of the blastocyst to the substrate.

34. The method of claim 32 or claim 33 comprising the step of removing the blastocyst from the zona pellucida.

35. The method of any one of claims 31 to 34 comprising the earlier steps of:

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

(b) culturing said embryo to blastocyst stage.

36. The method of claim 35, wherein the pre-blastocyst stage of development is a single cell embryo.

37. The method of claim 36, wherein the single cell embryo is a fertilised egg.

38. The method of claim 36, wherein the single cell embryo has been obtained by nuclear transfer.

39. The method of any of claims 31 to 38, wherein said embryo is cultured on a substrate from a pre-implantation stage of development to a post-implantation stage of development using only serum-free culture medium.

40. The method of any one of claims 31 to 38 comprising the steps of:

i. providing a first in vitro culture comprising said embryo in a first culture medium, wherein said first culture medium comprises fetal calf serum; and

ii. removing said first culture medium from said embryo and contacting said embryo with a second culture medium that is serum-free

to provide a second in vitro culture comprising said embryo in a serum-free culture medium.

41. The method of claim 40, wherein the step of removing said first culture medium from said embryo and contacting said embryo with a second culture medium that is serum-free is performed at the egg cylinder stage or embryonic disc stage.

42. The method of any of claims 31 to 41, wherein the post-implantation stage is the egg cylinder stage or embryonic disc stage.

43. The method of any one of claims 31 to 42 further comprising the step of removing one or more cells from said embryo.

44. The method of claim 43, wherein said one or more cells is taken from the inner cell mass.

45. The method of claim 44, wherein said cell is an epiblast cell.

46. The method of any of claims 43 to 45, wherein said cell is a pluripotent cell.

47. The method according to any of claims 31 to 46, wherein the substrate is a substrate as defined in any one of claims 15 to 23.

48. The method according to any one of claims 31 to 47, 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.

49. The method of claim 48, wherein each said culture comprises a plurality of embryos.

50. The method of claim 48 or claim 49, wherein each said culture has a volume of 15 μΐ to about 20 μΐ per embryo.

51. The method of any one of claims 31 to 50 comprising the step of recording one or more images of the embryo.

52. The method of any one of claims 31 to 51 comprising the steps of contacting said embryo with a test agent and determining the effect of said test agent on development of said embryo.

53. An in vitro method of culturing a mammalian embryo comprising contacting a mammalian embryo with a culture medium according to any one of claims 1 to 23, wherein said embryo is cultured on a substrate from a pre-implantation stage of development to produce a rosette of polarized cells.

54. The in vitro method of claim 53, wherein the substrate is as defined in any one of claims 15 to 23.

55. The in vitro method of claim 53 or claim 54, further comprising the steps of removing one or more cells from the rosette of polarized cells and culturing the cells to produce differentiated cells.

56. The in vitro method of claim 55, wherein the differentiated cells are selected from the group consisting of exocrine secretory epithelial cells, hormone secreting cells, cells of the integumentary system, cells of the nervous system, metabolism and storage cells, barrier function cells, extracellular matrix cells, contractile cells, blood and immune system cells, germ cells, nurse cells and interstitial cells.

57. A differentiated cell obtainable by the method of claim 55 or claim 56.

58. An in vitro method of culturing pluripotent stem cells, comprising contacting the stem cells with a culture medium according to any one of claims 1 to 23, wherein the stem cells are cultured on or in a substrate.

59. The in vitro method of claim 58, wherein the substrate is a matrix and wherein the method comprises the step of suspending the stem cells in the matrix.

60. The in vitro method of claim 58 or claim 59, wherein the substrate is as defined in any one of claims 15 to 23.

61. The in vitro method of any of claims 58 to 60, wherein the stem cells are embryonic stem cells.

62. The in vitro method of any one of claims 58 to 60, wherein the stem cells are induced pluripotent stem cells.

63. The in vitro method of any one of claims 58 to 62, wherein the stem cells are cultured to produce a sphere of cells having a lumen and apical-basal polarity.

64. The in vitro method of claim 63, further comprising the steps of removing one or more cells from the sphere of cells and culturing the cells to produce differentiated cells.

65. The in vitro method of claim 64, wherein the differentiated cells are selected from the group consisting of exocrine secretory epithelial cells, hormone secreting cells, cells of the integumentary system, cells of the nervous system, metabolism and storage cells, barrier function cells, extracellular matrix cells, contractile cells, blood and immune system cells, germ cells, nurse cells and interstitial cells.

66. A differentiated cell obtainable by the method of claim 64 or claim 65.

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

a. culturing a mammalian embryo using a culture medium according to any one of claims 1 to 23;

b. contacting the embryo with a test agent; and

c. determining the effect of said test agent on the embryo, optionally comprising comparing a phenotype or a genotype in the presence of said test agent with the phenotype or genotype in the absence of said test agent.

68. The method of claim 67 comprising contacting the embryo with the test agent before attachment of the embryo to the substrate.

69. The method of claim 67 comprising contacting the embryo with the test agent after attachment of the embryo to the substrate.

70. The method of claim 68 further comprising determining the subsequent effect on attachment of the embryo to the substrate.

71. The method of any one of claims 67 to 70 further comprising recording one or more images of the embryo.

72. Use of an in vitro culture medium according to any one of claims 1 to 23 for culturing a mammalian embryo on a substrate from a pre-implantation stage of development to a post-implantation stage of development.

73. Use of an in vitro culture medium according to any one of claims 1 to 23 for culturing stem cells on a substrate.

74. The use of claim 72 or claim 73, wherein the substrate is as defined in any one of claims 15 to 23.

75. The culture medium, culture medium supplement, kit, method or use according to any of the preceding claims, wherein the embryo or stem cells is/are non-human.