GB2517194A - Media and methods for culturing embryos and stem cells - Google Patents

Abstract

A serum-free culture medium capable of supporting in vitro culture of mammalian embryos on a substrate from a pre-implantation stage of development to a post-implantation stage of development comprises an insulin receptor agonist (e.g. insulin, IGF-I or IGF-II), an oestrogen receptor agonist (e.g. beta-estradiol) and a progesterone receptor agonist (e.g. progesterone). The medium may also comprise transferrin, sodium selenite, ethanolamine, L-glutamine, sodium pyruvate, amino acids, reducing agents and/or agonists of the activin type 1 or type 2 receptors. The insulin receptor agonist, oestrogen receptor agonist and progesterone receptor agonist may also be provided as a culture medium supplement. Methods of culturing mammalian embryos, and methods of testing the effects of agents on embryo development, are also provided. The medium may also be used for the culture of stem cells, such as embryonic stem cells.

Description

MEDIA AND METHODS FOR CULTURING EMBRYOS AND

STEM CELLS

FIELD OF THE INVENTION

[0001] The invention relates to cell culture, in particular media, kits and methods for culturing embryos and stem cells.

BACKGROUND TO THE INVENTION

[0002] 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 speeii'ing 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-inwlantation that have been described3'° have not been extensively used due to inherent irreproducibility. However, in 2012 Morris and co-workers described an in-vitro culture system that allowed ihc 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.

[0003] 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.

[0004] Reliable and reproducible methods for culturing embryos past the implantation stage in vitro would also greatly increase the availability of stem cells.

[0005] It is also an object of the present invention to provide in-vitro culture media and methods for culturing stem cells.

[0006] 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 consistcncy, 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 dunng 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

[0007] The invention provides an in vitro culture medium comprising: an insulin receptor agonist, an oestrogen receptor agonist, and progesterone rcccptor 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.

[0008] 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 [0009] The medium may comprise a basal medium. The basal medium may comprise water, salts, amino acids, a carbon source, vitamins, lipids and a buffer.

[0010] 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.

100111 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 hi prinrntes (e.g. humans). Other pre-implantation and post-implantation stages are described below.

[0012] The insulin receptor agonist may be one or more of insulin, JOE-I, and/or IGF-Il, 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/I to about 200 mg/I, about 0.5 mg/l to about 100 mg/l, about 1 mg/l to about 50 rng/l, about 2 mg/l to about 25 mg/l, or about 5 mg/l to about 12.5 mg/l e.g. about 10 mg/l. 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 nglml to about 200 ng/ml, about 1 ng/ml to about 150 ng/ml, about S ng/ml to about 100 ng/ml, or about 25 ng/rnl to about 75 ng/ml e.g. about 50 ng/ml.

[0013] The oestrogen receptor agonist may be one or more steroidal oestrogens, for example F-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 non-steroidal oestrogens, for example a xenoestrogen, a phytocstrogen and/or a niycoestrogen, 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 tiM to about 25 nM, about 4 nM to about 12.5 nM, about S nM to about 12 nM, about 6 n]VI to about 11 nM, about 7 riM to about 10 nM, or about 7.5 nM to about 9 nM e.g. about S nM.

[0014] 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 I ng/ml to about 2 Lg/ml, about 5 ng/ml to about 1.5 ig/ml, about 10 ng/ml to about I jtg/ml, about 20 ng/ml to about 750 ng/ml, about 50 ng/ml to about 500 ng/rnl, or about 100 ng/ml to about 300 ng/ml e.g. about 200 ng/ml.

[0015] The in vitro culture medium may be free of serum, substantially free of serum or essentially fl-cc of serum and may fbrther 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.

[0016] 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/l to about 500 mg/l, about 0.05 mg/l to about 250 rng/l, about 0.1 mg/l to about 100 mg/l, about 0.5 mg/l to about 25 mg/l, about 1 mg/l to about 10 mg/I, or about 2.5 mg/I to about 7.5 mg/I e.g. about 5.5 mg/I. The concentration of selenium (for example sodium selenite), or an analogue thereof, in the culture medium may be about 0.0001 mg'l to about 0.1 mg/l, about 0.0002 mg/l to about 0.05 mg/I, about 0.0005 mg/I to about 0.02 mg/I, about 0.001 mg/I to about 0.01 mg/I, or about 0.005 mg/I to about 0.0075 mg/I e.g. about 0.0067 mg/I. The concentration of ethanolamine, or an analogue thereof, in the culture medium may be about 0.01 mg/l to about 500 mg/I, about 0.025 mg/l to about 250 mg/I, about 0.05 mg/I to about 100 mg/I, about 0.1 mg/I to about 50 mg/I, about 0.25 mg/I to about 25 mg,l, about 0.5 mg/i to about 10 mg/I, or about 1 mg/I to about 5 mg/I e.g. about 2 mg/l.

[0017] The culture medium may comprise L-glutamine at a concentration as further defined herein. Preferably L-glutaniine is used at a concentration of about 2 mM.

[0018] Thc culture medium may comprisc sodium pyruvate at a concentration as ffirther defined herein. Preferably sodium pyruvate is used at a concentration of at about 1 mM.

[0019] The culture medium may comprise one, more than one or all components selected from die 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/i, L-alanine at a concentration of about 9 mg/l, L-asparagine at a concentration of about 13 mg/l, L-aspartic acid at a concentration of about 13 mg/I, L-glutamic acid at a concentration of about 14.5 mg/I, L-proline at a concentration of about 11.5 mg/I and L-serine at a concentration of about 10.5 mg/I.

[0020] The culture medium may fhrther comprise an agonist of the activin type I 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.

[0021] The culture medium may comprise a reducing agent, for example N-aeetyl-L-cysteine, glutathione, dithiothreitol (DTT) or 2-mercaptoethanol (3-mercaptoethanol), and/or an analogue or substitute thereof. The concentration of the reducing agent in the culture medium may be about 0.5 RM to about 250 M, about 5 R to about 200 RM, about 7.5 jil'.'l to about 150 iiM, about 10 RM to about 100 tM, about 15 t1v1 to about 50 RM, about 17.5 tM to about 40 RM, or about 20 jtM to about 30 jtM e.g. about 25 tM. Preferably the culture medium further comprises N-acetyl-L-cysteine at a concentration of at about 25 iM.

[0022] 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 andior gelatin. 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 endometriuni Preferably, the medium does not comprise a conditioned medium.

[0023] 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.

100241 The culture medium supplement may comprise insulin, or analogue thereof The medium may comprise ocstrogen, or an analogue thereof The medium may comprise progesterone, or an analogue thereof [0025] The culture medium supplement can be constituted such that when converted to the final medium for use in the in vitro cultunng 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.

100261 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 prc-implantation stage of development to a post-implantation stage of development.

[0027] For example, the culture medium supplement may comprise one or more components, or analogues thereot selected from transferrin; sodium selenite; ethanolanñne; sodium pyruvate; L-glutamine; L-glycinc; L-alanine; L-asparagine; L-aspartic acid; L-glutamic acid; L-proline; L-serine; and N-acetyl-L-cysteine.

[0028] 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 xSOO, about x25 to about x250, about xSO to about x200, or about xiS to about xl50 e.g. about x100.

[0029] The invention also provides a kit for culturing a mammalian embryo.

[0030] 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.

[0031] 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 (TI) one or more separate supplements comprising one or more of the components as defined herein. For example, separate supplements may coniprise 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-eysteine.

[0032] 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-eoated.

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, vitroneetin and/or gelatin. 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 coniprise uterine epithelial cells or uterine endometrium. The surface of the substrate may comprise one or more receptacles adapted to contain a culture comprising appropnate culture medium and one or more embryos. Preferably, the substrate is suitable for imaging, for example time-lapse imaging as discussed below.

[0033] The invention also provides an in vitro method of culturing a mammalian embryo, comprising contacting a nuinmalian embryo with a culture medium of the invention, wherein the embryo is cultured on or in a substrate from a pre-iniplaritation stage of development to a post-implantation stage of development.

[0034] 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.

[0035] The method may comprise the step of removing the blastocyst from the zona pellucida.

[0036] 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 perfonned 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.

[0037] 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-fl-ce cultui-e medium, or eultme medium substantially free of serum.

[0038] 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 ti'om the embryo and contacting the embryo with a second culture medium that is serum-free, or substantially fl-cc of serum, to provide a second iii vitro culture comprising the embryo in a serum-free culture medium (or a medium substantially free of serum).

[0039] 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%.

[0040] 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 fl-ce 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.

[0041] 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 exlraembryonic lineages c.g. the trophectodenn and/or the primitive endoderm, and/or derivatives of these lineages. The cell(s) may be unipotcnt or multipotent. For example, such cells may be useful for genotyping an embryo without the destruction of the embryo.

[0042] 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. 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 rl to about 20 l per embryo. Preferably, the substrate is suitable for imaging, for example time-lapse imaging as discussed below.

[0043] 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.

[0044] 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.

[0045] 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. 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 sm-face 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 il. Preferably, the substrate is suitable for imaging, for example time-lapse imaging as discussed below.

[0046] 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.

[0047] 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 -P5.5 mouse embryos in vivo.

[0048] The methods may thrther enable the culture of stem cells to form an embryo corresponding to one of the pre-implantation or post-implantation stages described below.

[0049] 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.

[0050] 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.

[0051] The method may comprise contacting the embryo with the test agent before attachment of the embryo to the substrate. Mternatively, 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.

[0052] The method nmy comprise recording one or more images of the embryo.

[0053] 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.

[0054] The invention also provides the use of an in vitro culture medium of the invention for culturing stern cells on a substrate.

100551 Examples of substrates that may be used are provided below.

[0056] 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.

[0057] The invention also provides an iniaging 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 ftirther comprise a fluorescent microscope. Additionally, or alternatively, an imaging apparatus may further comprise a confocal microscope.

BRIEF DESCRIPTION OF TUE DRAWINGS

[0058] Fig.1 Schematic representation of the early niouse development. During the first four days, starting from the fertilization, the embryo is free floating in the maternal reproductivc 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 pen-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.

[0059] Fig.2 Development of mouse blastoeyst 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.

[0060] F'ig.3 Similar organization of the mouse embryonic lineage in in vim recovered at E6.0 and in vitro cultured embryos (in accordance with Example 1) at day 4.

100611 F'ig.4 Development of mouse blastoeyst 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.

100621 F'ig.S Development of human blastoeyst beyond implantation outside the body of the mother using FCS-containing medium and defined (serunFfree) medium in accordance with Example 3.

[0063] 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-GEP 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

100641 The invention provides an in vitro culture medium comprising: an insulin receptor agonist, an oestrogen receptor agonisi, and progesterone receptor agonist.

[0065] 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-implanlation 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.

[0066] 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.

[0067] 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.

[0068] 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 activc 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.

[0069] The term "analogue" may rcfer to a compound which may be structurally related to the relevant molecule. The term "agonist" nrny 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 oftlie relevant molecule.

[0070] 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, inannitol, 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).

[0071] The components of Advanced DMEM/F12 are set out in Table 1 below.

Table 1

Components Molecular Weight 0?tb0 mM Amino Acids Glycine 75 18.75 0.25 L-Alanine 89 4.45 0.05 L-ginine hydrochloride 211 147.5 0.699 L-Asparagine-H20 150 7.5 0.05 L-Aspartic acid 133 6.65 0.05 L-Cysteine hydrochloride- 176 17.56 0.0998 H20 L-Cystine2FICl 313 31.29 0.1 L-Glutamic Acid 147 7.35 0.05 L-Histidine hydrochloride- 210 31.48 0.15 H20 L-Isoleucine 131 54.47 0.416 L-Leucine 131 59.05 0.451 L-Lysine hydrochloride 183 91.25 0.499 L-Methionine 149 17.24 0.116 L-Phenylalanine 165 35.48 0.215 L-Proline 115 17.25 0.15 L-Serine 105 26.25 0.25 L-Threonine 119 53.45 0.449 L-Tryptophan 204 9.02 0.0442 L-Tyrosine disodium salt 261 55.79 0.214 dihydrate L-Valine 117 52.85 0.452 Vita mill s Ascorbic Acid phosphate 289.54 2.5 0.00863 Biotin 244 0.0035 0.0000143 Choline chloride 140 8.98 0.0641 D-Calcium pantothenate 477 2.24 0.0047 Folic Acid 441 2.65 0.00601 Niacinamide 122 202 00166 Pyridoxine hydrochloride 206 2 0.00971 Riboflavin 376 0.219 0.000582 Thiamine hydrochloride 337 2.17 0.00644 Vitamin B12 1355 0.68 0.000502 i-Inositol 180 12.6 0.07 Lilorganic Salts Calcium Chloride (CaC12) 111 116.6 1.05 (anhyd.) Cupric sulfate (CuSO4- 250 0.0013 0.0000052 5H20) Ferric Nitrate 404 0.05 0.000124 (Fe(I'03)3'9H20) Ferric sulfate (FeSO4- 278 0.417 0.0015 7H20) Magnesium Chloride 28.64 0.301 (anhydrous) Magnesium Sulfate 48.84 0.407 (MgSO4) anhyd.) Potassium Chloride (KC1) 75 311.8 4.16 Sodium Bicarbonate 84 2438 29.02 NaHCO3) Sodium Chloride NaCl) 58 6995.5 120.61 Sodium Phosphate dibasic 142 71.02 0.5 Na2HPO4) anhydrous Sodium Phosphate monobasic (NaI-12P04-138 62.5 0.453 1120) Zinc sulfate (ZriSO4-71-120) 288 0.864 0.003 Proteins A1buIVIAX® II 400 cc Human Transferrin (fob) 7.5 cc Insulin Recombinant Full cc Chain Reducing Agents Glutathionc, 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 Compollents D-Glucose (Dexlrosc) 180 3151 17.51 Ethanolarnine 97.54 1.9 0.0195 FlypoxanthineNa 159 2.39 0.015 Linolcie Acid 280 0.042 0.000 15 Lipoic Acid 206 0.105 0.00051 PhenoiRed 376.4 8.1 0.0215 Putrescine 2HC1 161 0.08 1 0.000503 Sodium Pyruvatc 110 110 1 Thymidine 242 0.365 0.00151 [0072] The components of CMRL-1066 are set out in Table 2 below.

Table 2

Components Molecular Weight Th7ti0 mI Amino Acids Glycine 75 50 0.667 Hydroxy L-proline 131 10 0.0763 L-Alanine 89 25 0.281 L-Argininc hydrochloride 211 70 0.332 L-Aspartic acid 133 30 0.226 L-Cvsteine hydrochloride-H20 176 260 1.48 L-Cystine 240 26 0.108 L-Glutamic Acid 147 75 0.51 L-Histidine hydrochloride-H20 210 20 0.0952 L-Isoleucine 131 20 0.153 L-Lcucine 131 60 0.458 L-Lysine hydrochloride 183 70 0.383 L-Methionine 149 15 0.101 L-Pheriylalanine 165 25 0.152 L-Proline 115 40 0.348 L-Serine 105 25 0.23 8 L-Thrconinc 119 30 0.252 L-Tryptophan 204 10 0.049 L:Tyrosiiie disodium salt 262 58 0.221 di hydrate ____________________ _____________ __________ L-Valine 117 25 0.214 Vitamins Ascorbic Acid 176 50 0.284 Biotin 244 0.01 0.000041 Cholesterol 387 0.2 0.000517 Choline chloride 140 0.5 0.00357 D-alcium pantothenate 477 0.01 0.000021 Folic Acid 441 0.01 0.0000227 Niacinamide 122 0.025 0.000205 Nicotinic acid (Niacin) 123 0.025 0.000203 Para-Aminobenzoic Acid 137 0.05 0.000365 Pyridoxal hydrochloride 204 0.025 0.000123 Pyridoxine hydrochloride 206 0.025 0.000121 Riboflavin 376 0.01 0.0000266 Thiamine hydrochloride 337 0.01 0.0000297 k-1o5itol 180 0.05 0.000278 Inorganic Salts Calcium Chloride (CaC12) 111 200 1.8 (anhyd.) _____________________ ______________ ___________ Magnesium Sulfate 120 977 0 814 (MgSO4) (aiihyd.) ___________________ ____________ _________ Potassium Chloride (KC1) 75 400 5.33 Sodium Bicarbonate NaHCO3) 84 2200 26.19 Sodium Chloride (NaC1) 58 6365 109.74 Sodium Phosphate ____________________ _____________ __________ monobasic (NaFI2PO4-138 140 1.01 H20) ______________ _________ _______ Other Components 2'Deoxyadenosine 251 10 0.0398 2'Deoxyeytidinc 227 10 0.0441 2'Dcoxyguanosinc 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 (NAn) _____________________ ______________ ___________ FAD (flavin adeninc 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 0.0 178 Thymidine 242 10 0.0413 Thphosphopyridine 743 1 0 00135 Nucleotide (NADP) Tween 80® 5 Uridine 5'-triphosphate 484 1 0.00207 [0073] The in vitro culture medium or the basal medium may be supplemented with one or more components such as L-glutaniine, sodium pyruvate, non-essential amino acids (for example MEM NEAA, Gibco, 15070-063), penicillin and/or streptomycin.

[0074] 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 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.

100751 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 mlvi to about 5 mlvi, or about 0.5 mlvi to about 2.5 mM e.g. about 1 mlvi. Preferably, sodium pyruvate is included in the culture medium at a concentration of about 1mM.

[0076] Non-essential amino acids may be included in the culture medium, for example, comprising glycine (about 1 mg/l to about 25 mg/l or about 5 mg/l to about mg/l e.g. about 7.5 mg/l), L-alanine (about 1 mg/i to about 25 mg/l or about 5 mg/l to about 10 mg/l e.g. about 9 mg/l), L-asparagine (about S mg/l to about 30 mg/l or about 10 mg/I to about 15mg/I e.g. about 13.2 mg/I), L-aspartic acid (about 5mg/Ito about 30 mg/l or about 10 mg/l to about 15 mg/l e.g. about 13 mg/l), L-glutamic acid (about 5 mg/l to about 50 mg/l or about 10 mg/i to about 20 mg/l e.g. about 15 rng/l), L-proline (about 5mg/Ito about 30 mg/I orabout 10mg'! to about 15 mg/I e.g. about 11 mg/I) and/or L-serine (about 5 mg/I to about 30 mg/I or about 10 mg/I to about 15 mg/i e.g. about 11 mg/l). Preferably, the culture medium may comprise L-glycinc at a concentration of about 7.5 mg/l, L-alanine at a concentration of about 9 mg/l, L-asparagine at a concentration of about 13 mg/I, L-aspartic acid at a concentration of about 13 mg/l, L-glutamic acid at a concentration of about 14.5 mgIl, L-proline at a concentration of about 11.5 mg/l and L-serine at a concentration of about 10.5 mg/i.

[0077] Penicillin may be included in the culture medium at a concentration of about I 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/nil, or about 20 units/inl to about 30 units/nil e.g. about 25 units/mI. Streptomycin may be included in the culture medium at a concentration of about I g/rnl to about 500 g/ml, about 2 ig/ml to about 250 jxg/ml, about 5 jig'ml to about 100 tg/ml, about 10 g/ml to about 50 g/ml, or about 20 jig/ml to about 30 jtg/ml e.g. about 25 ig/ml. Preferably, the culture medium may comprise penicillin at a concentration of about 25 units/mI and/or streptomycin at a concentration of about 25 g/ml.

[0078] The culture medium may be fl-ce 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'TM Serum Repiacement, Invitrogen, 10828-0 10) and N2B27 (e.g. Invitrogen, MEIOOI37L1). Alternatively, the culture medium may comprise a serum replacement medium as described iii 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°A 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.

[0079] 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'l to about 25 mg/l), Transferrin (e.g. about 1 mg/l to about 10 mg/l), Selenium e.g. sodium selenite (e.g. about 0.001 mg/i to about 0.01 rng/l), Ethanolamine (e.g. about 0.5 mg/I to about 10 mg/I), an oestrogen receptor agonist e.g. -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 tM to about 40 M).

[0080] The culture medium may ftirther 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/l to about 10 mg/I), L-alanine (e.g. about 5 mg/I to about 10 mg/I), L-asparagine (e.g. about 10 mg/I to about 15 mg/l), L-aspartic acid (e.g. about 10 mg/i to about 15 mgJl), L-glutamic acid (e.g. about 10 mg/l to about 20 mg/l), L-proline (e.g. about 10 mg/l to about 15 mg/I) and/or L-serine (e.g. about 10mg/Ito about 15 mg/I) , Penicillin (e.g. about 10 units/mi to about 50 units/mI) and/or Streptomycin (e.g. about 10 pg/mI to about 50 g/ml).

[0081] 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.

[0082] The culture medium may consist ot or consist essentially of, a basal medium, an insulin receptor agonist, an oestrogen receptor agonist, a progesterone receptor agonist, a reducing agent, transferrin, selenium, etlianolamine and an albumin. The albumin may be provided by a non-human mammalian serum (e.g. FC S) 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.

[0083] In one embodiment there is provided a defined in vitro culture medium that is flee 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; Iransferrin, or analogue thereof; sodium selenite, or analogue or substitute thereof; ethanolamine, or analogue thereof; sodium pyruvate; L-glutamine; L-glycine; L-alaiiine; L-asparagine; L-aspartic acid; L-glutamic acid; L-proline; L-serine; N-aeetyl-L-eysteine; 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 aniounts 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.

[0084] 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 WC defined culture medium (final/worUn concentrations) Concentration Components (mg/L unless othenvise indicated) I Amino Acids Glycine 20.625 IL-Alanine 12.015 L-Argininc hydrochloride 103.25 L-Asparaginc-H20 18.45 L-Aspartic acid 17.955 L-Cystcinc hydrochloridc-H20 12.292 L-Cystine 2HC1 21.903 IL-Glutamic Acid 19.845 IL-Histidine hydrochloride-H20 22.036 IL-Isolcucinc 38.129 IL-Leucine 4L335 L-Lysine hydrochloride 63.875 L-Methionine 12.068 L-Phenylalanine 24.836 IL-Proline 23.575 IL-Serine 28.875 L-Thrconinc 37.415 L-Tryptophan 6.314 L-Tyrosinc disodium salt dihydrate 39.053 IL-Valine 36.995 IL-Glutaminc 2mM I Vitamins Ascorbic Acid phosphate 1.75 Biotin 0.00245 Choline chloride 6.286 ID-Calcium pantothenate 1.568 Folic Acid 1.855 Niacinamide 1.414 Pyridoxine hydrochloride 1.4 Riboflavin 0.1533 Thiamine hydrochloride 1.519 Vitamin B12 0.476 k-Inositoi 8.82 I Inorganic Salts Calcium Chloride (CaCI2) clanhyd.) 81.62 Cuprie sulfate (CuSO4-51120) 0.00091 Ferric Nitrate (Fe(N03)3'9H20) 0.035 Ferric sulfate (FeSO4-7H20) 0.2919 Magnesium Chloride (anhydrous) 20.048 Magnesium Sulfate (Mg504) (anhyd.) 34.188 Potassium Chloride (KC1) 218.26 Sodium Bicarbonate NaHCO3) 1706.6 Sodium Chloride (NaC1) 4896.85 Sodium Phosphate dibasic (Na2HPO4) 49 714 anhydrous ______________________________ Sodium Phosphate monobasic (NaH2PO4-H20) 43.75 Zinc sulfate (ZnSO4-7H20) 0.6048 I Proteins ________________________________ A1buMAXR II 280 Human Transferrin (Mob) 10.75 Insulin Recombinant Full Chain 17 -estradiol 8nM Progesterone 200 ng;ml Reducing Agents Glutathione, monosodium 0.7 Trace Elements Ammonium M etavaiiadate 0.00021 Manganous Chloride 0.000035 Sodium Selenite 0.0102 I Other Components ID-Glucose (Dextrose) 2205.7 Ethanolarninc 3.33 Hypoxanthine Na 1.673 Linoleie Acid 0.0294 Lipoic Acid 0.0735 Phenol Red 5.67 Putrescine 2HCI 0.0567 Sodium Pyruvate 187 Thymidine 0.25 55 N-acctyl-L-cysteine 2RM Penicillin 25 units/nil Streptomycin 25 ig/ml Serum replacement 30°/ (KSR KnockOut Serum Replacement) ___________________________________ 100851 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 EGE receptor agonist or an analogue thereof, such as EGF or an EGE substitute; an FGF receptor agonist or an analogue thereof, such as FGF or an FGF substitute; a LIE receptor agonist or an analogue thereof, such as LIP or a LIE substitute; a BMP receptor agonist or an analogue thereof; such as a BMP, or a BMIP substitute; a V/NT receptor agonist or an analogue thereof, such as V/NT or a WNT substitute.

100861 Terms such as "about" should be taken to mean within 10%, more preferably within 5%, of the specified value, unless the context requires otherwise.

[0087] 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, iii which a nucleus from a somatic cell is transferred into an enucleated egg.

Alternatively, the embryo may be produced from ooeytes and sperm derived from induced pluripotent stem cells.

[0088] 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 momla give rise to the inner cell mass and the outer cells to the trophectodenn.

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.

[0089] Theiler has established numbered stages of murine development. The earliest stages, as applied to (C57BLxCBA)F1 mice, arc described in thc "ernouse digital atlas" (http://www.emouseatlas.org) as follows: Theiler Stage dpc* (range) Cell number (CS7BLxCBA)FI mice 1 0-0.9 1 One-cell egg (0-2.5) 2 1 2-4 Dividillg egg (1-2.5) 3 2 4-16 Morula (1-3.5) (or 8-16) 4 3 16-40 Blastocyst, Inner cell mass (2-4) (or 16-32) apparent 4 Blastocyst (zona-free) (3-5.5) 6 4.5 Attachment of blastocyst, (4-5.5) primary endoderm covers blastocoelic surface of inner cell mass 7 5 Implantation and formation of (4.5-6) egg cylinder Ectoplacental cone appears, enlarged epiblast, primary endoderm lines mural trophcctoderrn 8 6 Differentiation of egg cylinder.

(5-6.5) Implantation sites 2x3 mm.

Ectoplacental cone region invaded by maternal blood, Reichert's membrane and proamniotic cavity form 9a 5.5 (6.25-Pre-streak(PS), advanced 7.25) endometrial reaction, ectoplacental cone invaded by blood, extraernbryonic ectoderm, embryonic axis visible 9b Early streak(ES), gastrulation starts, first evidence of mesoderm lOa 7(6.5-7.75) Mid streak (NIS), amniotic fold starts to form lOb Late streak, no bud (LSOB), exocoelorn lOc Late streak, early bud (LSEB), allantoic bud first appears, node, anmion closing lIa 7.5 (7.25-8) Neural plate (NP), head process developing, amnion complete 1 lb Late neural plate (LNP), elongated allantoic bud lie Early head fold (EHF) lid 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 TSI2 Absent 2nd arch, >7 somites 13 8.5 (8-9.25) Turning of the embryo, 1st branchial arch has maxillaiy and mandibular components, 2nd arch present Absent 3rd branchial arch 14 9(8.5-9.75) Formation & closure of ant.

neuropore, otic pit indcntcd but not closed, 3rd branchial arch visible Absent forelimb bud 9.5 (9-10.5) Formation of post. nenropore, forelimb bud, forebrain vesicle subdividcs Absent hindlimb bud, Rathke's p ouch 16 10(9.5-10.75) Posterior neuropore closes, Formation of hindlimb & tail buds, lens plate, Ratlike's pouch; the indented nasal processes start to form Absent thin & long tail 17 10.5 (10-Deep lens indentation, adv.

11.25) devcl. 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 hi I locks, 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 12(11.5-13) Earliest sign of fingers, (splayed-out), posterior footplatc 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 rcgion 23 15 Fingers & Toes separate, hair follicles also in cephalie 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 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.

[0090] Similarly, so-called "Carnegie stages" have been established to describe stages of human development. Each stage is defined by the development of specific structures, and can be used to define equivalent stages in development of other species. The earliest Carnegie stages are as follows: Carnegie stage Days since ovulation Characteristic (approx.) events/structures 1 fertilization; polar bodies 2 2-3 cleavage; morula; compaction 3 4-5 blastocyst and blastocoele; trophoblast and embryoblast 4 6 syncytiotrophoblast; cytotrophoblast; anchoring to endornetrium 5(a) 7-8 innplantation; embryonic disc; bilaminar germ disc; primary yolk sac; 5(b) 9-10 formation of trophoblast lacunae; complete penetration into endornetriurn; amniotic cavity; primary umbilical vesicle 5(c) 11-16 pro-chordal plate; extra-embryonic niesoblast; secondary yolk sac 6 17 primitive streak, primitive node, primitive groove: sccondary umbilical vesicle; primordial germ cells; body stalk [0091] 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.

[0092] The term "post-implantation stage" is used in this specification to rcfer to a stage of development later than the stage corresponding to Theiler stagc 7, Caniegie 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 FgfS; 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 Hand 1.

[0093] 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(e) and beyond (as described above); and corresponding stages in other species.

[0094] 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).

[0095] Screening may comprise determining the effect of a test agent on an embryo.

For exaniple, such a method niay 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 exaniple, comprise comparing a phenotype or a genotype in the presence of said tcst agent with the phenotype or genotype in the absence of said test agent.

[0096] 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 genolypic 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 cvtokinesis event or cell cycle.

[0097] TIme invention may be applied to embryos from ally 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 nianimals.

[0098] 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-hunian mammalian embryo on a substrate from a pre-implantation stage of development to a post-implantation stage of development [0099] Stem cells may be cultured using the media, kits and methods of the invention.

[0100] 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.

[0101] 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 obtaincd by nuclear reprogrammed somatic cells; totipotent stem cells and non-totipotent stem cells and the 111cc. Also, partially committed stem cells e.g. progenitor cells may be cultured using the media and according to the methods described herein.

[0102] In particular, "a pluripotcnt 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 (cctodcrm, mesoderm, endoderm)] (pluripotency); embryonic stem cells arc also included. "A pluripotent stem cell" may be obtained from a fertilized egg, clone embryo, reproductive stem cell, or steni 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.

[0103] Any of the stem cells as defined herein may be derived from diseased or non-diseased tissue.

[0104] 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; lagomoi-phs (including rabbits); cows; sheep; goats; horses; pigs,-and any other livestock, agricultural, laboratory or domestic mammals.

[0105] The invention may be applied to stem cells from any non-human mammal, including but not limited to those described above.

[0106] Examples of stem cells useful in the methods of the invention include embryonic stem cells of a manimal or the like established by culturing a pre-implantation early embryo, embryonic stem cells established by culturing an early embryo prepared by nuclear-transplanting thc nucleus of a somatic cell, induced plu-ripotent stem cells (iPS cells) established by transferring several different transcriptional factors to a somatic cell, and pluripotent stem cells prepared by modiMng a gene on a chromosome of embryonic stem cells or iPS cells using a gene engineering technique.

[0107] More specifically, embryonic stem cells include embryonic stem cells established from an inner cell mass that constitutes an early embiyo, 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 cctoderm), and cells obtained by culturing these cells.

[0108] 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 niore receptacles adapted to contain a culture comprising appropriate culture medium and one or more embryos.

[0109] The substrate may comprise a matrix. Thc matrix may comprise at least one extracellular matrix protein. The at least one extracellular matrix protein nrny comprise one or niore 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.

Folio] 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.

[0111] The gel may comprise or consist substaiitially of basement membrane matrix.

The basement membrane matrix may comprise one or more of laminin, collagen, heparan sulphate proteoglycan and entactin, amongst other components. The gel may be formed fi-om basement membrane extract, which may be isolated from a suitable basement membrane-secreting cell type, such as Engelbreth-HoIm-Swarm (EHS) mouse sarcoma cells. Bascmcnt mcmbranc cxtracts produced from EHS cclls arc commercially availablc 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 proteoglyean and cntactin. The skilled person is well awarc of how to form gels using such products, e.g. by following the manufacturer's instructions.

[0112] Alternatively, the gel may be a polyacrvlamide gel, e.g. a gel comprising a cross-linked polymcr matrix formed by polymerisation of aciylamide and bis-acrylamide (e.g. N,N'-methylencbisacrylamidc). Polymerisation may be initiated using aminonium persulphate and NNN' -M-tetramethylethyldiamine (TEMED).

[0113] Other suitable gel types may include alginate gels, polyethylene glycol (PEG) based gels and agarose gels.

[0114] Thc gel may be elastically deformable. It is appreciated that certain gels, including polyacrylamide gels, are not rigorously elastic. Rather, they are more properly descnbed 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 clasticily or stifThess. 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 detenmning the extent of stretching which results. E is calculated using the formula: E = FLJAQAL 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 onginal length of the web.

[0115] The gel substrate may, for example, have a Young's modulus of about 5x103 Nm2 to about 100x103 Nnf2, e.g. about 25x103 Nm? to about 100x103 Nn12, e.g. -, 3 -, -3 _-, about SOxift Nm to about lOOxlO Nrn, e.g. about 60x10 Nm to about lOOxift Nm2, e.g. about 70x 103 Nm2 to about 100x103 Nm2, e.g. about 75x 103 Nm2 to about 3 -2 3 -2 3 -2 3 -2 lOOxlO Nm, about 80x10 Nm to about lOOxlO Nm, e.g. about 90x10 Nm Measurement of Young's modulus for polyacrylamide gels is described, for example, in Pelham & Wang, PNAS USA, 94, 1366 1-13665, 1997, as corrected (PNAS USA 1998, 95(20): 12070).

[0116] Surface stifffiess, or compliance, may also be used to eharacterise 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 5x103 Nn12 to about 100x103 -2 3 -2 3 -2 3 -2 Nm, e.g. about 25x10 Nm to about lOOxlO Nm, e.g. about SOxlO Nm to about 100x103 Nm2, e.g. about 60x105 Nrn2 to about lOOxlO' Nn12, e.g. about 70x103 Nm2 to about 100x103 Nn12, e.g. about 75x103 Nm2 to about 100x103 Nm, about 80x103 Nm2 to about 100x103 Nn12, e.g. about 90x103 Nm?.

[0117] 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% N,N'-methylenebisacrylamide, e.g. 0.1-0.5% N,N'-methylenebisacrylamide, e.g. 0.2-0.4% N,N'-mcthylcnebisaciylamide, e.g. 0.25-0.35% N,N'-rnethylenebisaeiylamide, e.g. 0.3% N,N'-methylenebisacrylamide when polymensed with anmionium persulphate at a final concentration of 1/2000 v/v (i.e. 10 tl of 10% solution per 2 ml of polymer solution) and TEMED at a final conccntration of 1/2000 v/v (i.e. il per 2 ml of polymer solution).

[0118] 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.

[0119] Methods for coating gels with such proteins are well known in the art and illustrative methods are described in the examples below. Typically the exlraccllular 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.

[0120] It will be apparent that sonic gels (for example, those comprising or consisting of basement membrane matrix) will already contain suitable matrix proteins. For others (polyaeiylarnide, alginate, etc.) it may be necessary to incorporate suitable proteins into the gel components before gelatinisation or coal suitable proteins onto the gel surface after gelatinisation.

[0121] 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.

[0122] The gel substrate may be provided on a plastics material substrate, preferably conventional commercially available polystyrene cultureware, for example ibiTreat microscopy plastic t-plates. If desired, the surface may be coated with one or more extracellular matrix proteins as described above.

[0123] The substrate may be coated with at least one cxtracellular matrix protcin 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.

[0124] Whatever the nature of the substrate, it may have a surface topography which facilitates retention of one or more embiyos, or stcm cells, and their associated culture medium within a defined region of the substrate.

[0125] 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.

[0126] The receptacles may have any suitable shape as viewed from above. They may have approximately equal dimcnsions 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". Altenmtively 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".

[0127] 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 bc sloped such that thc top of thc rcccptaclc is broadcr than thc bottom.

[0128] The substrate may be formed in or upon a suitable template or mould in order to achieve the required topography.

[0129] Altcrnativcly the substrate may bc 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.

[0130] The substrate may comprise a single receptacle or a plurality of receptacles.

[0131] Each receptacle may have a depth of about 250 jim to about 400 jim, e.g. about 300 pin to about 350 jim. Additionally or alternatively, said plurality of receptacles may have a mean depth of about 250 jim to about 400 jim, e.g. about 300 iim to about 350 jini 101321 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.

[0133] Whatever the topography of the substrate, the substrate may cany 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.

[0134] 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 aplurality of embryos is cultured together. This effect may be mediated by factors secreted by the embiyos S 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.

[0135] For example, the culture volume per embiyo maybe about I p1 to about 50 p1, optionally about 2 il to about 40 tl, optionally about 5 p1 to about 30 p1, optionally about 10 p1 to about 25 p1, optionally about 12.5 p1 to about 22.5 p1 or about 15 p1 to about 20 p1 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 p1 or more.

[0136] 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.

[0137] 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.

[0138] The imaging apparatus may comprise microscopy apparatus, suitable recording apparatus, and optionally image processing apparatus.

[0139] Typically, fluorescent markers, such as fluorescent dyes or fluorescent marker proteins, are used in the imaging of embiyonic 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 mitochoridria.

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.

[0140] Thus, fluorescence imaging apparatus may be particularly suitable for the methods described. The imaging apparatus may thus comprise a fluorescence microscope, such as a eonfoeal microscope, that can include but is not limited to wide field, scanning and spinning disc eonfoeal, and light sheet microscope.

[0141] Confoeal 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 flvo or three dimensional image. For example, scanning a specimen in a single planc 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 confoeal laser scanning microscopy. Additionally, light sheet microscopy can also provide good imaging of embryonic development.

[0142] 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 exaniple, 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.

[0143] 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.

[0144] Such cultunng can be useflul as a source of stern 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 plunpotent stein 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.

[0145] 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.

[0146] 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.

[0147] 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. [0148] 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.

[0149] 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.

EXAMPLE S

Example 1

In vitro Culture UVC) system usin defined (serum-free) medium -Mouse em br'vos [0150] Wild-type F! (C57BL/6 x CBA) female mice (8-12 weeks old) were injected inlrapcritoncally with 7.5 IIJ of PMSG (Pregnant Mares Serum Gonadoiropin) followed by 7.5 IU injection of IiCG (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 coituin (d.p.c.) the females wcre 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, Tl788). The zona-free embryos were seeded on glass bottom plates coated with Matrigcl (BD, 356230) or on ibiTreat microscopy plastic a-plates (Ibidi) coated with Collagen (4 mg/ml, Sigma C.38671VL) / Fibronectin (I mg/n-il, Sigma, Fl 141) / Gelatin (0.1%) in proportion 1:2:13. The plates were filled with pre-warmed defined (serum-free) P/C-medium (Advanced DMEM/F12 (Gibco, 12634-010) supplemented with 2 mM L-glutamine (Gibco, 25030-024), 1 mlvi Sodium pyruvate (Gibco, 11360-039), 1 x MEM NEAA (7.5 mg/l glycine, 8.9 mg/I L-alanine, 13.2 mg/I L-asparagine, 13.3 mg/mI L-aspartic acid, 14.7 mg/l L-glutamie acid, 11.5 mg/l L-prolinc and 10.5 mg/l L-serine) (Gibco, 11140- 035), Penicillin (25 units/mi) / Streptomycin (25 ig/ml) (Gibco, 15070-063), 1 x ITS-X (10 mg/I Insulin, 5.5 mg/I Transferrin, 0.0067 mg/I sodium selenite, 2 mg/I Ethanolamine) (Invitrogen 5 1500-056), 8 nM 13-estradiol (Sigma, E8875), 200 ng/ml Progesterone (Sigma, P0130) and 25 RM N-acetyl-L-cysteine (Sigma, A7250), containing 30% KSR (KnockOut Serum Replacement (Invitrogen, 10828-010)).

During the next 36-48 horn-s 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) IYC-medium. At that time point the egg cylinders had already emerged. For the next 48 hours the embryos were cultured on defined (serunFfree) IVC-medium, supporting the egg-cylinder growth. The whole embryo culture was performed on 37°C and 5% CO2.

101511 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 t-p1ates (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 (YE) and extraembrvonic 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), YE (visceral endoderm), TE (trophectodent), EXE (extraembryonic ectoderm), CC (giant cells), PC (proanmiotic cavity).

[0152] Figure 3 shows the similar organization of the mouse embryonic lineage in in vn'o recovered at E6.0 and in vitro cultured embiyos (in accordance with the method described above) at day 4. C.onfocal images of embryos stained for the apical polarity marker ParÔ (gray) and the epiblast cell fate marker Oet4 (white).

Example 2

In vitro Culture (IVC) system using ECS-containing medium and defined (serum free) medium -Mouse embryos [0153] Wild-type Fl (C57BL/6 x CBA) female mice (8-12 weeks old) were injected intraperitoneally with 7.5 111 of PMSG (Pregnant Mares Serum Gonadotropin) followed by 7.5 IU injection of hCG (human Chorionic Conadotropin) 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 eoitum (d.p.e.) 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 i-plates (Ibidi) with no matrix coating, filled with pre-warmed P/C-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 nigh glycine, 8.9 mg/l L-alanine, 13.2 mg/i L-asparagine), 13.3 mg/ml L-aspartic acid, 14.7 mg/i L-glutamie acid, 11.5 mg/l L-proline and 10.5 mg/l L-serine) (Gibco, 11140- 035), Penicillin (25 units/mI) / Streptomycin (25 g/ml) (Gibco, 15070-063), ITS-X (10 mg/I Insulin, 5.5 mg/I Transferrin, 0.0067 mg/I Sodium selenite, 2 mg/I Ethanolamine) (Invitrogen 51500-056), 8 nM 3-estradioi (Sigma, E8875), 200 ng/ml Progesterone (Sigma, P0130) and 25 JIM N-acetyi-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) P/C-medium (as defined in Example 1), supporting the egg-cylinder growth. The whole embryo culture was performed on 37°C and 5% CO2.

[0154] Figure 4 shows the successful development of mouse biastocyst beyond impiantation outside the body of the mother using FC.S-eontaining 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 ri-plates (ibidi); B) Embryo attached to the sm-face with trophectodennal giant cells (OC) spreading out; C) Early egg cylinder emerges consisting of epiblast (EPI) surrounding proamniotie cavity (PC.) and two extraembryonic lineages -visceral endodenn (yE) 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 FF1 (epiblast), PrE (primitive endoderm), VE (visceral endoderm), TE (trophcctoderrn), EXE (extracmbiyonic cctoderrn), GC (giant cells), PC (proamniotic cavity).

S Example 3

In vitro Culture (F'C) system using ECS-containing medium and defined (serum free) medium -Human embryos [0155] 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 R-plates (Ibidi), with no matrix coating, filled with pre-warmed IVC-mcdium (Advanced DMEM/F12 (Gibco, 12634-0 10) supplemented with 2 mM L-glutamine (Gibco, 25030-024), 1 mM Sodium pyruvate (Gibco, 11360-039), 1 x MEM NEAA (7.5 mg/l glycine, 8.9 mg/l L-alanine, 13.2 mg/i L-asparagine), 13.3 mg/ml L-aspartic acid, 14.7 mg/l L-glutarnic acid, 11.5 mg/l L-proline and 10.5 mg/ L-serine) (Gibco, 11140-035), Penicillin (25 units/ml) / Streptomycin (25 g/ml) (Gibco, 15070-063), ITS-X (10 mg/I Insulin, 5.5 mg/I Transferrin, 0.0067 mg/I Sodium selenite, 2 mg/I Ethanolaminc) (Invitrogcn 51500-056), 8 nM 3-estradiol (Sigma, E8875), 200 ng/ml Progesterone (Sigma, P0130) and 25 JIM 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 TVC-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.

[0156] 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 thc method described above. The figure shows that between (lay 6 and day 9 the embryo attached to the surface. The embryo is cultured in vit;v up to day 13. The epiblast is marked with a dashed line.

Lineage annotations: EPI (epiblast), TB (trophoblast).

S Example 4

In vitro culture of mouse ES cells in 3D extracellular matrix [0157] 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 miii / 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 centrithging for 5 mm / 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 L-plates (Ibidi) and incubated for 5-10 miii until the matrigel solidified and formed a three dimensional gel. After that the plate was filled with pre-warmed defined (serum-free) TVC medium (as defined in Example 1) or pre-warmed PVC 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.

[0158] Figure 6 shows suecessllil culture of mouse ES cells in 3D exiracellular 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.

[0159] 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 documenits cited herein are expressly incorporated by reference.

REFERENCES

1. Perea-Gomez, A., et a!. Regionalization of the mouse visceral endoderm as the blastoeyst transforms into the egg cylinder. BMC Dcv Dial 7, 96 (2007).

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3. Hsu, Y.C. Differentiation in vitro of mouse embryos beyond the implantation stage.

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4. Hsu, Y.C. Differentiation in vitro of mouse embryos to the stage of early sornite.

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7. Hsu, Y.C. In vitro development of individually cultured whole mouse embryos from blastocyst to early somite stage. Dcv B/al 68, 453-461 (1979).

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Claims (63)

1. CLAIMS1. 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 developnient.
2. 2. The in vitro culture medium according to claim 1, wherein the medium comprises an albumin.
3. 3. The in vitro culture medium according to claim I or claim 2, wherein the medium comprises a serum replacement.
4. 4. The in vitro culture medium according to claim 3, wherein the medium comprises 30% serum replacement.
5. 5. 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.
6. 6. The in vitro culture medium according to any of claims Ito 5, wherein the insulin receptor agonist is selected from the group consisting of IGF-I or an analogue thereof and IGF-ll or an analogue thereof
7. 7. The in vitro culture medium according to any of claims 1 to 6, wherein the ocstrogcn receptor agonist is selected from the group consisting of 13-estradiol or an analogue thereof, estrone or an analogue thereof, estriol or an analogue thereof and estetrol or an analogue thereof.
8. S. 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. 9. The in vitro culture medium according to any of claims I to 8, wherein the medium comprises L-glutamine.
10. 10. The in vitro culture medium according to any of claims 1 to 9, wherein the medium comprises sodium pyruvate.
11. 11. The in vitro culture medium according to ally 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. 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. 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, dithiothrcitol (DTT) or 3-mercaptoethanol.
14. 14. The in vitro culture medium according to any of claims I to 13, wherein the medium docs not comprise a conditioned medium.
15. 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. 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 matenal or glass.
17. 17. The in vitro culture medium according to claim 16, wherein the substrate comprises a matnx.
18. 18. The in vitro culture medium according to claim 17, wherein the matrix comprises at least one extracellular matrix protein, or aiialogue thereof.
19. 19. The in vitro culture medium according to claim 18, wherein the cxtracellular matrix protein is collagen or analogue thereof, laminin or analogue thereof, fibronectin or analogue thereof and/or gelatin.
20. 20. The in vitro culture medium according to any of claims 1 to 19, wherein the substrate does not comprise a feeder-cell layer, preferably wherein the substrate does not comprise uterine epithelial cells or uterine endometrium.
21. 21. A culture medium supplement for producing the culture medium of any one of claims 1 to 20 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-implantatiori stage of development to a post-implantation stage of development.
22. 22. The culture medium supplement according to claim 21 comprising one or more components, or analogues thereo selected from transferrin; sodium selenite; ethanolanñne; sodium pvruvate; L-glutaminc; L-glycinc; L-alanine; L-asparagine; L-aspartic acid; L-glutamic acid; L-proline; L-serine; and N-acctyl-L-cysteine.
23. 23. A kit for culturing a mammalian embryo comprising: a. an in vitro culture medium of any one of claims I to 20; and b. a substrate as defined in any one of claims 15 to 20.
24. 24. A kit for culturing a mammalian embryo comprising: a. a culture medium supplement according to claim 21, and b. (I) a basal medium and/or (II) one or more separate supplements comprising one or more additional components, or analogues thereof, selected fiom transferrin; sodium selenite; ethanolaminc; 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.
25. 25. The kit according to claim 23 or 24, further comprising an additional separate supplement which is a serum replacement medium.
26. 26. The kit according to claim 24 or claim 25, further comprising a substrate as defined in any one of claims 15 to 20.
27. 27. The kit according to claim 26, 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.
28. 28. An in vitro method of culturing a mammalian embryo compnsing contacting a manmrnlian embiyo with a culture medium according to any one of claims 1 to 20, wherein said embryo is cultured on a substrate from a pre-implantation stage of development to a post-implantation stage of development.
29. 29. The method of claim 28, wherein the pre-implantation stage is the blastocyst stage.
30. 30. The method of claim 28, wherein the pre-implantation stage is prior to attachment of the blastocyst to the substrate.
31. 31. The method of claim 29 or claini 30 comprising the step of removing the blastoeyst from the zona pellucida.
32. 32. The method of any one of claims 28 to 31 comprising the earlier steps of (a) providing said embryo at a pre-blastocyst stage of development, and (b) culturing said embryo to blastocyst stage.
33. 33. The method of claim 32, wherein the pre-blastocyst stage of development is a single cell embryo.
34. 34. The method of claim 33, wherein the single cell embryo is a fertilised egg.
35. 35. The method of claim 33, wherein the single cell embryo has been obtained by nuclear tmnsfcr.
36. 36. The method of any of claims 28 to 35, 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.
37. 37. The method of any one ofclainis 28 to 35 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 culturc comprising said embryo in a serum-free culture medium.
38. 38. The method of claim 37, 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 perfonned at the egg cylinder stage or embryonic disc stage.
39. 39. The method of any of claims 28 to 38, wherem the post-implantation stage is the egg cylinder stage or embryonic disc stage.
40. 40. The method of any one of claims 28 to 39 frirther comprising the step of removing one or more cells from said embryo.
41. 41. The method of claim 40, wherein said one or more cells is taken from the inner cell mass.
42. 42. The method of claim 41, wherein said cell is an epiblast cell.
43. 43. The method of any of claims 40 to 42, wherein said ccli is a pluripotent cell.
44. 44. The method according to ally of claims 28 to 43, wherein the substrate is a substrate as defined in any one of claims 15 to 20.
45. 45. The method according to any one of claims 28 to 44, 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.
46. 46. The method of claim 45, wherein each said culture comprises a plurality of embryos.
47. 47. The method of claim 45 or claim 46, wherein each said culture has a volume of 15 p1 to about 20 p1 per embiyo.
48. 48. The method of any one of claims 28 to 47 comprising the step of recording one or more images of the embryo.
49. 49. The method of any one of claims 28 to 48 comprising the steps of contacting said embryo with a test agent and determining the effect of said test agent on development of said embiyo.
50. 50. 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 20, wherem the stem cells are cultured on or in a substrate.
51. 51. The in vitro method of claim 50, wherein the substrate is a matrix and wherein the method comprises the step of suspending the stem cells in the matrix.
52. 52. The in vitro method of claim 50 or claim 51, wherein the substrate is as defined in any one of claims 15 to 20.
53. 53. The in vitro method of any of claims 50 to 52, wherein the stem cells are embryonic stem cells.
54. 54. The in vitro method of any one of claims 50 to 52, wherein the stem cells are induced plunpotent stern cells.
55. 55. A method for investigating the efft of a test agent on embryo development comprismg die steps of: a. culturing a mammalian embryo using a culture medium according to any one of claims I to 20; b. contacting the embryo with a test agent; and c. determining the effect of said test agent on the embryo, optionally coniprising 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.
56. 56. The method of claim 55 comprising contacting the embryo with the test agent before attachment of the embryo to the substrate.
57. 57. The method of claim 55 comprising contacting the embryo with the test agent after attachment of the embryo to the substrate.
58. 58. The method of claim 56 ifirther comprising determining the subsequent effect on attachment of the embryo to the substrate.
59. 59. The method of any one of claims 55 to 58 thrthcr comprising recording one or more images of the embryo.
60. 60. Use of an in vitro culture medium according to any one of claims 1 to 20 for culturing a mammalian embryo on a substrate from a pre-implantation stage of development to a post-implantation stage of devclopmcnl.
61. 61. Use of an in vitro culture mediuni according to any one of claims I to 20 for culturing stem cells on a substrate.
62. 62. The use of claim 60 or claim 61, wherein the substrate is as defined in any onc of claims 15 to 20.
63. 63. The culture medium, culture medium supplement, kit, method or use according to any of the preccding claims, wherein the embryo or stem cells is/are non-human.