BACKGROUND

[0001] The disclosure is directed to methods, systems and compositions for isolating inner cell mass (ICM) from bovine blastocysts. More specifically, the disclosure is directed to methods for harvesting ICM from bovine blastocysts, the systems used to implement the methods and the compositions used to culture the harvested ICM for the formation of tissue culture.

[0002] In an effort to reduce the impact of animal agriculture and to improve people's nutrition, as well as for various other incentives, there is a need for alternatives to animal meat for development of novel protein sources containing viable cells culture(s) that correspond to the three-dimensional (3D) tissue, for instance, muscle tissue.

[0003] Embryonic stem (ES) cells are special kind of cells that can both duplicate themselves (in other words, divide and proliferate indefinitely in culture) and produce differentiated functionally specialized cell types. These stem cells are capable of becoming almost all of the specialized cells of the body and thus, may have the potential to generate cells for a broad array of tissues such as, for example, muscle, cartilage and the like.

[0004] Therefore, the need exists for methods, systems and compositions for harvesting the ES for the commercial production of tissue culture to form tissues that can be used as substitute for meat.

SUMMARY

[0005] Disclosed, in various embodiments, are methods, systems and compositions for harvesting ICM from bovine blastocysts.

[0006] In an embodiment provided herein is a method for establishing a tissue culture from the inner cell mass (ICM) of a bovine blastocyst, comprising the steps of: obtaining a plurality of blastocysts, each blastocyst having: a zona pellucida; a trophectoderm, and an inner cell mass; perforating each blastocyst; isolating the ICM from each blastocyst through the perforations; seeding the ICM on a predetermined substrate; and culturing the ICM to establish a bovine ICM tissue culture.

BRIEF DESCRIPTION OF THE FIGURES [0007] For a better understanding of the methods for isolating inner cell mass (ICM) from bovine blastocysts, with regard to the embodiments thereof, reference is made to the accompanying examples and figures, in which:

[0008] FIG. 1. is an embodiment of the primary method for isolating inner cell mass (ICM) from bovine blastocysts; and

[0009] FIG. 2, illustrating a schematic representation of a bovine blastocyst..

DETAILED DESCRIPTION

[00010] Provided herein are embodiments of methods, systems and compositions for isolating inner cell mass (ICM) from bovine blastocysts, cultured to emulate tissue and/or organs for (nonvegan) food consumption. At the early stages of development, the egg is surrounded by the zona pellucida. After fertilization, the egg cleaves to form a cluster of cells called the morula. After the 32 cell stage (in cattle), an internal cavity (blastocoel) forms and the cluster is called a blastocyst. One pole of the blastocyst has a thicker accumulation of cells, known as the inner cell mass (ICM). The entire embryo proper is derived from the inner cell mass.

[00011] Embryonic stem (ES) cells are found in the ICM of the bovine blastocyst, an early stage of the developing embryo lasting from about the 9 ^th to about 12 ^th day after fertilization. The blastocyst is the stage of embryonic development prior to implantation that contains two types of cells; namely: the Trophectoderm - the outer layer which gives extra embryonic membranes; and the Inner cell mass (ICM): which forms the embryo proper. ES cells (ESC) - like cells (referring to a pluripotent cell characterized by the properties of ESC such as, for example, proliferation without transformation, infinite replication, self-renewal and differentiation into all three germ layers; endoderm, mesoderm, and ectoderm), extracted from the ICM during the blastocyst stage, can be cultured in the laboratory and under the right conditions proliferate indefinitely. ES cells growing in this undifferentiated state retain the potential to differentiate into cells of all three embryonic tissue layers. Ultimately, the cells of the inner cell mass give rise to all the embryonic tissues. It is at this stage of embryogenesis, near the end of first week of development, that ES cells can be derived from the ICM of the blastocyst.

[00012] Accordingly and in an embodiment, provided herein is a method for establishing a tissue culture from the inner cell mass (ICM) of a bovine blastocyst, comprising the steps of: obtaining a plurality of blastocysts, each blastocyst having: a zona pellucida; a trophectoderm, and an inner cell mass; perforating each blastocyst; isolating the ICM from each blastocyst through the perforations; seeding the ICM on a predetermined substrate; and culturing the ICM to establish a bovine ICM tissue culture.

[00013] As illustrated in the flowchart of FIG. 1, the step of perforating the each blastocyst further comprises forming holes in at least one of: the zona pellucida (ZP) (see e.g., FIG. 2), and the trophectoderm (interchangeable with trophoblast). In the context of the disclosure, the “zona pellucida” refers to the embryo shell and is intended to include the complex extracellular glycoprotein matrix surrounding the blastocyst as described above. It is also intended to include the deglycosylated or deglycated proteins that are included in the matrix; "deglycosylated protein" means the protein backbone of a glycoprotein molecule lacking some or all of its carbohydrate moieties. The term "deglycated protein" may also be used to define the protein backbone of a glycoprotein molecule lacking some or all of its carbohydrate moieties. "Zona pellucida" is also intended to include polypeptides having essentially the same amino acid sequence as the naturally occurring and recombinant Zona pellucida protein(s) and any analogs thereof. The term "analogs" is intended to include proteins or polypeptides which differ from natural Zona pellucida protein by addition, deletion or substitution of one or more amino acids, providing that polypeptide demonstrates substantially the antigenic, biologic and growth stimulant activity of natural Zona pellucida. Additionally, “tophectoderm” (TE) refers, in the context of the disclosure to the outer layer of the blastocyst after differentiation of the ectoderm, mesoderm, and endoderm, when the outer layer is continuous with the ectoderm of the embryo

[00014] The step of perforating the ZR and TE, in the methods for harvesting ICM from a plurality of blastocysts described herein, can be done using at least one of: electroporation, partial cell digestion, drilling, laser ablation, and slitting. For example, electroporation meaning the temporary creation of holes or aqueous pores in the surface of a cell membrane can be done by an applying electrical potential and through which therapeutic agents may pass into and out of the cell depending on the existing chemical potential between the external and internal cell environment. In an example, Electroporation can be used also for thinning the ZP. Likewise, ZP drilling is done using, for example, a microneedle filled with acidic Tyrode’s solution are positioned in an example, by holding the needle tip very close to the ZP at the embryo’ s pole, or diametrically opposed to the embryo’ s pole and locally contacting the zona with the solution. Similarly, laser assisted cellular microsurgery can be used to create a hole in both the ZP and TE layers. For example, by using an argon fluoride excimer laser, in the deep ultraviolet (UV) region of the spectrum at about 193 nm wavelength, guided through a glass pipette filled with a positive air pressure, where the hole diameter is controlled by the pipette tip diameter (for example, between micropipette having a tip diameter between about 25 pm and about 45 pm). It is noted that some or all of the methods disclosed for perforating the ZP and TE, can be used for thinning the ZP as well. In an embodiment, at least one of: partial cell digestion, drilling, laser ablation, and slitting, is performed at each blastocyst’s pole (in other words, the location in the blastocyst exhibiting thicker accumulation of cells), or contralateral to the blastocyst’s pole

[00015] In an embodiment, the step of culturing (referring to the growth of cells in vitro in an artificial medium for commercial purpose), of the ICM to establish a bovine ICM tissue culture, in the methods, systems and compositions for harvesting ICM from a plurality of blastocysts described herein, can comprise culturing the cells of the ICM in the presence of (or in plating over) an embryonic stem cell medium and a feeder layer medium. In the context of the disclosure, the term “feeder layer” refers to a cell or cells used in co-culture to maintain stem cells as pluripotent. For bovine embryonic stem cell culture, feeder layers can include, for example, at least one of: mouse (murine) embryonic fibroblasts (MEFs), and bovine embryonic fibroblasts (BEF) that have been treated to prevent them from dividing in culture. The feeder layer can comprise for example a combination of the MEF and BEF at a ratio of between about 9:11 to about 11:9 MEF:BEF. In an embodiment, the ratio between MEF and BEF is 1:1. Furthermore, the embryonic stem cell medium, can be, for example, at least one of: a composition comprising: Dulbecco's modified Eagle's medium (DMEM) without Sodium pyruvate having glucose content of between about 70% and about 90%; between about 10% and about 30% Fetal bovine serum (FBS); 0 -mercaptoethanol (0.1 mM); about 1% of non-essential amino acids; E-Glutamine 2 mM; and basic fibroblast growth factor (BFGF), a composition comprising: Minimum Essential Medium Alpha (MEM-oc) with 10% inactivated fetal calf serum, and a composition comprising: DMEM; 15% Fetal bovine serum; Penicillin/ Streptomyocin; Glutamine; Non-essential amino acids; nucleosides; 0-mercaptoethanol; Sodium pyruvate; and leukaemia inhibitory factor (LIF).

[00016] Other embryo culture and maturation media routinely used for the collection and maintenance of embryos, used in connection with the methods disclosed, can be, for example, Ham's F-10+10% fetal calf serum (FCS), Tissue Culture Medium-199 (TCM-199)+10% fetal calf serum, Tyrodes-Albumin-Eactate-Pyruvate (TAEP), Dulbecco's Phosphate Buffered Saline (PBS), Eagle's and Whitten's media. For example TCM-199, and 1 to 20% serum supplement including fetal calf serum, newborn serum, estrual cow serum, lamb serum or steer serum. An example of maintenance medium can be TCM-199 with Earl salts, 10% fetal calf serum, 0.2 mM pyruvate and 50 pg/ml gentamicin sulphate. Any of the above may also involve co-culture with a variety of cell types forming a feeder layer such as, at least one of: granulosa cells, oviduct cells, BRL cells, and uterine cells.

[00017] The ICM harvested ESC-like cells may be subsequently cultured in differentiation medium, operable to cause the culture to differentiate to at least one of: a muscle cell, a cartilage cell, a fat cell, and a connective tissue cell. The term “differentiate” and its derivatives, as used with respect to cells in a differentiating cell system, refers to the process by which cells differentiate from one cell type (e.g., a multipotent, totipotent or pluripotent differentiable cell) to another cell type such as a target-differentiated cell (e.g., a muscle cell, a cartilage cell, a fat cell, a neuron cell, and the like). For example, differentiation into hematopoietic and endothelial cell lineages in the presence of (exposed to) a composition comprising: bFGF; stem cell factor; and oncostatin M. Myocytes differentiation can be done by, for example, by employing pharmacologic inhibitors and agonists (e.g., for upregulation of Mesogeninl) as well as isolated cytokines or other protein-based signals, for example, to cause overexpression of myogenic transcription factor MyoD. In other words, a process whereby the unspecialized ICM derived ESC-like cells acquires the features of a specialized cell such as a muscle cell. Differentiation is controlled by the interaction of the cells’ genes with the physical and chemical conditions outside the cell, for example, through signaling pathways involving proteins embedded in the cell surface.

[00018] In the methods for harvesting ICM from a plurality of blastocysts described herein, the step of seeding the ICM is preceded by forming a suspended mixture of the ICM. The suspended cells can be, for example, cells’ dispersion, solution, emulsion, suspension, hydrogel or liquid compositions.

[00019] Furthermore, culturing the isolated ICM ESC-like cell mass further comprises mechanically dissociating the ICM and re-plating the ICM on the feeder layer disclosed.

[00020] Under certain circumstances, the compositions used in the methods disclosed, can further comprise other additives that affect colonization, proliferation, adherence, inhibit apoptosis or other manipulation of the cells, retain native growth of the cells, and/or organ and/or tissue and other similar functions, for example: cells manipulation triggering compounds, (for example, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), bone morphogenic protein (BMP), insulin-like growth factor (IGF), glucoseaminoglycan (GAG), Transforming growth factor (TGF) or signaling compound composition comprising the foregoing), as well as therapeutically effective compounds, antimicrobial compounds, immunosuppressing compounds and the like. [00021] The term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

[00022] All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other. “Combination” is inclusive of blends, mixtures, alloys, reaction products, and the like. The terms “a”, “an” and “the” herein do not denote a limitation of quantity, and are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The suffix “(s)” as used herein is intended to include both the singular and the plural of the term that it modifies, thereby including one or more of that term (e.g., the cell(s) includes one or more cell). Reference throughout the specification to “one embodiment”, “another embodiment”, “an embodiment”, and so forth, when present, means that a particular element (e.g., feature, structure, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.

[00023] All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other. Furthermore, the term "about" means that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is "about" or "approximate" whether or not expressly stated to be such. For example, “about” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% or at least ±10% of the modified term if this deviation would not negate the meaning of the word it modifies.

[00024] Accordingly and in an exemplary implementation, provided herein is a method for establishing a tissue culture from the inner cell mass (ICM) of a bovine blastocyst, comprising the steps of: obtaining a plurality of blastocysts, each blastocyst having: a zona pellucida (ZP); a trophectoderm (TE), and an inner cell mass (ICM); perforating each blastocyst; isolating the ICM from each blastocyst through the perforations; seeding the ICM on a predetermined substrate; and culturing the ICM to establish a bovine ICM tissue culture, wherein (i) the step of perforating comprising forming holes in at least one of the ZP, and the TE, (ii) is performed by at least one of: electroporation, drilling, laser ablation, and slitting, wherein (iii) the blastocyst is preceded by a step of thinning the ZP, wherein (iv) the step of culturing comprises culturing the cells of the ICM in the presence of an embryonic stem cell medium and a feeder layer medium, wherein (v) the embryonic stem cell medium is a composition comprising: Dulbecco's modified Eagle's medium without Sodium pyruvate having glucose content of between about 70% and about 90%; between about 10% and about 30% Fetal bovine serum (FBS); □/?-mercaptoethanol (0.1 mM); about 1% of non-essential amino acids; L-Glutamine 2 mM; and basic fibroblast growth factor (BFGF), wherein (vi) the feeder layer medium is at least one of a murine embryonic fibroblast medium, and a bovine embryonic fibroblast medium, with (vii) a predetermined ratio of murine embryonic fibroblast medium to bovine embryonic fibroblast medium, (viii) the ratio is between about 9:11 and about 11:9, wherein (ix) the step of seeding the ICM is preceded by forming a suspended mixture of the ICM, wherein (x) the suspended system is at least one of: a gel, an emulsion, and a suspension, (xi) further comprising mechanically dissociating the ICM and re-plating the ICM on the feeder layer, wherein (xii) at least one of: partial cell digestion, drilling, laser ablation, and slitting, is performed at each blastocyst’s pole, or contralateral to the blastocyst’s pole, wherein (xiii) the step of thinning the ZP comprises partially digesting the ZP, comprising (xiv) contacting the ZP with a composition comprising at least one of: urokinase-type plasminogen activator (uPA) and plasmin, and pronase, wherein (xv) the embryonic stem cell medium is a composition comprising: Minimum Essential Medium Alpha (MEM-oc) with 10% inactivated fetal calf serum, wherein (xvi) the step of culturing the cells of the ICM in the presence of an embryonic stem cell medium and a feeder layer medium, is followed by a step of culturing the cells of the ICM in the presence of a differentiation medium, and wherein (xvii) the differentiation medium is operable to cause the culture to differentiate to at least one of: a muscle cell, a cartilage cell, a fat cell, and a connective tissue cell.

[00025] Although the foregoing disclosure for harvesting ICM from bovine blastocysts has been described in terms of some embodiments, other embodiments will be apparent to those of ordinary skill in the art from the disclosure herein. Moreover, the described embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions. Indeed, the novel methods, systems and compositions described herein may be embodied in a variety of other forms without departing from the spirit thereof. Accordingly, other combinations, omissions, substitutions and modifications will be apparent to the skilled artisan in view of the disclosure herein.