Pluripotent stem cell aggregates and derived microtissues for the cultivated meat industry

Abstract

Primed cells, pluripotent stem cell aggregates and microtissues produced therefrom, and their use to produce edible products.

Description

[Technical field]

[0001] [Related Applications] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 139,374, filed January 20, 2021, U.S. Provisional Patent Application No. 63 / 135,677, filed January 10, 2021, and U.S. Provisional Patent Application No. 63 / 256,679, filed October 18, 2021, each of which is incorporated by reference in its entirety. [Background technology]

[0002] The present invention, in some embodiments thereof, relates to pluripotent stem cell aggregates and microtissues derived therefrom for the cultured meat industry.

[0003] For the successful production of cultured meat or commonly called clean meat, three main considerations must be faced: i. the origin of the cells, ii. the culture conditions, and iii. scalability.

[0004] i. Cell Origin: Embryonic stem cells (ESCs) have always inspired scientists since their discovery due to their ability to self-renew and differentiate into virtually any cell type (6). Major advances in stem cell research have enabled the utilization of such cells in many scientific and medical applications and provided valuable data on the optimization of the necessary culture conditions (7). ESCs are derived from early embryos (e.g., mammalian blastocysts or avian stage X embryos) and can be cultured indefinitely as long as spontaneous differentiation is prevented (8). This characteristic is particularly attractive to the cultivated meat industry due to the large quantities of cells required to meet market demand. Following a growth period, ESCs can be differentiated into specific lineages (9) to recreate the desired taste and texture of specific animal tissues in the final product. While many of the above have been applied primarily for academic and medical purposes, the cultivated meat industry is still investing a lot of resources in developing new sources and cell line types that possess stem cell properties such as self-renewal and differentiation potential with rapid doubling times. Another cell type used for meat applications is primary and progenitor cells from adult tissues. Although these cells still maintain some self-renewal capacity, their efficacy is somewhat restricted to specific lineages ( 10 ).

[0005] ii.Culture conditions Cultured cells are typically grown in vitro in buffered media containing nutritional compounds (e.g., salts, fatty acids, amino acids, sugars, growth factors) and animal-derived serum. (13) Most available media for in vitro cell culture were originally optimized for small-scale applications. (14) Thus, other variables such as monetary cost, consumer sensitivity to animal components, and the ability of the medium to support high-density cell populations were ignored.

[0006] iii. Scalability Most, if not all, cells need to be adapted for high-density mass culture. The currently accepted methodology for achieving such culture is by using suspension cultures in monitored bioreactors with constant stirring, encouraging cells to grow in 2D as aggregates, as adherent cells on microcarriers (15). However, the most challenging part of this process will be to scale up cell differentiation or maturation, with or without co-culture, to produce tissue-like structures.

[0007] The case for cultured chicken cells is particularly attractive because chicken meat is considered nutritious with a high protein to muscle / fat ratio, and chicken is the most consumed organism on Earth (16). For these reasons, clean and sustainable alternatives for its production are most desirable. As used herein, "cultured meat" is interchangeable with "fresh meat."

[0008] Although an important model for embryo development research in the field of embryogenesis, the case of avian embryonic stem cells seems to have received much less attention compared to other models (e.g., mouse / human ESCs). Avian ESCs can be obtained from stage X-XIII germinal discs (17). These blastodermal cells exhibit all the characteristics of stem cells, including telomerase activity and expression of embryonic markers (e.g., alkaline phosphatase, SSEA1, Nanog, Oct4) (18). However, the easy access to avian blastodermal cells, together with their ability to self-renew in culture, has led to their application in the animal vaccine industry (20). Similar to the cultivated meat industry, vaccine production in culture also requires the cultivation of cells in high-density populations. Recently, several studies have demonstrated the ability to adapt avian ESC cells of duck and chicken origin to suspension-based, large-volume culture conditions (21). Summary of the Invention

[0009] According to an aspect of some embodiments of the present invention there is provided a method of producing pluripotent stem cell aggregates comprising the steps of: (a) providing a non-human pluripotent stem cell line of non-human pluripotent stem cells grown under conditions of matrix adherence; (b) gradually removing the matrix-adherent non-human stem cell line to obtain aggregates comprising non-human pluripotent stem cells, wherein the non-human pluripotent stem cells of the aggregates exhibit a doubling time of 12 hours or less in an undifferentiated state for more than 60 passages, are capable of differentiating into muscle, fat and connective tissue upon induction of differentiation, and exhibit lower cell-cell adhesion than embryoid bodies (EBs) as determined by reduced expression of adhesion molecules selected from the group consisting of COL6A2, CD44, COL6A1, ANXA1, ANXA2 and S100A11, A method is provided, wherein steps (a) and (b) are carried out in the presence of a growth factor.

[0010] According to an aspect of some embodiments of the present invention there is provided a method of producing microtissues comprising one or more cell types, the method comprising the steps of: (a) forming aggregates in suspension without matrix attachment comprising non-human pluripotent stem cells, wherein the non-human pluripotent stem cells of the aggregates exhibit a doubling time of 12 hours or less when undifferentiated for more than 60 passages, are capable of differentiating into muscle, fat and connective tissue upon induction of differentiation, grow in the presence of growth factors, and exhibit lower cell-cell adhesion than embryoid bodies (EBs) as determined by reduced expression of adhesion molecules selected from the group consisting of COL6A2, CD44, COL6A1, ANXA1, ANXA2 and S100A11, as compared to said EBs; (b) producing microtissues by transferring the aggregates to a bioreactor and growing them in suspension in the absence of growth factors and without matrix attachment; A method is provided that includes:

[0011] According to an aspect of some embodiments of the present invention there is provided a method of producing microtissues comprising one or more cell types of interest, the method comprising the steps of: (a) producing a microtissue as described herein; (b) subjecting the microtissues in suspension to differentiation conditions, thereby producing microtissues containing one or more cell types of interest; A method is provided, comprising:

[0012] According to some embodiments of the invention, the values ​​shown are given under optimal conditions for cell growth of that type and developmental stage as a non-human pluripotent stem cell.

[0013] According to some embodiments of the invention, the non-human stem cells are embryonic stem cells.

[0014] According to some embodiments of the invention, the embryonic stem cells are from an embryonic stem cell line.

[0015] According to some embodiments of the invention, the non-human pluripotent stem cells are livestock pluripotent stem cells.

[0016] According to some embodiments of the invention, the non-human pluripotent stem cells are selected from the group consisting of avian pluripotent stem cells, bovine pluripotent stem cells, porcine pluripotent stem cells, caprine pluripotent stem cells, ovine pluripotent stem cells, shrimp pluripotent stem cells and fish pluripotent stem cells.

[0017] According to some embodiments of the invention, the avian pluripotent stem cells are selected from the group consisting of chicken pluripotent stem cells and duck pluripotent stem cells.

[0018] According to some embodiments of the invention, the avian pluripotent stem cells are chicken pluripotent stem cells.

[0019] According to some embodiments of the present invention, the microtissues are between 30 and 500 μm in diameter.

[0020] According to some embodiments of the present invention, the microtissues are between 30 and 500 μm in diameter.

[0021] According to some embodiments of the invention, the non-human pluripotent stem cells of the aggregates exhibit an average diameter of between 80 and 120 μm.

[0022] According to some embodiments of the invention, the non-human pluripotent stem cells of the aggregates exhibit alkaline phosphatase expression.

[0023] According to some embodiments of the invention, the non-human pluripotent stem cells of the aggregates exhibit telomerase gene expression.

[0024] According to some embodiments of the invention, the non-human pluripotent stem cells of the aggregates are SSEA4-, LIN28+, ENS-1+, NANOG+, OCT4+, and TRA-I-60+.

[0025] According to some embodiments of the invention, the aggregates or microtissues exhibit the organoleptic properties of a native meat product.

[0026] According to some embodiments of the invention, one of the plurality of tissue types is selected from the group consisting of muscle cells, fat cells, and connective tissue cells.

[0027] According to some embodiments of the invention, the matrix attachment is selected from feeder cells, and natural or synthetic matrix molecules.

[0028] According to some embodiments of the invention, the matrix molecule comprises gelatin.

[0029] According to some embodiments of the invention, the growth factor is selected from the group consisting of IGF-1, IL6, sIL6Rα, hLIF and stem cell factor (SCF).

[0030] According to some embodiments of the invention, the growth factors include IGF-1, IL6, sIL6Rα, hLIF and stem cell factor (SCF).

[0031] According to some embodiments of the invention, each step of the method is devoid of animal components other than the non-human pluripotent stem cells.

[0032] According to some embodiments of the invention, the aggregates exhibit gene expression approximately similar to that of the stem cell line from which they were derived, with the exception of expression levels of genes associated with cell motility and migration.

[0033] According to some embodiments of the invention, the microtissues are NANOG-OCT4-LIN28-, SSEA3+.

[0034] According to an aspect of some embodiments of the present invention there is provided a microtissue obtainable by the method described herein.

[0035] According to an aspect of some embodiments of the present invention there is provided a pluripotent stem cell aggregate obtainable by the methods described herein.

[0036] According to an aspect of some embodiments of the present invention there is provided a microtissue comprising one or more cell types, the microtissue having a diameter of 30-500 μm, and wherein the cells of the microtissue are NANOG-OCT4-LIN28-, SSEA3+.

[0037] According to some embodiments of the present invention, the microtissues exhibit the organoleptic properties of a native meat product.

[0038] According to some embodiments of the invention, the one or more cell types include adipocytes and muscle cells.

[0039] According to one aspect of some embodiments of the present invention, there is provided a pluripotent stem cell aggregate comprising non-human pluripotent stem cells, wherein the non-human pluripotent stem cells of the aggregate exhibit a doubling time of 12 hours or less when undifferentiated for more than 60 passages, are capable of differentiating into muscle, fat and connective tissue upon induction of differentiation, and exhibit lower cell-cell adhesion than embryoid bodies (EBs) as determined by reduced expression of adhesion molecules selected from the group consisting of COL6A2, CD44, COL6A1, ANXA1, ANXA2 and S100A11.

[0040] According to some embodiments of the invention, the pluripotent stem cell aggregates are characterized by: (i) average diameter of 80–120 μm; (ii) the non-human pluripotent stem cells of the aggregates exhibit alkaline phosphatase expression; (iii) the aggregate non-human pluripotent stem cells exhibit telomerase gene expression; (iv) the non-human pluripotent stem cells of the aggregates are SSEA4-, LIN28+, ENS-1+, NANOG+, OCT4+, and TRA-I-60+; (v) exhibiting the organoleptic properties of natural meat products; (vi) the aggregates exhibit gene expression approximately identical to that of the stem cell line from which they were derived, with the exception of expression levels of genes associated with cell motility and migration; At least one of the following is further illustrated:

[0041] According to some embodiments of the invention, the non-human pluripotent stem cells are selected from the group consisting of avian pluripotent stem cells, bovine pluripotent stem cells, porcine pluripotent stem cells, caprine pluripotent stem cells, ovine pluripotent stem cells, shrimp pluripotent stem cells and fish pluripotent stem cells.

[0042] According to some embodiments of the invention, the avian pluripotent stem cells are selected from the group consisting of chicken pluripotent stem cells and duck pluripotent stem cells.

[0043] According to some embodiments of the invention, the avian pluripotent stem cells are chicken pluripotent stem cells.

[0044] According to an aspect of some embodiments of the present invention there is provided a food product comprising the microtissues or aggregates described herein.

[0045] According to an aspect of some embodiments of the present invention there is provided a method of producing a food product comprising combining the microtissues or aggregates described herein with an edible composition for human consumption.

[0046] According to an aspect of some embodiments of the present invention, there is provided a method of producing cells comprising culturing stem cells having an adipocyte fate while retaining a proliferative phenotype in the presence of a fatty acid at a concentration of greater than 100 μM for a sufficient time to allow differentiation into adipocytes.

[0047] According to some embodiments of the present invention, culturing stem cells with an adipocyte fate is performed for 5 to 10 days.

[0048] According to an aspect of some embodiments of the present invention there is provided a cell obtainable by the method described herein.

[0049] According to an aspect of some embodiments of the present invention there is provided a microtissue obtainable by the method described herein.

[0050] According to an aspect of some embodiments of the present invention there is provided a pluripotent stem cell aggregate obtainable by the methods described herein.

[0051] According to an aspect of some embodiments of the present invention there is provided a food product comprising the cells as described herein or the microtissues or aggregates as described herein.

[0052] According to an aspect of some embodiments of the present invention there is provided a method of producing a food product comprising combining a cell or microtissue or aggregate of the claims described herein with an edible composition for human consumption.

[0053] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, controls. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. [Brief description of the drawings]

[0054] [Figure 1A] Isolation of cESCs. a. Isolated cESCs plated on a feeder cell layer generating cell cluster. b. Typical established stem cell colony grown on feeder cells. c. Morphology of cESCs plated without the addition of a feeder layer. d. Typical stem cell high nucleus / cytoplasm ratio in proliferating cells. e. Growth of a colony of cESCs on a feeder-free culture dish. [Figure 1B] Isolation of cESCs. a. Isolated cESCs plated on a feeder cell layer generating cell cluster. b. Typical established stem cell colony grown on feeder cells. c. Morphology of cESCs plated without the addition of a feeder layer. d. Typical stem cell high nucleus / cytoplasm ratio in proliferating cells. e. Growth of a colony of cESCs on a feeder-free culture dish. [Figure 1C] Isolation of cESCs. a. Isolated cESCs plated on a feeder cell layer generating cell cluster. b. Typical established stem cell colony grown on feeder cells. c. Morphology of cESCs plated without the addition of a feeder layer. d. Typical stem cell high nucleus / cytoplasm ratio in proliferating cells. e. Growth of a colony of cESCs on a feeder-free culture dish. [Figure 1D] Isolation of cESCs. a. Isolated cESCs plated on a feeder cell layer generating cell cluster. b. Typical established stem cell colony grown on feeder cells. c. Morphology of cESCs plated without the addition of a feeder layer. d. Typical stem cell high nucleus / cytoplasm ratio in proliferating cells. e. Growth of a colony of cESCs on a feeder-free culture dish. [Figure 1E] Isolation of cESCs. a. Isolated cESCs plated on a feeder cell layer generating cell cluster. b. Typical established stem cell colony grown on feeder cells. c. Morphology of cESCs plated without the addition of a feeder layer. d. Typical stem cell high nucleus / cytoplasm ratio in proliferating cells. e. Growth of a colony of cESCs on a feeder-free culture dish. [Figure 2A] Adaptation of cESCs for growth in suspension: a. EB morphology of cESC colonies transferred to suspension. b. Single cell morphology of suspension-adapted cESCs. c. Early stage aggregates of suspension-adapted cESCs. f. Mature cell aggregates of suspension-adapted aggregates. [Figure 2B] Adaptation of cESCs for growth in suspension: a. EB morphology of cESC colonies transferred to suspension. b. Single cell morphology of suspension-adapted cESCs. c. Early stage aggregates of suspension-adapted cESCs. f. Mature cell aggregates of suspension-adapted aggregates. [Figure 2C] Adaptation of cESCs for growth in suspension: a. EB morphology of cESC colonies transferred to suspension. b. Single cell morphology of suspension-adapted cESCs. c. Early stage aggregates of suspension-adapted cESCs. f. Mature cell aggregates of suspension-adapted aggregates. [Figure 2D] Adaptation of cESCs for growth in suspension: a. EB morphology of cESC colonies transferred to suspension. b. Single cell morphology of suspension-adapted cESCs. c. Early stage aggregates of suspension-adapted cESCs. f. Mature cell aggregates of suspension-adapted aggregates. [Figure 2E]Adaptation of cESCs for growth in suspension: a. EB morphology of cESC colonies transferred to suspension. b. Single cell morphology of suspension-adapted cESCs. c. Early stage aggregates of suspension-adapted cESCs. f. Mature cell aggregates of suspension-adapted aggregates. [Figure 2F] Adaptation of cESCs for growth in suspension: a. EB morphology of cESC colonies transferred to suspension. b. Single cell morphology of suspension-adapted cESCs. c. Early stage aggregates of suspension-adapted cESCs. f. Mature cell aggregates of suspension-adapted aggregates. [Diagram 3] Doubling time of aggregate cells growing in suspension. The graph shows three separate experiments of aggregate suspension growth. The average doubling time was calculated for runs ranging from 75 to 120 hours. The calculated doubling times ranged from 10 to 12 hours, with an average doubling time of 11.9 hours / cycle. [Figure 4] Expression of stem cell markers in aggregate cells. Immunofluorescence staining revealed high and global expression of stem cell markers Oct4, Nanog, in28, Tra-1-60 and ENS-1 (chicken stem cell marker). Surface markers SSEA 3 / 4 were found to be negative. [Figure 5A] Self-renewal of aggregates: a, aggregates stain positive for alkaline phosphatase, b, high magnification of AP-positive aggregates, c, representative examples of telomerase activity in three populations of aggregates. [Figure 5B] Self-renewal of aggregates: a, aggregates stain positive for alkaline phosphatase, b, high magnification of AP-positive aggregates, c, representative examples of telomerase activity in three populations of aggregates. [Figure 5C] Self-renewal of aggregates: a, aggregates stain positive for alkaline phosphatase, b, high magnification of AP-positive aggregates, c, representative examples of telomerase activity in three populations of aggregates. [Figure 6]Expression levels of representative oncogenes and tumor suppressor genes in aggregate samples compared to chicken cES. Analysis demonstrated that aggregates retained expression levels of oncogenes compared to cES. Tumor suppressors were either unchanged or elevated compared to chicken ES. [Figure 7A] Differentiation of aggregate cells into adipose-accumulating cells. (A) a. Initial culture of undifferentiated aggregates. b. Mature aggregates of aggregates 3 days in culture before addition of differentiation medium. c. Change in morphology of aggregate cells after 4 days of treatment with adipogenic differentiation medium. d. Single aggregate cell after 4 days of differentiation medium treatment, demonstrating accumulation of droplets within the cytoplasm of the cell. (B) Verification of lipid droplet accumulation in differentiated aggregates. a. Differentiated aggregates stained positive for BODIPY staining (evidence of lipid accumulation). b. Higher magnification of differentiated adipose-accumulating cells. [Figure 7B] Differentiation of aggregate cells into adipose-accumulating cells. (A) a. Initial culture of undifferentiated aggregates. b. Mature aggregates of aggregates 3 days in culture before addition of differentiation medium. c. Change in morphology of aggregate cells after 4 days of treatment with adipogenic differentiation medium. d. Single aggregate cell after 4 days of differentiation medium treatment, demonstrating accumulation of droplets within the cytoplasm of the cell. (B) Verification of lipid droplet accumulation in differentiated aggregates. a. Differentiated aggregates stained positive for BODIPY staining (evidence of lipid accumulation). b. Higher magnification of differentiated adipose-accumulating cells. [Figure 8] Differentiation of aggregates into fibroblasts. After treatment, aggregates attached to the plate surface (top left), followed by disintegration of the aggregates (top right) and adoption of a monolayer of epithelial fibroblasts. [Figure 9] Rhythmic contractions of the aggregates undergo muscle differentiation: contractions occur once every 1.5-2 seconds. [Figure 10] Culture of microtissues in serum-free defined medium. Representative example of the ability of cells to adapt to different culture conditions. The clones shown were cultured and grown in GRO-I® (Merck) medium supplemented with Ex-Cell® (Merck) chemically defined hydrolysate. [Figure 11]Expression of stem cell and ECM markers in microtissues is shown. Oct4, Nanog and Lin28 are lost due to the differentiation process. However, cells gained expression of SSEA3 (upper panel). Furthermore, enhanced expression of ECM and collagen family members in microtissues is found positive for Col2A1 and Col9a2, as well as high abundance of ECM markers laminin and HSPG. [Figure 12A] Muscle differentiation within defined regions of the microtissue. a. Naive aggregates grown in suspension. b. Transfer of the aggregates to differentiation conditions leads to the appearance of cell protrusions in a reproducible manner. c&d. Initiation of muscle differentiation (as shown by MyHC staining). e. Formation of muscle differentiation areas in several locations of the microtissue. [Figure 12B] Muscle differentiation within defined regions of the microtissue. a. Naive aggregates grown in suspension. b. Transfer of the aggregates to differentiation conditions leads to the appearance of cell protrusions in a reproducible manner. c&d. Initiation of muscle differentiation (as shown by MyHC staining). e. Formation of muscle differentiation areas in several locations of the microtissue. [Figure 12C] Muscle differentiation within defined regions of the microtissue. a. Naive aggregates grown in suspension. b. Transfer of the aggregates to differentiation conditions leads to the appearance of cell protrusions in a reproducible manner. c&d. Initiation of muscle differentiation (as shown by MyHC staining). e. Formation of muscle differentiation areas in several locations of the microtissue. [Figure 12D] Muscle differentiation within defined regions of the microtissue. a. Naive aggregates grown in suspension. b. Transfer of the aggregates to differentiation conditions leads to the appearance of cell protrusions in a reproducible manner. c&d. Initiation of muscle differentiation (as shown by MyHC staining). e. Formation of muscle differentiation areas in several locations of the microtissue. [Figure 12E] Muscle differentiation within defined regions of the microtissue. a. Naive aggregates grown in suspension. b. Transfer of the aggregates to differentiation conditions leads to the appearance of cell protrusions in a reproducible manner. c&d. Initiation of muscle differentiation (as shown by MyHC staining). e. Formation of muscle differentiation areas in several locations of the microtissue. [Figure 13A]Expression of muscle progenitor markers in avian stem cell microtissues cultured in suspension. [Figure 13B] Expression of muscle progenitor markers in avian stem cell microtissues cultured in suspension. [Figure 14A] A. Formation of microtissues in suspended floating aggregate cultures. Protrusions emerge from aggregates with elevated expression of MyHC (myosin heavy chain) and show a clear muscle identity. B. Maturation of muscle fibers differentiated from microtissues in the adherent state. Higher magnification of CB. [Figure 14B] A. Formation of microtissues in suspended floating aggregate cultures. Protrusions emerge from aggregates with elevated expression of MyHC (myosin heavy chain) and show a clear muscle identity. B. Maturation of muscle fibers differentiated from microtissues in the adherent state. Higher magnification of CB. [Figure 14C] A. Formation of microtissues in suspended floating aggregate cultures. Protrusions emerge from aggregates with elevated expression of MyHC (myosin heavy chain) and show a clear muscle identity. B. Maturation of muscle fibers differentiated from microtissues in the adherent state. Higher magnification of CB. [Figure 15A] A. Differentiation of microtissues into mature muscle cells. (a)-(c): Expression of troponin-T indicates commitment to the cardiomyocyte lineage. B: Transfer of cells to adherent conditions promotes muscle fiber maturation and elongation. [Figure 15B] A. Differentiation of microtissues into mature muscle cells. (a)-(c): Expression of troponin-T indicates commitment to the cardiomyocyte lineage. B: Transfer of cells to adherent conditions promotes muscle fiber maturation and elongation. [Figure 16] Extensive accumulation of lipid droplets in adipocytes within differentiated microtissues following exposure to high doses of oleic acid (upper panel) and combined oleic and linoleic acid treatment (lower panel). [Figure 17A] Differentiation of the microtissues into mutually exclusive populations of adipocytes and muscle cells. (a) Troponin-T staining highlights differentiation into muscle cells at the microtissue periphery, while bodipy staining demonstrates the formation of an adipose-accumulating cell population within the microtissue core. The two populations are mutually exclusive. (b) Magnification of the squared area in panel a. [Figure 17B] Differentiation of the microtissues into mutually exclusive populations of adipocytes and muscle cells. (a) Troponin-T staining highlights differentiation into muscle cells at the microtissue periphery, while bodipy staining demonstrates the formation of an adipose-accumulating cell population within the microtissue core. The two populations are mutually exclusive. (b) Magnification of the squared area in panel a. [Figure 18A] Parallel differentiation of microtissues into distinct adipocyte and muscle cell populations: (a) Typical arrangement of muscle cells skeletal muscle within the microtissue population (phalloidin staining). (b) Nuclear staining of muscle fibers highlights the formation of multinucleated fibers by microtissue cells. (c) Typical arrangement of fat-storing cells (adipocytes) microtissue population (phalloidin staining). (d) Lipid staining (BODIPY) growth highlights fat accumulation within the differentiating microtissue. (e) Parallel differentiation of microtissue cells into adipocyte and muscle within the same culture highlights that adipocyte and muscle cells are derived from mutually exclusive populations. [Figure 18B] Parallel differentiation of microtissues into distinct adipocyte and muscle cell populations: (a) Typical arrangement of muscle cells skeletal muscle within the microtissue population (phalloidin staining). (b) Nuclear staining of muscle fibers highlights the formation of multinucleated fibers by microtissue cells. (c) Typical arrangement of fat-storing cells (adipocytes) microtissue population (phalloidin staining). (d) Lipid staining (BODIPY) growth highlights fat accumulation within the differentiating microtissue. (e) Parallel differentiation of microtissue cells into adipocyte and muscle within the same culture highlights that adipocyte and muscle cells are derived from mutually exclusive populations. [Figure 18C] Parallel differentiation of microtissues into distinct adipocyte and muscle cell populations: (a) Typical arrangement of muscle cells skeletal muscle within the microtissue population (phalloidin staining). (b) Nuclear staining of muscle fibers highlights the formation of multinucleated fibers by microtissue cells. (c) Typical arrangement of fat-storing cells (adipocytes) microtissue population (phalloidin staining). (d) Lipid staining (BODIPY) growth highlights fat accumulation within the differentiating microtissue. (e) Parallel differentiation of microtissue cells into adipocyte and muscle within the same culture highlights that adipocyte and muscle cells are derived from mutually exclusive populations. [Figure 18D]Parallel differentiation of microtissues into distinct adipocyte and muscle cell populations: (a) Typical arrangement of muscle cells skeletal muscle within the microtissue population (phalloidin staining). (b) Nuclear staining of muscle fibers highlights the formation of multinucleated fibers by microtissue cells. (c) Typical arrangement of fat-storing cells (adipocytes) microtissue population (phalloidin staining). (d) Lipid staining (BODIPY) growth highlights fat accumulation within the differentiating microtissue. (e) Parallel differentiation of microtissue cells into adipocyte and muscle within the same culture highlights that adipocyte and muscle cells are derived from mutually exclusive populations. [Figure 18E] Parallel differentiation of microtissues into distinct adipocyte and muscle cell populations: (a) Typical arrangement of muscle cells skeletal muscle within the microtissue population (phalloidin staining). (b) Nuclear staining of muscle fibers highlights the formation of multinucleated fibers by microtissue cells. (c) Typical arrangement of fat-storing cells (adipocytes) microtissue population (phalloidin staining). (d) Lipid staining (BODIPY) growth highlights fat accumulation within the differentiating microtissue. (e) Parallel differentiation of microtissue cells into adipocyte and muscle within the same culture highlights that adipocyte and muscle cells are derived from mutually exclusive populations. [Figure 19] 1 shows a schematic diagram of a process according to some embodiments of the present invention. The embodiments of the present invention also refer to parts of the process, such as 1-7, 8b-9a, 9b-a, 8b, 9b, etc. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] The present invention, in some embodiments thereof, relates to methods of producing microtissues for the cultivated meat industry.

[0056] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by way of examples, as the invention is capable of other embodiments or of being practiced or carried out in various ways.

[0057] The self-renewal capacity of embryonic stem cells (ESCs) and chicken ESCs in particular makes them highly applicable to the cultivated meat industry.

[0058] The novel platform is designed for optimal meat production. The platform offers several unique features that make it well positioned to serve as a powerful industrial manufacturing tool for the meat and food industry with an inexhaustible non-genetically modified (GMO) production source. The cells are naturally immortal and have been shown to retain stable and consistent growth and characteristics over 100 passages. This eliminates the need to replenish animal cell banks and completely removes animals from the production process. The platform is based on the addition of xeno-free factors and is completely devoid of animal components other than the cells themselves. The platform is highly proliferative in nature and supports a continuous production process enabling a cost-efficient and high-yield manufacturing process.

[0059] Efficient production platform - aggregates and microtissue lines showed rapid self-renewal capability and could reach doubling times as fast as 8 hours. Cell densities in these cultures exceed 10^8 cells / ml. These lines retain high plasticity that supports the maturation process to form native microtissue structures that express a heterogeneous composition of fat, muscle and connective tissue cells in a controllable manner. Industry standard tests showed high similarity between the nutritional composition and profile of cultured and conventionally produced chicken meat. The platform can be used to produce raw materials or to construct products with organoleptic properties comparable to conventionally produced meat. Industry standard assays as well as multiple taste panels show high similarity to conventionally produced chicken meat in terms of flavor profile and consumer experience.

[0060] Thus, according to one aspect of the invention there is provided a method of producing pluripotent stem cell aggregates comprising the steps of: (a) providing a non-human pluripotent stem cell line of non-human pluripotent stem cells grown under conditions of matrix adherence; (b) gradually removing the matrix-adherent non-human stem cell line to obtain aggregates comprising non-human pluripotent stem cells, wherein the non-human pluripotent stem cells of the aggregates remain undifferentiated and exhibit a doubling time of 12 hours or less for more than 60 passages, are capable of differentiating into muscle, fat and connective tissue upon induction of differentiation, and exhibit lower cell to cell adhesion than embryoid bodies (EBs), as determined by reduced expression of adhesion molecules selected from the group consisting of COL6A2, CD44, COL6A1, ANXA1, ANXA2 and S100A11, as compared to said EBs; Including, A method is provided, wherein steps (a) and (b) are carried out in the presence of a growth factor.

[0061] As used herein, "pluripotent stem cells" refer to non-human cells that can differentiate into all three embryonic germ layers, i.e., ectoderm, endoderm and mesoderm, or can remain in an undifferentiated state. Pluripotent stem cells include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS).

[0062] The phrase "embryonic stem cells" refers to embryonic cells that can differentiate into cells of all three embryonic germ layers (i.e., endoderm, ectoderm and mesoderm) or can remain undifferentiated. The phrase "embryonic stem cells" can include cells obtained from embryonic tissue (e.g., blastocyst) formed after conception prior to implantation of the embryo (i.e., pre-implantation blastocysts), expanded blastocyst cells (EBCs) obtained from blastocysts at the post-implantation / pre-gastrulation stage (see WO 2006 / 040763), embryonic germ (EG) cells obtained from fetal reproductive tissue at any time during conception, and cells derived from unfertilized eggs stimulated by parthenogenesis (parthenogenesis).

[0063] The main source of avian embryonic stem cells is the unincubated fertilized egg (day 0). At this stage, the embryo consists of 60-100K pluripotent cells locked in an arrested state. The arrested stage is important to allow hens to synchronize the hatching of several eggs that had been laid on different days. In vitro proliferation of these cells occurs upon incubation at 39 °C (e.g., Pokharel, N et al. Poult Sci. 2017 Dec 1;96(12):4399-4408. doi:10.3382 / ps / pex242. PMID:29053871).

[0064] Induced pluripotent stem cells (iPS; embryonic-like stem cells) are cells obtained by dedifferentiation of adult somatic cells that have been rendered pluripotent (i.e., capable of differentiating into the three embryonic germ cell layers, i.e., endoderm, ectoderm and mesoderm). According to some embodiments of the present invention, such cells are obtained from differentiated tissues (e.g., somatic tissues such as skin) and undergo dedifferentiation by genetic manipulation that reprograms the cells to acquire embryonic stem cell characteristics. According to some embodiments of the present invention, induced pluripotent stem cells are formed by inducing the expression of Oct-4, Sox2, Kfl4 and c-Myc in somatic stem cells.

[0065] As used herein, "aggregates" refer to groups of proliferative, non-differentiated, i.e., pluripotent, cells that are attached to one another via secretion of adhesion molecules (e.g., ECM). Sizes can be 30-1000 mm, e.g., 50-500 um, e.g., 30-100 um, 100-500 um, 100-400 um, 200-500 um, 300-400 um, 100-200 um, 50-200 um, 500-1000 um, 700-1000 um. Where sizes are given throughout this specification, it will be understood that the size refers to the average size in a population of aggregates or microtissues.

[0066] Markers: Oct4+, Lin28+, SSEA1+, SSEA4-, ENS1+, Tra-1-60+.nanog+. According to a particular embodiment, the markers are Oct4+, Lin28+, SSEA4-, ENS1+, Tra-1-60+.Nanog+.

[0067] According to certain embodiments, the aggregates are grown in the presence of growth factors (e.g., IGF1, SCF, IL6, IL6Rα, LIF hLIF, combinations thereof, or additionally or alternatively, IWR1, FGF2, etc.) or signaling inhibitors such as inhibitors of Rho (e.g., Y27632), MEK, GSK3, FGFR3, N2B27-3, or as further exemplified below.

[0068] According to certain embodiments, about 90%-100% of the cells in the aggregates are pluripotent stem cells.

[0069] As mentioned, the cells are non-human pluripotent stem cells.

[0070] According to certain embodiments, the non-human pluripotent stem cells are livestock pluripotent stem cells.

[0071] According to certain embodiments, the non-human pluripotent stem cells are selected from the group consisting of avian pluripotent stem cells, bovine pluripotent stem cells, porcine pluripotent stem cells, caprine pluripotent stem cells, ovine pluripotent stem cells, shrimp pluripotent stem cells and fish pluripotent stem cells.

[0072] As used herein, the term "avian" refers to any species, subspecies or variety of organisms of the taxonomic Class Ayes, including, but not limited to, organisms such as chickens, turkeys, ducks, geese, quails, pheasants, parrots, finches, hawks, crows, and ratites such as ostriches, emus and cassowaries. The term includes the various known strains of Gallus gallus (chicken), such as White Leghorn, Brown Leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island, Australorp, Cornish, Minorca, Amrox, California Gray, Italian Partidge-colored, as well as turkeys, pheasants, quails, ducks, hens, guinea fowl, scoopers, ostriches and other poultry strains commonly raised in commercial quantities.

[0073] In certain embodiments, the avian cells are chicken cells.

[0074] In one example, the cells are derived from the avian embryo-derived stem cell lines EB14 (chicken) or EB66 (duck) (WO2005042728).

[0075] According to one embodiment, the cells throughout the method are non-genetically modified.

[0076] In one embodiment, the pluripotent stem cells are a stem cell line.

[0077] Pluripotent stem cells are naturally adherent and therefore grow under conditions of cell adhesion, also called two-dimensional culture (2D).

[0078] According to certain embodiments, 2D culture refers to growth on a two-dimensional matrix (without a feeder layer) or on feeder cells.

[0079] Thus, pluripotent stem cells can be cultured in the presence of a medium, with the aid of an extracellular matrix (e.g., gelatin, fibronectin, MatrigelRTM The cells can be grown (proliferated) on a solid surface such as a cellular surface (e.g., erythrocyte, endothelial cell, or laminin).

[0080] According to a particular embodiment, the surface is gelatin.

[0081] According to certain embodiments, the cells are grown on a feeder layer.

[0082] According to a particular embodiment, the feeder cells are mouse embryonic fibroblasts (MEFS).

[0083] According to certain embodiments, the cells are cultured in the presence of the factors and optionally serum (e.g., fetal bovine serum, horse serum and / or fish serum from trout) or serum replacements (e.g., yeast or vegetable hydrolysates, e.g., soy). Other factors, e.g., sodium pyruvate, sodium selenite, amino acids, 2-mercaptoethanol, may be included at this stage. The cells are grown and passaged every 24-72 hours.

[0084] According to certain embodiments, the growth factor is selected from the group consisting of IGF-1, IL6, sIL6Rα, hLIF and stem cell factor (SCF).

[0085] According to certain embodiments, the growth factors include IGF-1, IL6, sIL6Rα, hLIF and stem cell factor (SCF).

[0086] According to some embodiments of the invention, when the cells reach passages 3-5, they are gradually removed of material attachment (e.g., feeder layer) and grown for several passages to select stable feeder-free clones. According to some embodiments, factors are still present at this stage.

[0087] In particular, after the feeder layer harvesting stage, the cells tend to form less compact stem cell colonies composed of large nucleated cells, since they are not bound by fibrous cells (Fig. 1c-e). According to certain embodiments, at this stage, the cells show an expected doubling time of about 24 hours per cycle. This stage is also referred to as "gradually removing matrix attachments from non-human stem cell lines".

[0088] As used herein, "gradually remove" refers to depletion from matrix attachments (not from GFs).

[0089] To do this, the cells are gradually adapted to grow in suspension rather than as adherent cells.

[0090] As used herein, the phrase "suspension culture" refers to a culture in which pluripotent stem cells are suspended in medium rather than attached to a surface.

[0091] Thus, the cultures of the invention are "matrix attachment deficient" in that pluripotent stem cells can grow without attachment to external substrates such as components of the extracellular matrix, glass microcarriers or beads.

[0092] According to some embodiments, the cells are gradually transferred from the adhesive surface (e.g., one containing an adhesive matrix, e.g., gelatin, laminin, fibronectin, poly-L-lysine) and a slight shaking is imposed (e.g., 50-100 rpm) and, optionally, mechanical dissociation of the aggregates is performed. The shaking may be gradually increased with each passage. According to certain embodiments, by successive selection over a period of about 2-3 months, the cells are prompted to downregulate different adhesion molecules while expressing others, allowing the formation of 3D loose raspberry-like aggregates over time, with a clear definition of each cell that composes the aggregates, in contrast to the structure of embryoid bodies. At this stage, the cells become very stable and the doubling time is reduced to 10-20 hours per cycle, e.g., 18-20 hours, 10-12 hours, 12-14 hours, 12-16 hours, 10-16 hours, 12-18 hours, as in the case of avian embryonic stem cells.

[0093] According to certain embodiments, the aggregates are those of the aggregates that form cell lines. Thus, following adaptation of the cells to growth in suspension, the cells are adapted to continue rapid growth in a reproducible manner, such as in a stirred bioreactor environment, to ensure the cells are capable of being suitable for industrial scale-up. For this, clones are tested for the generation of cell lines that grow as aggregate cells at high proliferation rates, optionally with high agitation (tip speed 200-400 rpm) in a stirred bioreactor, while maintaining the integrity and stem cell properties of the aggregates. Following this process of optimization, according to some embodiments of the invention, several cell lines are produced that possess all the desired properties, and these cells are named "SMCMC". These cells exhibit the desired morphology, differentiation potential, and doubling times of 10-12 hours per cycle, with some growth conditions exhibiting 8 hours per cycle (Figure 3).

[0094] Such aggregates forming a cell line can be stored in a cell bank.

[0095] According to one aspect of the present invention, a pluripotent stem cell aggregate is provided.

[0096] According to certain embodiments, the aggregates are obtainable by the methods described herein.

[0097] According to a particular embodiment, the aggregates exhibit an average diameter between 80 and 120 μm.

[0098] According to certain embodiments, the non-human pluripotent stem cells of the aggregates exhibit alkaline phosphatase expression.

[0099] According to certain embodiments, the non-human pluripotent stem cells of the aggregates exhibit telomerase gene expression.

[0100] According to certain embodiments, the non-human pluripotent stem cells of the aggregates are SSEA4-, LIN28+, ENS-1+, NANOG+, OCT4+, and TRA-I-60+, e.g., as determined at the RNA level.

[0101] According to certain embodiments, the human pluripotent stem cells of the aggregates do not exhibit oncogenic transformation.

[0102] According to one aspect of the present invention, there is provided a pluripotent stem cell aggregate comprising non-human pluripotent stem cells, wherein the non-human pluripotent stem cells of the aggregate exhibit a doubling time of 12 hours or less in an undifferentiated state for more than 60 passages, and upon induction of differentiation are capable of differentiating into muscle, fat and connective tissue, and exhibit lower cell-cell adhesion than embryoid bodies (EBs), as determined by reduced expression of adhesion molecules selected from the group consisting of COL6A2, CD44, COL6A1, ANXA1, ANXA2 and S100A11, as compared to said EBs.

[0103] According to certain embodiments, the pluripotent stem cell aggregates comprise: (i) average diameter of 80–120 μm; (ii) the non-human pluripotent stem cells of the aggregates exhibit alkaline phosphatase expression; (iii) the aggregate non-human pluripotent stem cells exhibit telomerase gene expression; (iv) the non-human pluripotent stem cells of the aggregates are SSEA4-, LIN28+, ENS-1+, NANOG+, OCT4+, and TRA-I-60+; (v) exhibiting the organoleptic properties of natural meat products; (vi) the aggregates exhibit gene expression approximately identical to that of the stem cell line from which they were derived, with the exception of expression levels of genes associated with cell motility and migration; At least one of the following is further illustrated:

[0104] According to certain embodiments, the aggregates exhibit combinations of i+iiI+ii+iii, i-iv, iv, i-vi, ii-iii, ii-iv, ii-v, ii-vi, iii-iv, iii-v, iii-vi, iv-v, iv-vi, v-vi.

[0105] According to certain embodiments, the non-human pluripotent stem cells are selected from the group consisting of avian pluripotent stem cells, bovine pluripotent stem cells, porcine pluripotent stem cells, caprine pluripotent stem cells, ovine pluripotent stem cells, shrimp pluripotent stem cells and fish pluripotent stem cells.

[0106] According to certain embodiments, the avian pluripotent stem cells are selected from the group consisting of chicken pluripotent stem cells and duck pluripotent stem cells.

[0107] According to a particular embodiment, the avian pluripotent stem cells are chicken pluripotent stem cells.

[0108] Exemplary aggregates according to some embodiments of the present invention are shown in Figures 4-6 (avian, eg, chicken).

[0109] According to certain embodiments, the aggregates exhibit gene expression that is approximately the same as that of the stem cell line from which they were derived, with the exception of expression levels of genes associated with cell motility and migration, e.g., as determined at the RNA level (see Examples section) or protein level (e.g., immunostaining).

[0110] According to certain embodiments, the cells of the aggregates and microtissues maintain a normal karyotype.

[0111] According to certain embodiments, the above covers steps 1 to 7 of Figure 19. According to certain embodiments, these steps are carried out in the presence of serum, although as mentioned above, serum can be replaced with serum substitutes or other substitutes such as yeast or vegetable hydrolysates.

[0112] Cultivated meat production places a particular emphasis on the media used to culture the cells. Tissue culture media have traditionally been designed for other purposes (e.g., research, pharmaceutical, clinical production, etc.) and therefore carry several caveats from the perspective of cultivated meat production. The cost of the culture media is generally the largest economic burden for cultivated meat production. Furthermore, most available culture media contain fetal calf (or other animal-derived) serum (FCS). The use of FCS has the inherent drawback of being highly batch-to-batch variability and undefined (in terms of chemical composition). Furthermore, the use of such materials in cultivated meat production will most likely shy away from public acceptance due to the problematic way in which it is produced. To overcome these obstacles, the inventors set out to adapt the aggregates and microtissues (as described further herein below) to grow in serum-free media without compromising the quality, safety, and inherent characteristics of the microtissues.

[0113] Figure 19 shows such options.

[0114] To achieve this, the microtissues (as described further below) or the aggregates after they have been formed (under shaking conditions) are adapted to grow in serum-free medium.

[0115] Adaptation to serum-free medium is performed after growing ES cells as aggregates in shake flasks (Figure 19, step 7b) either by further growth in flasks using serum-free medium (Figure 19, step 7b) and then transfer to stirred bioreactors (Figure 19, step 8b) or by direct seeding in bioreactors using serum-free medium (Figure 19, step 8b). Adaptation to serum-free medium is performed by collecting highly proliferative aggregate populations and introducing them into serum-free medium (Figure 19, step 7b). Gradual adaptation is performed by decreasing the percentage of serum, e.g. FBS, in the serum-free medium over time (e.g. 5%-2.5%-1%, until complete recovery). In most cases, the period of gradual adaptation is 2-4 weeks. Another possibility to generate serum-free (SF) cultures for large-scale production is to transfer aggregates directly into serum-free medium. In this procedure, cells with the ability to survive and adapt quickly to the changed environment are collected. The length of such a process is about 2 weeks. According to some embodiments of the present invention, either possibility is concluded by the generation of a cell bank for future use (step 8a in FIG. 19).

[0116] Thus, as shown below, the cells were adapted to new medium in the absence of serum for about 14 days, resulting in the recovery of the original 12-hour doubling time and retaining a high level of cell viability (Figure 10). Similarly, we were also able to adapt the cells to grow in other combinations of basal media supplemented with yeast, plant peptones and hydrolysates. Among the mixtures successfully used to adapt the cells to serum-free media, we were able to identify DMEM (high glucose) supplemented with Ex-Cell lysate, DMEM / F12 supplemented with Ex-Cell lysate, DMEM (low glucose) supplemented with Ex-Cell lysate, and DMEM (high glucose) supplemented with a combination of soy and yeast lysates produced by the KERRY group. Successful lysates tested with this process were either full or partial combinations of these four products as follows: Hypep 1510 (ID: S-2048780, Item: U1-5X99023), SHEFF-VAX PLUS ACF (ID S-2048778, U1-5X00484.K1G), SHEFF-VAX PF ACF (ID: S-2048777, U1-5X01143.K1G), SHEFF-VAX PLUS PF ACF VP (ID: S-2048776, U1-5X01090). We have also successfully adapted microtissues to routinely used basal media (DMEM HG / LG, DMEM / F12, RPMI 1640) in combination with soy and yeast hydrolyzing enzymes manufactured by FUJIFILM-IRVINE Scientific (Ultrafiltered Soy Hydrolysate #IR-96857E; Ultrafiltered Yeast Hydrolysate #IR-96863E). Additionally, microtissues were adapted to grow in basal media (DMEM HG / LG, DMEM / F12, RPMI 1640) supplemented with various combinations of DIFCO Select Soytone (#15ABP196), Bacto Yeast Extract (#15ABP197), BBL Phytone Peptone (#15ABP195), BBL Yeast Extract (#15ABP202), Bacto Malt Extract (15ABP201) and Bacto TC yeastolate (#15ABP163).For this purpose, yeast-derived lysates were combined with one or several plant-derived lysates. In all experiments, the medium was also supplemented with ethanolamine (20ng / L), insulin (100ug / L), selenium (50ng / L) and transferrin (55ug / L). The medium was also supplemented with 1X MEM NEAA (Biological industries, 01-340-1B), 2mM L-alanine / L-glutamine (Biological industries-03-022-1B) and 1mM sodium pyruvate (Biological industries). The results presented here highlight the ability of the microtissues to adapt multiple combinations based on basal media supplemented with soy and plant hydrolysates as a replacement for serum in the culture medium.

[0117] It will be understood that not all steps are required to practice an embodiment of the invention, thus, for example, aggregates may be provided from a cell bank or commercial vendor and further used in the food industry as described below.

[0118] Therefore, regardless of production, the aggregates can be used in food production as such, or can be further subjected to further development processes to generate micro-tissues.

[0119] Thus, according to one aspect of the invention there is provided a method of producing microtissues comprising one or more cell types, comprising the steps of: (a) forming aggregates in suspension without matrix attachment comprising non-human pluripotent stem cells, wherein the non-human pluripotent stem cells of the aggregates are undifferentiated and exhibit a doubling time of 12 hours or less for more than 60 passages, are capable of differentiating into muscle, fat and connective tissue upon induction of differentiation, grow in the presence of growth factors and exhibit lower cell-cell adhesion than embryoid bodies (EBs) as determined by reduced expression of adhesion molecules selected from the group consisting of COL6A2, CD44, COL6A1, ANXA1, ANXA2 and S100A11, as compared to said EBs; (b) producing microtissues by transferring the aggregates to a bioreactor and growing them in suspension in the absence of growth factors and without matrix attachment; A method is provided that includes:

[0120] According to an alternative embodiment, there is provided a method of producing microtissues comprising one or more cell types, comprising: (a) providing a suspension culture comprising aggregates comprising non-human pluripotent stem cells, wherein the non-human pluripotent stem cells of the aggregates remain undifferentiated and exhibit a doubling time of 12 hours or less for more than 60 passages, are capable of differentiating into muscle, fat and connective tissue upon induction of differentiation, grow in the presence of growth factors, and exhibit lower cell-cell adhesion than embryoid bodies (EBs) as determined by reduced expression of adhesion molecules selected from the group consisting of COL6A2, CD44, COL6A1, ANXA1, ANXA2 and S100A11; (b) producing microtissues by transferring the aggregates to a bioreactor and growing them in suspension in the absence of growth factors and without matrix attachment; A method is provided that includes:

[0121] At this stage, the aggregates are transferred to a bioreactor in the absence of growth factors, for example as described above.

[0122] As used herein, a bioreactor refers to a vessel, device or system designed to grow cells, aggregates or tissues in a cell culture context. Such bioreactors are described in Popovic et al. BIOTECHNOLOGY-Bioreactoes and Cultivation Systems for Cell and Tissue Culture-MK Popovic, Ralf Portner Encyclopedia of Life Support Systems (EOLSS) and further herein below and in the Examples section below.

[0123] The selection of culture equipment for production is based on scale. For large-scale production, it is preferable to use dedicated equipment. Continuous cell culture systems are reviewed in Furey (2000) Genetic Eng.News 20:10. Suitable bioreactors that can be used according to the present teachings include, but are not limited to, Sartorius Biostat STR, Sartorius Biostat BDCU, Thermo Fisher HyPerforma DynaDrive SUB, Eppendorf Bioflo 510.

[0124] Such systems can also be used for the production of microtissues when used in the food industry.

[0125] As used herein, the term "microtissue" refers to mesodermal cells that are grown in the absence of growth factors or signaling inhibitors and are either fully non-differentiated or terminally differentiated.

[0126] As used herein, "in the absence of growth factors" refers to maturation medium in which growth factors (over and above those in serum, if present) are not added that cause the cells to lose important pluripotency markers while gaining mesodermal characteristics, including specific markers, and elevated expression of connective tissue proteins, e.g., compared to the cell aggregates of step 7 of Figure 19. Due to their early differentiation stage and the specific culture conditions in the bioreactor, these microtissues can still grow rapidly and extensively and propagate for at least 150 population doublings.

[0127] According to certain embodiments, growth in the bioreactor is controlled by defined set points of bioreactor running parameters such as agitation in the bioreactor and gassing at tip speeds ranging from 0.25 to 1.70 m / sec with an optimal tip speed of 0.85 m / sec. These parameters in turn control the size of the microtissues. The size of the microtissues is important for the ability to differentiate multiple cell types in a single microtissue.

[0128] According to certain embodiments, the microtissues are grown in serum-free medium, as described herein above.

[0129] According to another embodiment, the microtissues are grown in the presence of serum.

[0130] This maturation step (step 8 in Figure 19) can continue for extended periods of time, such as over 150 passages.

[0131] As used herein, "maturation" refers to a process that involves the withdrawal of GFs (i.e., the absence of growth factors, also called "factor free") that allows pluripotent stem cells to initiate mesodermal lineage commitment.

[0132] Various maturation protocols can be used, some of which are illustrated in the Examples section below: for example, maturation of bESCs in suspension in a monitored stirred bioreactor in factor-free medium (DMEM(HG) / 10-20% FBS, 10U / 1U penicillin / streptomycin, 2mM glutamine); or maturation of pESCs in suspension in a stirred bioreactor performed in factor-free medium (DMEM(HG) / 10-20% FBS, 10U / 1U penicillin / streptomycin, 2mM glutamine).

[0133] The process without the presence of growth factors can last, for example, from 7 days to 12 months, during which time the cells can be expanded, harvested, stored, or further differentiated into specific cells of interest (e.g., muscle, fat, or a combination thereof).

[0134] Thus, a microtissue is provided.

[0135] Thus, according to one aspect of the present invention there is provided a microtissue comprising one or more cell types, the microtissue having a diameter of 30-500 μm, and wherein the cells of the microtissue (approximately 90%) are NANOG-OCT4-LIN28-, SSEA3+.

[0136] According to some embodiments of the present invention, the microtissue exhibits organoleptic properties of natural meat products.

[0137] According to some embodiments of the invention, the one or more cell types include adipocytes and muscle cells.

[0138] Unless subjected to a specific differentiation protocol, the microtissues are fully undifferentiated microtissues and are not pluripotent.

[0139] As used herein, "non-fully differentiated microtissues" refers to aggregates of cells that contain mesodermal lineage multipotent cells, i.e., cells that have the ability to develop into different types of mesodermal tissues. The size of the microtissues can be 30-1000 μm, e.g., 50-300 uM (markers: Pax3+, Pax7+, MyoD+, SSEA4+, SSEA1-, oct4-), as determined at the RNA level (see, e.g., Examples section). According to certain embodiments, the aggregates of step 7 are smaller than the microtissues of steps 8b-9b of FIG. 19.

[0140] Such microtissues may be incorporated into the food industry itself or may be subjected to further differentiation processes.

[0141] Thus, at this stage, covered by step 8b of FIG. 19 illustrating a non-limiting embodiment of the present invention, non-fully differentiated microtissues can be harvested.

[0142] Alternatively, these microtissues can be subjected to further differentiation.

[0143] Thus, according to one aspect of the present invention there is provided a method of producing microtissues comprising one or more cell types of interest, comprising the steps of: (a) producing (not fully differentiated) microtissues; (b) subjecting the microtissue in suspension to differentiation conditions, thereby producing a microtissue containing one or more cell types of interest.

[0144] Specific protocols for differentiation are provided in the Examples section below, as well as in WO 2018 / 189738, which is incorporated by reference in its entirety.

[0145] According to a particular embodiment, the differentiated microtissue is NANOG-OCT4-LIN28-, SSEA3+.

[0146] Thus, "differentiated microtissues" refer to cellular aggregates consisting of cells differentiated into mature muscle (Troponin T+, Myosin+), adipocytes (oil-red and Bodipy stain positive) and collagen-secreting cells (COL9+COL1A+, COL2A+). Sizes can range from 30 to 5000 μm. See, for example, Figures 16-18A-E for exemplary embodiments.

[0147] According to a particular embodiment, the microtissues are between 30 and 500 μm in diameter.

[0148] As mentioned above, mature microtissues or aggregates can be subjected to differentiation using differentiation protocols well known in the art.

[0149] Alternatively, the differentiation protocol for the microtissues or aggregates can be a multi-step process.

[0150] Thus, according to one aspect of the present invention, there is provided a method for priming cells towards an adipocyte fate, comprising culturing pluripotent or multipotent stem cells in the presence of fatty acids at a concentration not exceeding 100 μM for a time sufficient to obtain stem cells having an adipocyte fate whilst retaining muscle cell and proliferative phenotypes.

[0151] As used herein, "adipocyte fate" refers to "preadipocytes," which are mesenchymal stem cells that have adopted a commitment to adipogenic fate prior to terminal differentiation. Preadipocytes tend to express Sca-1, Cd-90, and cd29. Preadipocytes also adopt a fibroblast morphology and usually show positive reaction for BODIPY staining around the nucleus.

[0152] As used herein, "priming" refers to achieving an adipocyte fate by culturing in the presence of low doses (e.g., less than 100 uM, e.g., 30-50 uM, 30-80 uM, 10-90 uM) of FAs (e.g., oleic acid, palmitic acid) or their precursors to drive cells into the adipocyte lineage.

[0153] In the presence of low levels of fatty acids, i.e. less than 100 uM, e.g. 30-50 uM, 30-80 uM, 10-90 uM, the cells acquire a muscle phenotype, as seen in Figure 9b. However, increasing the levels, i.e. above 100 uM, will drive the cells towards adipocytes.

[0154] "Priming" step: The priming step involves conditioning (e.g., avian) stem cells for growth in the presence of low doses of different fatty acids. In this process, we add a specific or several fatty acids to the cell culture medium at a final concentration not exceeding 100 uM (e.g., 10-100 uM or 10-50 uM).

[0155] Differentiation step: Priming the cells for adipogenic differentiation followed by terminal differentiation. Differentiation of the cells involves increasing the level of FAs, e.g. to 100uM or more, e.g. to 500uM, for up to 14 days, e.g. 1-7 days, 1-10 days, 5-10 days.

[0156] According to certain embodiments, the multipotent stem cells are adult stem cells.

[0157] According to certain embodiments, the adult stem cells are mesodermal.

[0158] According to certain embodiments, the mesodermal stem cells are mesenchymal stem cells.

[0159] According to certain embodiments, priming and / or differentiation is performed in suspension.

[0160] According to a particular embodiment, the culture for priming is for a period of 2 to 5 weeks.

[0161] According to a particular embodiment, the multipotent stem cells have been obtained by ex vivo maturation of pluripotent stem cells, as described above.

[0162] According to certain embodiments, ex vivo maturation is carried out in suspension in the absence of growth factors.

[0163] According to certain embodiments, the pluripotent and / or multipotent stem cells are in aggregates, optionally in aggregates of between 30 μm and 500 μm.

[0164] Priming the cells allows them to undergo terminal differentiation.

[0165] Thus, a method of cell differentiation is provided, comprising culturing stem cells having an adipocyte fate while retaining a proliferative phenotype in the presence of a fatty acid at a concentration of greater than 100 μM for a sufficient period of time to allow differentiation into adipocytes.

[0166] Combining the priming and differentiation protocols can result in the formation of microtissues containing both fat and muscle cells. Depending on the length of each step, the composition can be controlled with respect to the relative ratio of muscle cells to fat cells. If the process ends with the priming step, the composition is more muscle-like.

[0167] According to certain embodiments, either the aggregates or the microtissues exhibit the organoleptic properties of natural meat products.

[0168] As used herein, the term "organoleptic properties" refers to those aspects of a food product experienced by the consumer through the senses, including taste, texture, sight, smell, or touch.

[0169] For example, adipocytes can be used to provide non-meat foods that have a meaty taste and / or texture (eg, beef or chicken) when cooked or grilled.

[0170] Muscle or cardiomyocytes can be used to impart meat texture as well as meat taste (eg bitter / metallic) to non-meat foods.

[0171] Red blood cells can provide non-meat foods with the color of meat (eg, beef) as well as the taste of grilled meat (eg, beef or chicken).

[0172] Hepatocytes can not only enrich meat flavor but also give non-meat foods the color of meat (e.g., beef).

[0173] The term "meat" is meant to encompass any animal flash eaten as food (eg, beef, pork, poultry, fish, as well as additional examples provided below).

[0174] In this case the sensory attribute is that of meat, for example when referring to taste the sensory attribute may be umami.

[0175] As used herein, "nutritional properties" refers to compositions of meat that are valuable to a subject consuming it.

[0176] For example, muscle tissue is very high in protein, contains all of the essential amino acids, and is almost always a good source of zinc, vitamin B12, selenium, phosphorus, niacin, vitamin B6, choline, riboflavin, and iron. Some forms of meat are also high in vitamin K. Muscle tissue is very low in carbohydrates and contains no dietary fiber. Protein, vitamins, and minerals are available and generally consistent from nearly any meat source.

[0177] The fat content of meat can vary widely depending on the species and breed of the animal, how the animal was raised (including what it was fed), the anatomical part of the body, and the method of meat cut and cooking. Wild animals such as deer are typically leaner than livestock, so those concerned about fat content choose game meats such as venison. Fat deposits present with muscle fibers in meat tenderize the meat when cooked and improve flavor through chemical changes initiated by heat that allow protein and fat molecules to interact. Fat, when cooked with meat, makes the meat appear juicier. However, the nutritional contribution of fat is primarily calories, as opposed to protein. As the fat content increases, the nutritional contribution of meat decreases. In addition, there is cholesterol associated with fat around meat. Cholesterol is a lipid associated with the type of saturated fat found in meat.

[0178] Table A below compares the nutritional content of several types of meat (per 110 gr). Each type of meat has roughly the same content of protein and carbohydrates, but a very wide range in fat content.

[0179] [Table 1]

[0180] Other key nutrients and their sources are provided below. Vitamin B12 is found primarily in fish, meat, poultry and dairy products. Vitamin D is found in oily fish, eggs and dairy products. Docosahexaenoic acid (DHA) is an essential omega-3 fat found in fatty fish. Heme iron is found primarily in meat, especially red meat. Zinc is found primarily in animal protein sources such as beef, pork and lamb.

[0181] Thus, the improved / enhanced nutritional profile can be any of the protein, calories, fat, vitamins, minerals as listed herein.

[0182] As used herein, "enhancement," "increase," or "augmentation" refers to an addition of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% (e.g., by weight or score) of a sensory or nutritional attribute compared to the same food product without the aggregates or microtissues or in which a portion has been replaced with an equivalent amount of the aggregates or microtissues.

[0183] Thus, there is provided a food product (also referred to as a "foodstuff") comprising the microtissues or aggregates described herein. The food product may comprise meat and / or non-meat portions.

[0184] Additionally or alternatively, there is provided a method of producing a food product comprising combining the microtissues or aggregates described herein with an edible composition for human consumption, such as meat and / or non-meat parts.

[0185] According to a particular embodiment, the non-meat is a plant-origin substance(s).

[0186] According to certain embodiments, the non-meat is a non-plant origin material (e.g., mineral, synthetic material(s)).

[0187] According to particular embodiments, the non-meat is selected from the group consisting of material(s) of plant origin and material(s) of non-plant origin.

[0188] According to a particular embodiment, the food is a vegetarian food.

[0189] According to a particular embodiment, the food is a vegan food.

[0190] According to certain embodiments, the foodstuff comprises a meat substitute or is commonly consumed as a meat substitute (plant-based).

[0191] According to certain embodiments, the hybrid food does not include bodily fluids such as saliva, serum, plasma, mucus, urine, feces, tears, milk, and the like.

[0192] Hereinafter, the term "foodstuff" refers to any substance that has a food value. The term further refers to food ingredients before, during and after processing, food or parts thereof (e.g. coatings for edible articles such as schnitzels), by-product(s) and end products (e.g. sausages, minced meat, schnitzels, etc.). In the present invention, three families of foodstuffs are provided: foodstuffs and substances thereof, and aggregates and microtissues, and foodstuffs of plant origin.

[0193] According to one embodiment of the present invention, foodstuffs are final manufactured products consumed by human or non-human subjects, or aggregates and microtissues consumed by the food industry in the process of preparing food products.

[0194] Examples of meat substitutes: natural, traditional and commercially produced that are contemplated according to some embodiments of the present invention: Alpro and Provamel (both commonly known for their plant milk ranges) also offer different veggie meat substitutes; Beanfeast; Beyond Meat; Boca Burger; Falafel, traditional Middle Eastern bean fritters (thought to have been created by the ancient Copts as a meat substitute during Lent); Fistulina hepatica (commonly known as liver mushroom); Ganmodoki: a traditional Japanese tofu-based dish similar to a veggie burger; Gardein; Gardenburger; Glamorgan sausages; Goshen Alimentos; Green Slice vegetarian (organic and soy-free hot dogs and deli slices); Impossible Foods; jackfruit (a fruit whose flesh when cooked has a texture similar to pulled pork); Koya tofu (freeze-dried tofu common in seitan dishes that, when prepared, has a meat-like taste and texture); Laetiporus (also called chicken of the forest mushroom); Leaf protein concentrate; LightLife; Linda McCartney Foods; Hatakeshimeji (Lyophyllum decastes) (also known as the fried chicken mushroom); Meat extenders; Meatless; Mock duck; Morningstar Farms; Muscolo di grano (Wheat's muscle) (seitan prepared according to an Italian recipe); Nut roast; Oncom; Paneer (used in dishes such as paneer tikka); Quorn; Soy protein; Soy pulp (used in veggie burger patties and croquettes); Tempeh; Textured vegetable protein; Tofu (not traditionally seen as a meat substitute in Asia, but is widely used for that purpose in the Western Hemisphere): Tofurkey; Tofurky; Turtle Island Foods; vegetarian bacon; vegetarian hot dogs; veggie burger; veggie chicken patty; veggie chicken cutlet, breaded (schnitzel); Veggie Chicken Frankfurter (Hot Dog); Viana (one of the largest vegan food producers in Germany, offering a variety of veggie burgers, croquettes, sausages, mock minced meats, up to vegan doners, vegan gyros and deli slices for sandwiches); Wheat gluten.

[0195] All of the above examples are independent embodiments not necessarily associated with any particular vendor.

[0196] It will be understood that food refers to food or feed (animal feed).

[0197] The process of producing the food product may include any of the following: rising, kneading, extruding, molding, shaping, cooking, soaking, boiling, baking, baking, frying, and any combination thereof.

[0198] Also provided is a method of providing nutrition to a subject in need thereof, the method comprising providing a foodstuff as described herein to the subject.

[0199] According to certain embodiments, the subject is at risk of nutritional deficiency.

[0200] According to certain embodiments, the control is a healthy control (eg, not suffering from a nutrition / absorption related disease).

[0201] It will be understood that, according to certain embodiments, the indicated values ​​are given under optimal conditions for cell growth of that type and developmental stage as non-human pluripotent stem cells.

[0202] As used herein, the term "about" refers to ±10%.

[0203] The terms "comprises," "comprising," "includes," "including," "having" and their conjugations mean "including but not limited to."

[0204] The term "consisting of" means "including and limited to."

[0205] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, but only if the additional components, steps, and / or moieties do not materially change the basic and novel characteristics of the claimed composition, method, or structure.

[0206] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0207] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0208] Whenever a numerical range is given herein, it is meant to include any recited numbers (fractional or integer) within the given range. The phrases "ranging / ranges between" a first designated number and a second designated number and "ranging / ranges from...to" a first designated number are used interchangeably herein and are meant to include the first designated number and the second designated number and all fractional and integer numbers therebetween.

[0209] As used herein, the term "method" refers to manners, means, techniques and procedures for accomplishing a given task, including, but not limited to, manners, means, techniques and procedures that are known to those of skill in the art of chemistry, pharmacology, biology, biochemistry and medicine, or that are readily developed from known manners, means, techniques and procedures by those of skill in the art.

[0210] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination, or as appropriate in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0211] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[0212] Reference is now made to the following examples which, together with the above descriptions, illustrate certain embodiments of the invention in a non-limiting manner.

[0213] In general, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are fully explained in the literature, see, e.g., "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley&Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. New York (1998); methods described in U.S. Patents Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Culture of Animal Cells-A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", WHFreeman and Co., New see, York (1980); available immunoassays are widely described in the patent and scientific literature, e.g., U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Immunoassays," in "Synthesis" Gait, MJ, ed. (1984); "Nucleic Acid Hybridization" Hames, BD, and Higgins SJ, eds. (1985); "Transcription and Translation" Hames, BD, and Higgins SJ, eds. (1984); "Animal Cell Culture" Freshney, RI, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al.See, "Strategies for Protein Purification and Characterization--A Laboratory Course Manual," CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

[0214] Materials and Methods [Isolation of chicken embryonic stem cells (cESCs)] Isolation of cESCs from freshly laid chicken (Gallus) eggs was performed according to a previously published protocol (26). Briefly, the shells of disinfected fertilized eggs were carefully removed to separate the yolk. The yolk was placed in a 1500 mm dish and the embryo was placed directly on top. A small perforated ring of sterile 3M paper was placed directly on the embryo to allow the embryo to detach. The collected embryos were immersed in cold PBS by taking care to place the ring perpendicular to the liquid surface, preventing the embryo from detaching itself. Excess yolk was removed by gently shaking the embryo. The clarified embryos were placed in a fresh dish containing ES medium (see below). The collected embryos were plated into wells of a 6-well plate coated with gelatin and MEFs-2 embryos / well. The embryos were incubated at 39 °C under 10% CO2. The medium was changed every 24 h. The eggs were at stage X (day 0).

[0215] ES medium composition: DMEM / F-12, 10% fetal bovine serum, 1x MEM non-essential amino acid concentrate, 1mM sodium pyruvate, 10U / 1U penicillin / streptomycin, 2mM glutamine, 15uM β-mercaptoethanol, 5ng / mL IGF1, 1ng / mL SCF, 1ng / mL IL6, 1ng / mL sIL6Rα, 20ng / mL (1,000U / mL) hLIF.

[0216] [Adaptation and scale-up for growth in suspension] Adaptation of cESCs to growth in suspension included propagation of stem cells on MEFS seeded on gelatin-coated plates or by direct cell seeding on gelatin-coated plates. After 2-3 passages, the feeder layer, if present, was gradually removed. After establishment of a feeder-free population of cESCs, the cells were transferred to a non-treated culture plate and placed on an orbital shaker at 75 RPM. After several passages (30-90 passages) with daily mechanical disruption of the aggregates, the cells began to form loose aggregates of 10-5000 cells per aggregate, with a size of 30-300 microns. The aggregates were then transferred to 125 ml flasks supplemented with 50 ml of ES medium. After several passages (3-5), the cells were transferred to larger flasks (up to 2 L) and incubated at 2-6X10 6 A concentration of 100-300 million cells / ml was reached. Several clones were then tested in a stirred bioreactor setup to obtain cell lines with high proliferation rates while maintaining aggregate integrity and stem cell properties. After several passages, selected clones exhibited the desired morphology, differentiation potential, and reached a doubling time of 8-12 hours per cycle. At this point (following step 7 in Figure 19), the cells were ready to be inoculated into a stirred bioreactor containing factor-free medium by Sartorius, Eppendorf, Solaris, Thermofisher, New Brunswickm, or Applikon (AMBR 250 and Biostat A / B / B-DCU (2L, 5L / 10L, respectively)), which allows the cells to form microtissues and reach a population density of approximately 100-300 million cells / ml in factor-free medium under perfusion conditions. Factor-free medium composition: DMEM 10% fetal bovine serum, 10U / 1U penicillin / streptomycin, 2mM glutamine.

[0217] [Isolation, suspension adaptation and differentiation of bovine ESCs (bESCs)] (Isolation of bovine ESCs (according to Bogliotti et al. 2018)) Fertilized bovine embryos were placed on a feeder layer (gamma-irradiated MEFS) in 12-well plates and allowed to attach and grow in CTFR-bESC medium (custom-made mTESR*) supplemented with 20 ng / ml FGF2 and 2.5 uM IWR1. Cells were passaged every 48 hours using TrypLE (12563011; Gibco) and replated on MEFS in the presence of 10 uM of the rho-kinase inhibitor Y-27632. After 5 passages, the use of Y-27632 was exhausted. At this stage, cells were cryopreserved in a cell bank of MEF-dependent bESCs. Adaptation to suspension growth was performed as follows. MEF-dependent bESCs were transferred to MEF-free 6-well plates in either CTFR-bESC or mTESR medium supplemented with 20 ng / ml FGF2 and 2.5 uM IWR1 with or without the addition of Y-27632. The cell plates were placed in a shaker incubator at 37C / 5%CO2 with gentle agitation (75RPM). To inhibit cell attachment to the well surface, cells were gently pipetted manually every 24 hours. After 3-5 passages, cells were transferred to 10mM plates in a shaker incubator at 37C / 5%CO2 for 14 days with gentle agitation (75RPM). Media was replenished every 48 hours. After 14 days, cells were transferred to a 125ml shaker flask in a total volume of 30ml mTESR supplemented with 20ng / ml FGF2 and 2.5uM IWR1. Cells adapted to these conditions were cryopreserved in a cell bank of suspension adapted bESCs. Selection of aggregates was performed by replenishing media every 48 hours leaving 30-500um aggregates in the flask. The expansion steps of the bulk and differentiated populations were performed as follows: after gradually increasing the volume flask (up to 0.5 L for 2 L flasks) for up to 14 days, the cells were transferred for maturation in bioreactors. Expansion of bESCs in suspension in monitored stirred bioreactors in factor-free medium (DMEM(HG) / 10-20% FBS, 10U / 1U penicillin / streptomycin, 2 mM glutamine).For muscle differentiation: grow cells in large bioreactors supplemented with 5-50uM OA / LA with or without supplementation of FGF2 / 50uM hydrocortisone / 5% horse serum / Gsk inhibitor CHIR99021 (see brown et al. 2014, Adhikari et al., 2019, lian et al. 2013) for 7-21 days prior to harvest. For adipogenesis: grow cells in large bioreactors supplemented with 200-400uM OA / LA for 4-7 days prior to harvest.

[0218] (Isolation of porcine ESCs (Burrell et al., 2019))

[0219] Fertilized porcine embryos were placed on a feeder layer (gamma-irradiated MEFS) in a 12-well plate and incubated in N2B27-3i medium (1:1 ratio of DMEM / F12 / Neurobasal medium (21103049; Gibco), 0.5 × N2 (17502048; Thermo Fisher), 0.5 × B27 (17504044; Thermo Fisher), 1,000 U / mL human leukemia inhibitory factor (hLIF, L5283; Sigma)), 3i cocktail [30 mM CHIR99021 (GSK3 inhibitor), 40 mM PD0325901 (MEK inhibitor), 10 mM PD173074 (FGFR3 inhibitor)], 0.1 mM Cells were allowed to adhere and grow in 0.5× GlutaMAX (35050-061; Gibco), 1× MEM non-essential amino acids (MEM NEAA, 11140050; Gibco), 0.01% bovine serum albumin (BSA) (A7906; Sigma) supplemented with b-mercaptoethanol (M3148; Sigma).

[0220] Cells were passaged every 48 h using TrypLE (12563011; Gibco) and replicated on MEFS in the presence of 10 uM of the rho-kinase inhibitor Y-27632, and were depleted using Y-27632 after 5 passages (other factors were included in mTESR medium).

[0221] At this stage, cells were cryopreserved in a cell bank of MEF-dependent fESCs. (Adaptation of pESCs to suspension growth was performed as follows:) MEF-dependent pESCs were transferred to MEF-free 6-well plates in mTESR medium (85850; STEMCELL Technologies) with or without the addition of Y-27632. Plates were placed in a shaker incubator at 37C / 5% CO2 with gentle agitation (75 RPM). Cells were gently pipetted manually every 24 hours to inhibit cell attachment to the well surface. After 3-5 passages, cells were transferred to 10mM plates in a shaker incubator at 37C / 5% CO2 with gentle agitation (75 RPM) for 14 days. Medium was replenished every 48 hours. After 14 days, cells were transferred to a 125 ml shaker flask in a total volume of 30 ml mTESR. Cells adapted to suspension conditions were cryopreserved in a cell bank of suspension-adapted pESCs. Selection for aggregate growth was performed by leaving large aggregates in the flask and replenishing the medium every 48 hours.

[0222] (Bulk pESC and differentiated populations were expanded as follows:) After gradually increasing the volume of flasks (up to 0.5 L for 2 L flasks) for up to 14 days, the cells were transferred for maturation in bioreactors. Growth of pESCs in suspension in stirred bioreactors was performed in factor-free medium (DMEM(HG) / 10-20% FBS, 10U / 1U penicillin / streptomycin, 2 mM glutamine). For muscle differentiation: growth of cells in large bioreactors supplemented with 5-50 uM OA / LA with or without supplementation of (FGF2 / 50 uM hydrocortisone / 5% horse serum) (see brown et al. 2014, Adhikari et al., 2019) for 7-14 days before harvest. For adipogenesis: growth of cells in large bioreactors supplemented with 200-400 uM OA / LA for an additional 4-7 days before harvest.

[0223] (Isolation of shark, tuna, salmon, herring, and sardine ESCs (according to Hong et al., 1996):) Fertilized embryos were collected after 6–8 h in vitro (blastulae) and trypsinized into a single cell suspension. The cell suspension was resuspended in 20 mM HEPES (pH 7.4), 10 U / 1 U penicillin / streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, 2 nM sodium selenite, 1 mM non-essential amino acids, 50 pM 2-mercaptoethanol, 10 ng / ml The cells were placed in gelatin-coated 6-well dishes in ESM medium (DMEM (HG) supplemented with bFGF, 10 ng / ml human recombinant LIF, 15% fetal bovine serum, and fish serum from trout (FS, 1%). The cells were expanded and passaged every 48-72 hours. At this stage, the cells are used for the generation of a cell bank of fESCs. (Adaptation to suspension growth was performed as follows:) fESCs were transferred to uncoated 6-well plates in ESM1 medium with or without the addition of Y-27632 (mTESR already contains other factors reported by the manufacturer). The cell plates were placed in a shaker incubator at 37C / 5% CO2 with gentle agitation (75 RPM). To inhibit cell attachment to the well surface, the cells were gently pipetted manually every 24 hours. After 3-5 passages, the cells were transferred to uncoated 6-well plates in ESM1 medium with or without the addition of Y-27632 (mTESR already contains other factors reported by the manufacturer). Cells were transferred to 10 mM plates in a shaker incubator (200 rpm) at 37C / 5% CO2 for 14 days. Medium was replenished every 48 hours. After 14 days, cells were transferred to a 125 ml shaker flask in a total volume of 30 ml mTESR. Cells adapted to suspension growth were cryopreserved in a cell bank of suspension adapted fESCs.

[0224] Selection of aggregates, performed by replenishing the medium every 48 h, leaves large aggregates in the flasks as described above. (Propagation of bulk and differentiated populations was performed as follows:) After gradually increasing the volume flask (up to 0.5 L for 2 L flasks) for up to 14 days, cells are prepared for maturation in bioreactors. Propagation of fESCs is performed in suspension stirred bioreactors in factor-free (DMEM(HG) / 10-20% FBS, 10U / 1U penicillin / streptomycin, 2 mM glutamine). For muscle differentiation: growth of cells in large bioreactors supplemented with 5-50uM OA / LA with or without supplementation of FGF2 / 50uM hydrocortisone / 5% horse serum (see brown et al. 2014, Adhikari et al., 2019) for 7-14 days prior to harvesting. For adipogenesis: Grow cells in large bioreactors supplemented with 200-400uM OA / LA for 4-7 days prior to harvest.

[0225] [Detection of marker expression] Aggregates were collected in 15 ml vials and precipitated by centrifugation (800G / 5 min). After precipitation, cells were washed twice with PBS X 1. Cells were fixed in 4% PFA for 20 min and washed twice in PBSX1. After fixation, cell membranes were permeabilized for 15 min in PBSX1 / 0.01% Triton X-100). Epitope blocking was performed by incubating cells for 1 h in PBSX 1 / 5% goat serum. After blocking, antibodies (Ab) were added to cells and samples were incubated overnight at 4C with gentle shaking. AB was washed twice from cells and secondary fluorescent AB was added. Cells were incubated with secondary AB for another 2 h and washed three times before microscopy.

[0226] [Table 2]

[0227] [Telomerase activity assay] Telomerase activity was detected in aggregates using the TRAPeze® Telomerase Detection Kit (S7700 Merck) according to the manufacturer's protocol. Briefly, aggregates were isolated and suspended in Chaps lysis buffer. Lysates were added to TS (telomerase substrate) master mix and incubated at 37°C for 30 minutes. In the next step, samples were taken for PCR amplification using kits for specific factors. Telomerase activity was detected by gel electrophoresis of PCR products, and heated samples (95°C) were used as negative controls. Positive control samples provided by the kit (abcam#ab83369).

[0228] [Alkaline phosphatase assay] Detection of alkaline phosphatase in the aggregates was performed using the Alkaline Phosphatase Assay Kit (abcam#ab83369) according to the manufacturer's protocol. RNA of the aggregates was extracted by HyLabs and RNA sequencing was performed by the Weizmann Institute.

[0229] [RNA Seq.] RNA was extracted from cell pellets using an outsourced service (Hylabs), Sequencing INCPM mRNA-seq. Briefly, total RNA was fragmented followed by reverse transcription and second strand cDNA synthesis. Double stranded cDNA was subjected to end repair, base addition, adapter ligation and PCR amplification to generate libraries. Libraries were assessed by Qubit and TapeStation. Sequencing libraries were constructed with barcodes to allow multiplexing of 12 samples in half lanes with Illumina NextSeq High output 75 cycles, yielding approximately 20 million single-end 82bp reads per sample. Bioinformatics: PolyA / T stretches and Illumina adapters were trimmed from the reads using cutadapt, and resulting reads shorter than 30bp were discarded. Reads were mapped to the G. gallus reference genome GRCg6a using STAR (End To End option and utFilterMismatchNoverLmax set to 0.04) supplied with gene annotations downloaded from Ensembl. Using gtf as above, the expression level of each gene was quantified using htseq-count. Differentially expressed genes were identified using DESeq2 with betaPrior, cooksCutoff, and independent filtering parameters set to False. Raw P values ​​were adjusted for multiple testing using the Benjamini and Hochberg procedure. The pipeline was run using snakemake.

[0230] [Growth in factor-free conditions (general description of step 8b in Figure 19)] For the growth and maturation of pluripotent aggregates into microtissues with traits desirable for meat production, the aggregates were transferred to factor-free medium in a stirred bioreactor environment. Under these conditions, the forming microtissues decreased the expression of pluripotency markers (OCT4, Nanog, Lin28) while accumulating the expression of members from the collagen family (Col9a2, Col9, Col2a) and ECM factors (HSPG, CSPG, laminin). For the production of cultured chicken feedstock, the cells were harvested, centrifuged, and washed three times with PBS. The microtissues were then stored at -20C until use.

[0231] [Differentiation of microtissues into adipose-accumulating cells (general description of step 9a in FIG. 19)] To achieve adipocyte differentiation with induced triglyceride synthesis and lipid droplet accumulation, microtissue cells were subjected to oleic acid treatment. Oleic acid was dissolved in 70% ethanol or conjugated to fatty acid-free BSA. Dissolved or conjugated oleic acid was added to factor-free medium to a final concentration of >100uM up to 500μM (preferably 315μM). Microtissues were grown with serum for 3-6 days before harvesting.

[0232] [Differentiation of microtissues into muscle cells (general description of step 9b in FIG. 19)] For muscle cell differentiation, microtissues were grown in the presence of 20-50 μM oleic acid / linoleic acid (or a combination of both) for at least 14 days. The medium was changed every 72-96 h.

[0233] [Adaptation of microtissues to serum-free medium (general description of steps 7-9 in FIG. 19)] Adaptation of the microtissues for growth in serum-free medium was performed in two parallel ways:

[0234] Direct selection: aggregated cells were placed directly into serum-free medium at concentrations of 500K to 1M cells / ml. The medium was replenished every 72 hours. A single cell was removed from the culture each time the medium was replenished.

[0235] Gradual adaptation: The aggregated cells were placed in a medium containing DMEM supplemented with 10% FCS with 10% serum medium mixed with serum-free medium in a 1:1 ratio (e.g., 250 ml serum / 250 ml serum-free). Over the next few weeks, the serum-containing medium was reduced by 50% after 7 days of culture until a complete withdrawal from serum-free medium was reached. After 7 days, the portion of serum medium in the mixture was reduced by half every week (e.g., 125 ml serum / 375 ml serum-free). We gradually reduced the serum medium by half until the cells were fully adapted to serum-free medium).

[0236] For experiments using the GRO-I / EX-CELL system, the medium was prepared according to the manufacturer's instructions. For all other experiments, the lysate was dissolved in the medium at a concentration of 1gr / L. In all experiments, the medium was also supplemented with ethanolamine (20ng / L), insulin (100ug / L), selenium (50ng / L) and transferrin (55ug / L). The medium was also supplemented with 1xMEMNEAA (Biological industries, 01-340-1B).

[0237] 2 mM L-alanine / L-glutamine (Biological Industries-03-022-1B) and 1 mM sodium pyruvate (Biological Industries).

[0238] Different combinations of yeast / soy lysates were used to replace serum. The lysates used in these experiments were either:

[0239] KERRY's:Hypep 1510(ID:S-2048780,Item:U1-5X99023), SHEFF-VAX PLUS ACF(ID S-2048778,U1-5X00484.K1G), SHEFF-VAX PF ACF(ID:S-2048777,U1-5X01143.K1G), SHEFF-VAX PLUS PF ACF VP(ID:S-2048776,U1-5X01090).

[0240] DIFCO: DIFCO Select Soytone (#15ABP196), Bacto Yeast Extract (#15ABP197), BBL Phytone Peptone (#15ABP195), BBL Yeast Extract (#15ABP202), Bacto Malt Extract (15ABP201) and Bacto TC yeastolate (#15ABP163)

[0241] DIFCO Select Soytone (#15ABP196), Bacto Yeast Extract (#15ABP197), BBL Phytone Peptone (#15ABP195), BBL Yeast Extract (#15ABP202), Bacto Malt Extract (15ABP201) and Bacto TC yeastolate (#15ABP163)

[0242] IRVINE Scientific (Ultrafiltered Soybean Hydrolyzate #IR-96857E; Ultrafiltered Yeast Hydrolyzate #IR-96863E)

[0243] [Fatty acid composition] The fatty acid composition of the cells was analyzed using an outsourced service (HUJI). Volatile organic compound analysis was performed using VOC Analysis-Outsource Services (HUJI). Nutritional value analysis was performed using an outsourced service (Bactochem).

[0244] <Example 1> [Recovery and proliferation of cESCs] The aim was to establish stem cell-derived cell lines capable of long-term replication and growth in complete suspension while retaining the ability to differentiate into different cell types with good organoleptic and nutritional properties, as well as various engineering-related attributes that would allow supporting large-scale production.Since embryonic stem cells (ESCs) retain unlimited replicative potential and lineage plasticity, the aim was to establish cell lines derived from chicken embryonic stem cells (cESCs) following published isolation protocols (26) to obtain early cESCs that could then undergo further selection, adaptation and differentiation processes.

[0245] cESC isolation was performed based on the protocol of Aubel & Pain, 2013, with some modifications (1). Primary cultures were obtained by isolating blastoderm from stage X germinal discs of newly laid chicken eggs. Embryonic cells were then plated onto feeder cells and cultured in stem cell support medium at 39°C in a 5% CO2 incubator for 10 days until colonies were easily visible (Figure 1a).

[0246] Colonies were passaged for at least 10 passages by mechanical dissociation. Only clones showing stability of cell identity and morphology were used further (Figure 1b).

[0247] <Example 2> [Feeder removal] After successful isolation of cESCs, cells were gradually transferred from the feeder layer and grown for several passages to select stable feeder-free clones. Notably, after the feeder layer shedding stage, cells tended to form less compact stem cell colonies composed of large nucleated cells because they were not confined by fibrous cells (Figure 1c-e). At this stage, cells showed an expected doubling time of approximately 24 h per cycle.

[0248] <Example 3> [Suspension compatibility and scale-up conditions] In order to be able to grow large quantities of cells in a way that allows the process to be upscaled to the levels required for the food industry, it was necessary to establish cell lines that could grow in complete suspension while maintaining their self-renewal and differentiation capacity while developing new properties advantageous for food production. To do this, the cells were gradually adapted to grow in suspension rather than as adherent cells. When ESCs were transferred to suspension conditions, they naturally formed embryoid bodies (EBs), which became more compact as they grew, thus blocking the flow of nutrients and signals from the surface to the inner cell layer, creating a changing gradient condition from the outside to the center of the sphere. This causes the cells to differentiate and lose their proliferation capacity. The aim was therefore to select cells that did not form compact EBs when grown in complete suspension (see EBs in Figure 2a) and still retained the desired stem cell properties underlying the process. To do this, the cells were gradually transferred from the adherent surface and subjected to slight shaking. Again, successive selection over a period of approximately 2-3 months prompted the cells to downregulate different adhesion molecules while expressing others, allowing the formation of 3D loose raspberry-like aggregates over time, with clear definition of each cell that composes the aggregates (Figure 2F). At this stage, the cells became highly stable, and the doubling time was reduced to 18-20 h per cycle.

[0249] Following the adaptation of the cells to growth in suspension, we aimed to identify and optimize the growth conditions necessary to support continuous rapid growth in a reproducible manner in a stirred bioreactor environment to ensure the cells' ability to be suitable for industrial scale-up. To this end, several clones were tested for the generation of cell lines that grow as aggregate cells at high proliferation rates with fast agitation (tip speed 200-400 rpm) in a stirred bioreactor while maintaining the integrity and stem cell properties of the aggregates. Following this process of optimization, several cell lines were produced that possessed all the favorable properties, and these cells were named "SMCMCs". These cells exhibited the desired morphology, differentiation potential, and reached doubling times of 10-12 hours per cycle, with some growth conditions exhibiting 8 hours per cycle (Figure 3).

[0250] <Example 4> [Characterization of aggregate cells – immortality] The self-renewal capacity of the aggregates (stage 7 in FIG. 19) was tested according to four well-established criteria.

[0251] A. Pluripotency marker expression. Expression of canonical stem cell factors such as NANOG, OCT4 and TRA-I-60 was determined by using specific immunofluorescent antibody staining as well as other key pluripotency markers such as SSEA4, LIN28 and ENS-1. Aggregates were found to be positive for all pluripotency markers except SSEA4, confirming the presence of essential self-renewal factors common to all pluripotent stem cells (Figure 4).

[0252] B. Alkaline phosphatase activity. Alkaline phosphatase staining was found to be positive in the aggregates, indicating their proliferative nature (FIG. 5a-b).

[0253] C. Long-term passaging (>60) with the ability of the cells to reach full confluence after each passage / dilution was also tested. Aggregates were grown continuously for at least 180 passages, demonstrating stability of growth and marker expression.

[0254] D. Telomerase activity. Stem cells uniquely express the telomerase gene, which allows them to maintain the integrity of chromosomes, so the detection of telomerase activity is also considered an important criterion in their self-renewal capacity. The aggregates show telomerase activity using the TRAPeze Telomerase Detection Kit (Figure 5c).

[0255] <Example 5> [Characterization of cell aggregate stability] Stem cells naturally activate multiple self-renewal pathways and can grow indefinitely when deprived of the appropriate environment, either by positive signaling or by blocking differentiation-inducing stimuli. The aggregate cells display key characteristics of pluripotent cells, marking them as stable stem cell lines with natural immortality and self-renewal capabilities. However, because these cells were established to provide large yields for food production, the inventors sought to verify that these cells had not been transformed at some point along the establishment process.

[0256] To address this issue, RNA-seq analysis was performed to compare the transcriptional profile of the aggregates with the initial adherent cESC lines, focusing on key cell cycle and metabolic genes. Comparative detailed analysis showed that genes related to RNA synthesis and processing, protein translation, binding and activity, all important metabolic and cell cycle processes, remained relatively unchanged. Furthermore, no differences in telomerase activity or DNA repair mechanisms were observed compared to the initial stem cell lines. Expression levels of classical oncogenes such as MYC, AKT, EGFR, ELK, KRAS and CDX2 were also analyzed, showing no changes in expression levels, with some even being downregulated in the aggregates. Expression levels of tumor suppressor genes were also analyzed. Genes such as p53, APC, BRCA, MSH2 and WT1 showed similar expression levels between the two cell groups (Figure 6). Looking at the invasive behavior, a decrease in the expression levels of genes related to cell motility and migration was observed.

[0257] Overall, the present data suggest that the aggregates are naturally immortalized by activation of stem cell regulatory gene pathways for self-renewal, without overt evidence of oncogenic transformation.

[0258] <Example 6> [Differentiation potential of aggregate cell lines] Finally, to confirm that the aggregates retained the ability to differentiate into different cell types with possible value for the food industry, several differentiation protocols aimed at producing cells of mesodermal lineage were tested.

[0259] To test the ability of the aggregates to synthesize and store lipid, cells were supplemented with adipocyte differentiation medium for up to 4 days. Rapid changes in cell morphology could be detected as early as 12 h after the change in medium composition, as cells gradually began to accumulate lipid droplets in an enhanced manner (Figure 7A). BODIPY staining also confirmed the presence of lipid droplets within the differentiated aggregates (Figure 7B).

[0260] The ability of the aggregates to form fibroblasts was also tested, since fibroblasts can secrete different proteins of connective tissue. Differentiation of the aggregates into fibroblasts was performed using differentiation medium containing fibroblast support factors. The cells were supplemented with fibroblast medium for 72 hours, after which basal growth medium was added. Within the next 48 hours, a typical epithelial morphology was observed (Figure 8). The cells could be maintained and retain their morphology for more than 30 passages. Fibroblasts could also be frozen and thawed with the ability to maintain the fibroblast phenotype.

[0261] Finally, the ability of the aggregates to differentiate into muscle cells was also tested. Aggregates were grown for 72 h in muscle differentiation medium, and then the cells were transferred to a non-stirred environment supplemented with basal growth medium. Over the next 3-8 days, the aggregates loosely attached to the plate, grew in size, and rhythmic contractions were observed. Muscle differentiation and contractions could be achieved even in fully floating aggregates (Figure 9), supporting the contribution of these cells to a scalable process for meat production.

[0262] <Example 7> [Production process] (See FIG. 19 for illustrative, but non-limiting, avian stem cells) Following the successful isolation and establishment of chicken stem cell derived lines that can be expanded and grown in large quantities and exhibit advantageous properties for food manufacturing, the inventors created a cell bank to provide a well-defined and immutable seed train for the first stage of the production process. The next challenge was to use the aggregate platform to produce chicken-flavored ingredients in a scalable process.

[0263] This task involved careful media preparation, mechanical process development, and regular sensory testing.

[0264] [Seed Train Expansion Stage] The first step in the production process involves thawing a new batch of aggregates from a cell bank. These cells are then rapidly grown to the appropriate concentration to feed downstream bioreactors for production, where the aggregates turn into microtissues and continue to grow and change their identity to suit the needs of the food industry.

[0265] [Microtissue formation stage] To achieve the desired chicken flavor, several attributes must be present. These include fat amount and composition, expression of different proteins, and the presence of specific sugars. By manipulating the maturation medium with careful taste testing and chemical analysis, the inventors have succeeded in developing a process to obtain microtissues grown in complete suspension with enhanced savory chicken flavor. The maturation medium does not add growth factors, causing the cells to lose important pluripotency markers, while gaining mesodermal characteristics including specific markers and elevated expression of connective tissue proteins. Due to their initial identity and the specific culture conditions in the bioreactor, these microtissues can still grow rapidly and extensively and can be propagated for at least 150 population doublings.

[0266] Control of these characteristics is achieved through defined set points for bioreactor running parameters, such as agitation within the bioreactor and gassing at tip speeds ranging from 0.25 to 1.70 m / s with an optimal tip speed of 0.85 m / s.

[0267] <Example 8> Adaptation of aggregates and microtissues for growth in serum-free medium. A key challenge in cultivated meat production concerns the media used to culture the cells. Tissue culture media were originally designed for other purposes (e.g., research, pharmaceutical, clinical production, etc.) and therefore come with some caveats from the perspective of cultivated meat production. The cost of the culture media is generally the largest economic burden for cultivated meat production. Furthermore, most available culture media contain fetal calf (or other animal-derived) serum (FCS). The use of FCS has the inherent drawback of being highly variable between batches and undefined (in terms of chemical composition). Furthermore, the use of such material in cultivated meat production will most likely shy away from public acceptance due to the problematic way in which it is produced. To overcome these obstacles, the inventors set out to adapt the aggregates and microtissues to grow in serum-free media without compromising the quality, safety, and inherent characteristics of the microtissues. To achieve this, the microtissues were adapted to grow in several combinations of serum-free media. As an initial step, the inventors adapted the microtissues to the commercially available serum-free system GRO-I / EX-CELL from SIGMA-Merck. The system is based on cell growth in a basal medium (GRO-I) supplemented with a chemically defined yeast / plant hydrolysate (EX-CELL) as an alternative agent to FCS. The adaptation process was carried out either as a preliminary step for seeding the bioreactor (7b) or at the very seeding of the cells in the stirred bioreactor (Figure 19, step 8B), by complete drop-off of serum from the culture medium or by gradually depleting the serum concentration over time. In both cases, the cells ((aggregates) or microtissues) were able to adapt to grow in serum-free medium (in flasks and bioreactors). For about 14 days, the cells were adapted to the new medium, restoring the original 12-h doubling time and retaining a high level of cell viability (Figure 10). Similarly, we were also able to adapt the cells to grow in other combinations of basal medium supplemented with yeast, plant peptone and hydrolysate.Among the mixtures successfully used for the adaptation of cells to serum-free media, the inventors were able to identify DMEM (high glucose) supplemented with Ex-Cell lysate, DMEM / F12 supplemented with Ex-Cell lysate, DMEM (low glucose) supplemented with Ex-Cell lysate, DMEM (high glucose) supplemented with a combination of soy and yeast lysates produced by the KERRY group. Successful lysates tested with this process were either full or partial combinations of these four products: Hypep 1510 (ID: S-2048780, Item: U1-5X99023), SHEFF-VAX PLUS ACF (ID S-2048778, U1-5X00484.K1G), SHEFF-VAX PF ACF (ID: S-2048777, U1-5X01143.K1G), SHEFF-VAX PLUS PF ACF VP (ID: S-2048776, U1-5X01090). We have also successfully adapted microtissues to routinely used basal media (DMEM HG / LG, DMEM / F12, RPMI 1640) in combination with soy and yeast hydrolyzing enzymes manufactured by FUJIFILM-IRVINE Scientific (Ultrafiltered Soy Hydrolysate #IR-96857E; Ultrafiltered Yeast Hydrolysate #IR-96863E). Additionally, cells were adapted to grow in basal media (DMEM HG / LG, DMEM / F12, RPMI 1640) supplemented with various combinations of DIFCO Select Soytone (#15ABP196), Bacto Yeast Extract (#15ABP197), BBL Phytone Peptone (#15ABP195), BBL Yeast Extract (#15ABP202), Bacto Malt Extract (15ABP201) and Bacto TC yeastolate (#15ABP163). For the purpose of the latter process, yeast derived lysates were combined with one or several plant derived lysates. In all experiments, media was also supplemented with ethanolamine (20ng / L), insulin (100ug / L), selenium (50ng / L) and transferrin (55ug / L).The medium was also supplemented with 1X MEM NEAA (Biological industries, 01-340-1B), 2 mM L-alanine / L-glutamine (Biological industries-03-022-1B) and 1 mM sodium pyruvate (Biological industries). The results presented herein highlight the ability of the microtissues to accommodate multiple combinations based on basal media supplemented with soy and vegetable hydrolysates as replacements for serum in the culture medium.

[0268] <Example 9> [Molecular characterization of microtissues] During the microtissue formation stage (8b in Figure 19), the cells lose some of their pluripotency markers such as NANOG, OCT4, LIN28, but not SSEA3 (Figure 11), and acquire some mesodermal properties such as fat storage and secretion of extracellular matrix proteins (ECM) that surround each aggregate and form a specific niche that creates the microtissues. These ECM proteins also contribute to the microtissues developing their enhanced chicken flavor and ability to form chunks when cooked together.

[0269] Microtissues derived from microtissue cells exhibit distinct mesodermal characteristics. These include structural proteins such as fibronectin, laminin, and collagen, which constitute the ECM and organize cells during tissue development. The cells also express SNAI1, a master regulator of the formation and maintenance of embryonic mesoderm. Finally, the cells have the ability to synthesize and store triglycerides. Molecular function analysis and KEGG analysis also revealed that microtissues are enriched for Wnt canonical and non-canonical associated factors. Wnt signaling is closely associated with the acquisition of mesenchymal identity, as suggested by gene expression analysis.

[0270] One important factor in the ability of cell cultures to recapitulate the experience of meat consumption depends on the expression of certain molecules that contribute significantly to the texture and taste of these cells. The main contribution to these aspects is due to the presence of ECM molecules anchored to the membrane of the cells or secreted by the cells. In fact, most of the variability between the texture of different muscle tissues is a direct result of the collagen content in their fibers (32). The collagen superfamily consists of 28 members with numerous derivatives. Collagens attach to fibrils and provide both mechanical support as well as numerous biological activities through interactions with other ECM and functional factors. Notably, RNA-seq analysis identified upregulation of collagen-family members and collagen-associated proteins, such as Col2A, Col8A2, and Col9A2. Furthermore, RNA-seq analysis revealed significant enrichment with additional ECM and cell adhesion molecules. This finding holds particular importance since collagen and proteoglycan interactions, such as HSPGS, and CSPGs, and laminins, all of which are upregulated in microtissues, underlie cellular matrix organization, structure, and function.

[0271] To confirm the expression of key ECM factors, an ECM-related antibody specific screen was performed. The results of the screen confirmed the enrichment of distinct collagens and other ECM molecules in the microtissues (Figure 11 illustrates step 8b of Figure 19).

[0272] <Example 10> [Microtissue differentiation for augmentation of fat and muscle cells] In order to control the exact parameters of the final meat product, the next step was to develop a process that would allow directing and adjusting the rate of differentiation into either fat, muscle or connective tissue. To this end, a process was developed that involved culturing several cell lines with different combinations of fatty acids at different concentrations.

[0273] Treatments included growing cells with differentiation media supplemented with either 20um-300um oleic acid (OA) or linoleic acid (LA) and combinations of the two. Findings revealed that prolonged growth at fatty acid (FA) concentrations below 50uM allowed proliferation of cells. Cultivation of microtissues in low concentrations of fatty acids (FA) resulted in the microtissues developing discernible protrusions in a highly reproducible manner within 96 hours (Figure 12A-E). Further analysis revealed that the initiation of muscle differentiation occurred as early as 4-7 days after introduction into FA, prior to the appearance of protrusions by immunostaining using MyHC antibodies (Figure 12A-E). Primed cells began to express early markers of muscle differentiation as early as 4-7 days after maturation, although the stage could be extended to several weeks, e.g., 3 weeks, since the cells were still proliferative. Figures 12A-E and 13A-B are taken from stage 9b and show the initiation of muscle differentiation beginning with the addition of GFs. Thus, if this stage was extended, more muscle cells were evident (Fig. 14A-C) or higher concentrations of FA were added to enhance fat accumulation. MyHC staining (Fig. 12A-E) indicates muscle differentiation. To confirm the presence of muscle precursors on the microtissue protrusions, we also stained the cells using AB against muscle precursor markers Pax7 and L4. In both cases, cells within the protrusions stained positive (Fig. 13A-B). Further staining of microtissues cultured in the presence of low doses of FA could confirm muscle maturation on top of the microtissues while still in suspension growth conditions (MyHC staining, Fig. 14A-C). Notably, the presence of mature contracting muscle on top of the microtissues was demonstrated by recording synchronous contraction of microtissues growing for 14 days in the presence of FA (data not shown). Furthermore, the ability of muscle precursors within the microtissues to eventually differentiate into mature muscle was determined by troponin-T staining in both suspended and attached conditions (Fig. 15A-B).

[0274] In addition to the appearance of muscle precursor sites in different locations of the microtissue. FA concentrations between 50 and 200 uM allow fat storage within the microtissue, except in areas where muscle differentiation occurs. Culturing cells with higher amounts of FA (>200 uM) leads to a global and terminal differentiation of the microtissue into fat-depleting tissue, ultimately leading the culture to growth arrest.

[0275] We found that chronic treatment with low doses of either oleic acid or linoleic acid or a combination not only promoted muscle differentiation, but also simultaneously drove a portion of the microtissue cells to accumulate fat (step 9b in Figure 19). The microtissues were double stained with troponin T AB along with a lipid stain (Bodipy) and showed striking mutually exclusive populations within the microtissues that differentiated into either the adipose or muscle lineage (Figure 16). Furthermore, transferring the microtissues to adherent growth conditions resulted in the differentiation of both mature adipocytes and mature delayed muscle fibers (see Figures 17A-B). The two populations can be easily distinguished based on cytoskeleton organization (Figures 18A-E). Further staining for fat accumulation clearly shows the ability of the microtissues to differentiate in parallel into both adipose and muscle tissue (Figures 18A-E). These characteristics make the differentiation process of the microtissues highly favorable for cultivated meat production, as it allows for control of the characteristics of the feedstock by varying the fat and protein ratios as needed.

[0276] <Example 11> [Sensory and nutritional properties of microtissues] During cooking, different chemical reactions take place between different components of food. One of the most important and complex of these reactions is the Maillard reaction, in which amino acids react with reducing sugars to form new molecular compounds that are responsible for deep meaty flavors and aromas.

[0277] Another major factor that controls the flavor delivery and unique aroma that each meat develops during cooking is the fat content and composition.

[0278] The medium compositions (factor-free medium and differentiation medium supplemented with FA) allow the formation of desired components within the microtissues and produce similar amino acid and fatty acid profiles for the development of chicken flavor visible from amino acid composition analysis as well as fatty acid profiling by gas chromatography-mass spectrometry (Tables 1-3).

[0279] Multiple tastings of the product were conducted by trained tasters and chefs and reported enhanced and pleasant chicken flavor tasting experiences. To demonstrate this analytically, a preliminary volatile compound (VOC) analysis was performed on the cooked product and cooked chicken breasts. The results showed a highly parallel pattern of VOC emissions, indicating similar aroma evolution between the two samples (Figure 19).

[0280] Finally, we wanted to verify the nutritional value of the product. All the main components of the food were analyzed: moisture, carbohydrates, protein, fat, and minerals. All values ​​show that the overall nutritional composition of the microtissues is very similar to that of chicken meat (Table 3).

[0281] [Table 3]

[0282] [Table 4]

[0283] [Table 5]

[0284] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0285] It is the intention of the applicant(s) that all publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety, as if each individual publication, patent, or patent application was specifically and individually indicated as being incorporated herein by reference. Furthermore, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting. Additionally, any priority document(s) of this application are incorporated herein by reference in their entirety.

Claims

1. 1. A method for producing a microtissue comprising one or more cell types, comprising: (a) forming aggregates in suspension without matrix attachment comprising non-human pluripotent stem cells, wherein the non-human pluripotent stem cells of the aggregates exhibit a doubling time of 10-20 hours undifferentiated for over 60 passages, are capable of differentiating into muscle, fat and connective tissue upon induction of differentiation, grow in the presence of growth factors and exhibit lower cell-cell adhesion than embryoid bodies (EBs) as determined by reduced expression of adhesion molecules selected from the group consisting of COL6A2, CD44, COL6A1, ANXA1, ANXA2 and S100A11, as compared to said EBs; (b) transferring the aggregates to a bioreactor and growing them in suspension in the absence of growth factors and without matrix attachment to produce microtissues; A method comprising:

2. 1. A method for producing microtissues comprising one or more cell types, comprising: (a) providing a suspension culture comprising aggregates comprising non-human pluripotent stem cells, wherein the non-human pluripotent stem cells of the aggregates exhibit a doubling time of 10 to 20 hours undifferentiated for over 60 passages, are capable of differentiating into muscle, fat and connective tissue upon induction of differentiation, grow in the presence of growth factors, and exhibit lower cell-cell adhesion than embryoid bodies (EBs) as determined by reduced expression of adhesion molecules selected from the group consisting of COL6A2, CD44, COL6A1, ANXA1, ANXA2 and S100A11, as compared to said EBs; (b) transferring the aggregates to a bioreactor and growing them in suspension in the absence of growth factors and without matrix attachment to produce microtissues; A method comprising:

3. 1. A method for producing microtissues containing one or more cell types of interest, comprising: (a) producing a microtissue according to claim 1 or claim 2; (b) subjecting said microtissues in suspension to differentiation conditions, thereby producing said microtissues containing one or more cell types of interest; A method comprising:

4. The method according to any one of claims 1 to 3, wherein the indicated values ​​are given under optimal conditions for cell growth of the type and developmental stage as said non-human pluripotent stem cells.

5. 5. The method of any one of claims 1 to 4, wherein the non-human pluripotent stem cells are selected from the group consisting of avian pluripotent stem cells, bovine pluripotent stem cells, porcine pluripotent stem cells, caprine pluripotent stem cells, ovine pluripotent stem cells, shrimp pluripotent stem cells, and fish pluripotent stem cells.

6. The method according to any one of claims 1 to 5, wherein the microtissue has a diameter of 30 to 500 μm.

7. 6. The method of any one of claims 1 to 5, wherein the non-human pluripotent stem cells of the aggregates exhibit alkaline phosphatase expression, exhibit telomerase gene expression, and / or are SSEA4-, LIN28+, ENS-1+, NANOG+, OCT4+, and TRA-I-60+.

8. A method according to any one of claims 3 to 7, wherein subjecting the microtissue to differentiation conditions comprises priming the microtissue in the presence of a fatty acid at a concentration not exceeding 100 μM for a time sufficient to obtain stem cells having an adipocyte fate whilst retaining a muscle cell and proliferative phenotype, and optionally thereafter differentiating in the presence of a fatty acid at a concentration of 100 μM or greater.

9. The method according to any one of claims 1 to 8, wherein the one or more cell types are selected from the group consisting of muscle cells, fat cells and connective tissue cells.

10. 10. The method of claim 9, wherein the one or more cell types comprise fat cells and muscle cells.

11. The method of any one of claims 1 to 10, wherein each step of the method is devoid of animal components other than the non-human pluripotent stem cells.

12. The method of any one of claims 1 to 11, wherein the microtissue is NANOG-OCT4-LIN28-, SSEA3+.

13. The method of claim 1, wherein the doubling time does not exceed 12 hours.

14. Microtissue obtainable by the method according to any one of claims 1 to 13.

15. 1. A microtissue comprising one or more cell types, said microtissue having a diameter of 30-500 μm, and cells of said microtissue are NANOG-OCT4-LIN28-, SSEA3+, and optionally, said one or more cell types comprise adipocytes and muscle cells.

16. A food comprising the microtissue described in claim 14 or claim 15.

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