From fertilised oocyte to cultivated meat – harnessing bovine embryonic stem cells in the cultivated meat industry

Pluripotent stem cells as cell source for cultivated meat

Pluripotent stem cells (PSCs) are natural candidates as a cell source for the cultivated meat industry. These cells are able to self-renew and have indefinite proliferative capabilities with a high growth rate, both essential qualities for the development of an efficient, cost-effective cultivated meat production process (Edelman et al. 2005; Lavon et al. 2018; Mattick 2018; Ben-Arye and Levenberg 2019; Lavon 2022). Bovine PSCs can give rise to the mesoderm germ layer and further differentiate to myocytes, adipocytes, and fibroblasts, which together define the structure and organoleptic properties of cultivated meat products. The clear advantages of using PSCs in the cultivated meat industry stands out when considering the establishment of cell banks as a cellular reservoir for process production, where one vial of cells can be used in several production batches and one cell bank can yield indefinite amounts of meat. In addition, due to their size and morphology, PSCs can reach high cell density (cells/cm²) when grown in 2D culture vessels, and can be cultured in suspension, form aggregates and even grow on microcarriers (Singh et al. 2010; Kwok et al. 2017; Lavon et al. 2018; Lipsitz et al. 2018; Manstein et al. 2019; Bodiou et al. 2020), attributes that are needed for 3D culture which simplify upscaled procedures.

PSCs can be sourced as either embryonic stem cells (ESCs) from the inner cell mass, or as induced pluripotent stem cells (iPSCs) from somatic cells. In various papers, stem cells were shown to be produced from ovine, porcine, chicken, fish and bovine (Collodi et al. 1992; Wakamatsu et al. 1994; Pain et al. 1996; Fuet and Pain 2017; Kinoshita et al. 2021). However, unlike human or mouse species, limited studies were conducted to study the derivation of chicken, bovine, ovine and fish PSCs. Bovine ESCs (bESCs), which are used as a source of cells for the meat product developed by Aleph Farms (Lavon 2022), are derived from the inner cell mass (ICM) of a blastocyst, formed 7–8 days post fertilisation, and collected in a non-surgical procedure from the donor cow (Evans and Kaufman 1981; Martin 1981; Bogliotti et al. 2018). Once the blastocyst is transferred to the lab, the ICM is isolated, either surgically or enzymatically, and cultured to produce bESCs. Specific medium and growth factors are required to maintain the propagation and the pluripotency of the derived cells, and to inhibit their spontaneous differentiation (Chan et al. 2013; Gafni et al. 2013; Valamehr et al. 2014; Ware et al. 2014). iPSCs are produced by reprogramming somatic cells to express OCT4, KLF4, C-MYC and SOX2 transcription factors, also known as the Yamanaka factors. These cells were shown to be produced from porcine, chicken, fish and bovine sources (Ezashi et al. 2009; Wu et al. 2009; Fuet and Pain 2017; Peng et al. 2019; Su et al. 2021). The Yamanaka factors can be expressed using either viral vectors, episomes, mRNA transfection or any other method of expression (Takahashi and Yamanaka 2006; Seki and Fukuda 2015; Karagiannis et al. 2019). As for ESCs, culture conditions and medium supplements must be maintained to prevent spontaneous differentiation of iPSCs, and using specific inducers they can be differentiated to any cell type. While ESCs and iPSCs have similar propagation and differentiation abilities, epigenetic traits of iPSCs may affect these capabilities. Moreover, reprogramming of somatic cells to iPSCs may also introduce genetic modification to the produced cell line and as a result they may be labelled as genetically modified products (Rosselló et al. 2013; Pillai et al. 2019). For the above reasons, we chose bESCs as our source of cells.

Cell sourcing for cultivated meat industry

The cellular composition of cultivated meat is of mainly skeletal myocytes with integration of adipocytes and fibroblasts. In the production process of cultivated meat, PSCs (either ESCs or iPSCs) are differentiated to mesenchymal stem cells (MSCs), which can be found for instance in bone marrow, adipose, and skeletal tissues. In turn, MSCs are further differentiated to adipocytes, fibroblasts and to some skeletal muscle cells (Prockop 1997; Okamura et al. 2018). Upon selection of cell source for the cultivated meat industry, several parameters, such as genetic background, species breed and gender, need to be considered. The genetic background of cells utilised to produce ESCs or iPSCs can in turn affect their differentiation potential. For instance, differentiation potential of iPSCs sourced from adipose- derived MSCs differs from bone marrow–derived iPSCs (Dai et al. 2021; Zagury et al. 2022). However, in regards of ESCs, it is yet unclear if the origin of the breed can affect the pluripotency or differentiation potential of the ESCs.

In this article review we describe the approaches taken to derive Aleph Farms’s bESCs and the means we took to characterise them. We show our ability to differentiate our bESCs to MSCs and discuss their potential differentiation to the cell types required for cultivated meat production. We finally outline our strategy for preparation of Master and Working Cell Banks that allow us to indefinitely expand and grow our bESCs and show our ability to grow our cells in suspension in amounts suitable for mass production. All the above highlights the potential use of harnessing bESCs to promote the production process of a high quality, nutritional, and economically feasible cultivated meat.

Collection of flushed embryos

Bovine heifers were superovulated 8–12 days post oestrus by injection of FSH and prostaglandins (PG), then inseminated 5 days post initiation of superovulation. Seven days post insemination, the embryos were collected by non-surgical uterine flush using complete flush medium (Agtech, ME, USA). At the farm, under binocular microscope, blastocysts were collected to a holding cap and transferred to 5-well plates (minitube culture dish, WI, USA) containing holding medium (Agtech, ME, USA). Embryos were then counted and examined for their stage and grade, then transferred to Aleph’s labs.

Derivation of bovine embryonic stem cell line

At the laboratory, embryos were placed in a biological safety cabinet and carefully washed in CSC-NXc medium (Irvine Scientific, CA, USA) to remove residual holding medium. The stage and grade of the embryos were re-examined under a binocular microscope. Then, they were carefully transferred to CSC-NXc drops, covered in oil for embryos (Irvine Scientific, CA, USA) and incubated overnight at 38.5°C, 5% CO₂, to allow their maturation. The following day, in the biological safety cabinet under binocular microscope, embryos at the blastocyst stage were surgically cut to isolate their ICM. Embryos were then carefully transferred to 12-well plates pre-seeded with feeder layer in mTeSR1 medium (StemCell technologies, BC, CA) supplemented with growth factors and incubated at 38.5°C, 5% CO₂ for 48 h. Medium of isolated ICMs was replaced daily and the wells were examined daily until detection of initial colonies.

Cell culture maintenance

The cells of the initial colonies were incubated at 38.5°C, 5% CO₂ and grown in mTeSR1 medium (StemCell technologies, BC, CA) supplemented with growth factors and replaced daily. Cells were allowed to grow and propagate and were transferred to a new culture growth plate when they reached full confluency. Each transfer is considered as one passage.

Cell bank preparation

Following several propagation cycles, a seed stock of 30 vials was cryopreserved, each containing 1 × 10⁶ cells. Vials were frozen in designated containers allowing cooling of 1°C/min at (−80°C) and transferred to liquid nitrogen for storage 1–3 days following freezing. To create a Master Cell Bank (MCB), one vial from the seed stock was thawed and propagated for several passages, in an increasing vessel size and volume. Once reaching sufficient cell numbers, cells were detached and 200 vials, each containing 5 × 10⁶ cells, were frozen and stored as described. To create a Working Cell Bank (WCB), one vial from the MCB was thawed and propagated for several passages, in an increasing vessel size and volume. Once reaching sufficient cell numbers, cells were detached and >200 vials, each containing 5 × 10⁶ cells, were frozen and stored as described.

Immunofluorescence staining

Cells were carefully washed in phosphate-buffered saline (PBS), then fixed with 4% paraformaldehyde (PFA) soultion (Biolab, IL). PFA was then discarded, and the cells were washed in PBS. Further blocking was done in 5% bovine serum albumin (BSA), and 0.2% Triton X-100 in PBS followed by incubation with primary antibodies in PBS, containing 1% BSA: PE-OCT4 (Abcam, MA, USA), Brachyury (R&D Systems MN, USA).

Next, cells were washed in PBS. In case the primary antibody was not conjugated, cells were incubated with a secondary antibody, goat-anti-mouse Alexa fluoro-594 (Abcam, MA, USA). Finally, cells were washed in PBS followed by nuclei stained using DAPI (4′,6-diamidino-2-phenylindole) (Sigma Aldrich, MO, USA). Imaging was performed using EVOS fluorescence microscopy.

Flow cytometry analysis

Cells were washed in PBS and incubated with anti-SSEA-4-FITC (R&D Systems MN, USA) in FACS (fluorescence-activated cell sorting) staining buffer (R&D Systems) and then washed with PBS. Data acquisition was done using Attune NxT flow cytometer (Thermo Scientific, MA, USA). Data analysis was performed using Kaluza software.

Real-time PCR

Total RNA was extracted from cells using NucleoSpin RNA purification kit (MACHEREY-NAGEL, DE). One microgram of total RNA was reverse transcribed by RevertAid First Strand cDNA Synthesis Kit (ThermoScientific, MA, USA) using the Thermal Cycler SimpliAmp device (ThermoScientific, MA, USA). Real-time PCR was performed using specific primers with TaqMan Fast Advanced Master Mix (ThermoScientific, MA, USA) using a QuantStudio 5 Real-time PCR device (ThermoScientific, MA, USA). Readouts were normalised to a housekeeping level. Data is presented as mean fold change using the 2^(−ΔΔCT) method.

Derivation of bovine embryonic stem cell lines

bESCs were derived from the ICM of blastocysts. At the farm, superovulated female bovines heifers were artificially inseminated. Then, 7 days post insemination, embryos were non-surgically flushed, collected and visualised using binocular microscope to determine their stage and grade. Embryos were then transferred to Aleph Farms’s laboratory and incubated in a medium that allows their recovery and maturation (Fig. 1a). The following day, embryos were examined under the microscope and their maturation into the blastocyst stage was determined. Mature embryos were then individually transferred to a culture plate pre-seeded with feeder-layer, and each embryo was surgically dissected to isolate its ICM. Isolated ICMs attached and adhered 24 h post seeding (Fig. 1b) and initial colonies showing ES-like morphology were visualised 5 days post seeding (Fig. 1c).

Fig. 1.

Derivation of bovine embryonic stem cell line. (a) Embryos flushed 7 days post in vivo insemination, imaged following recovery, and maturation. (b) Isolated inner cell mass (ICM) attached to feeder layer (images acquired 8-days post seeding using bright field EVOS-Fl microscope (×10 and 20 magnification).

Bovine embryonic stem cell line characterisation

The initial colony was allowed to grow and proliferate on feeder layer for five passages. Then, the pluripotency features of the newly derived bESCs were characterised. Immunofluorescence staining against the pluripotency marker OCT4, showed its presence in over 90% of the imaged colony cells (Fig. 2a). This finding was confirmed by flow cytometry analysis using the pluripotency marker SSEA4, showing positive staining of 97% of the gated population (Fig. 2b). We next employed real-time PCR analysis on two derived cell lines that showed the expression of the pluripotent markers NANOG, SALL4, DNMT3B and OCT4 (Fig. 2c).

Fig. 2.

Bovine embryonic stem cell line characterisation. (a) Morphological features of bESC line, imaged in brightfield (right) or using immunofluorescence staining using anti-OCT4 antibody and DAPI (left and centre) for nuclei visualisation (unstained cells presented in lower panel, n = 3). (b) Flow cytometry analysis of cells positive to stage-specific embryonic antigen-4 (SSEA4). (c) Real-time PCR analysis of two derived cell lines shows the expression of pluripotent markers: NANOG, SALL4, DNMT3B and OCT4 (expression level relative to non-pluripotent cell line – bovine embryonic fibroblasts (BEFs)).

Differentiation of bESCs to mesoderm committed cells

PSCs can be differentiated to the three germ layers: endoderm, ectoderm, and mesoderm. We evaluated the potential of our cells to differentiate to mesoderm committed cells (MCCs). To test the potential use of our derived bESCs, we cultured them in media either maintaining their pluripotency or i inducing their differentiation to MCCs. Real-time PCR analysis showed that while OCT4 and NANOG expression was high in medium maintaining pluripotency, it was significantly reduced following incubation in differentiation medium. In addition, accompanied with OCT4 and NANOG downregulation, we noticed an increase in the expression of the mesenchymal markers Brachyury (TBXT) and TBX6 following induction of differentiation (Fig. 3a). Increase in the expression of Brachyury was also observed at the protein level using immunofluorescence staining (Fig. 3b).

Fig. 3.

Differentiation of bESCs to mesoderm committed cells. (a) Following incubation with differentiation medium, real-time PCR analysis shows the downregulation of pluripotent markers OCT4 and NANOG and the upregulation of mesoderm markers Brachyury (TBXT) and TBX6 (expression level relative to bESCs) (b) Immunofluorescence staining against mesenchymal marker Brachyury (TBXT), shows its expression in differentiated cells (upper panel) and not in pluripotent cells (lower panel) (n = 3).

Cell bank preparation and characterisation

The main advantage of bESCs is their indefinite proliferative capability and high growth rate, both essential for the high population doubling levels required throughout the production process of cultivated meat. In addition, by harnessing bESCs for the establishment of cell banks, one can avoid repeated biopsy collection. Cell bank samples are extensively characterised for their growth profiles, characteristics, and assurance that they are free of adventitious agents, ensuring a safe, reproducible, and consistent production process. Furthermore, bESCs are cost-effective since one vial of thawed cells from an MCB is sufficient for the establishment of an entire WCB, each composed of at least 200 vials. In turn, each vial of WCB can be used for an entire production process (Fig. 4a). Thus, from a single derivation procedure using one blastocyst, more than 40 000 batches of production processes can be performed, giving rise to thousands of tonnes of cultivated meat products. After establishing our cell banks, we tested our bESCs for their growth and pluripotency during extended passages, correlating to entire production process. Growth rate, represented as the population doubling time (PDT) of cells was found to be ~24 h on average, demonstrating the high proliferative growth rate of our bESCs. Flow cytometry analysis showed that >80% of cells were expressing SSEA4, representing the high pluripotency of our bESCs. Both growth rate and pluripotency were found to be stable along 65 population doubling levels (PDLs), demonstrating the potential use of bESCs in the cultivated meat industry (Fig. 4b, c). In addition, we employed RNA sequence-based digital karyotyping across different generations to test genomic stability along 15 passages. RNA expression per chromosome was calculated based on the expression level of genes located on each chromosome. The average fold change between the early and late passages was calculated and found to be of similar expression (Fig. 4d). Thus, all employed analyses confirmed the stability of our derived cell lines.

Fig. 4.

Cell bank preparation and characterisation. (a) Schematic representation of Master Cell Bank (MCB) and Working Cell Bank (WCB) preparation, each containing >200 vials proved and tested to be free of adventitious agents. (b, c) Cells were tested for their growth and pluripotency during extended passages correlating to the entire cultivated meat production process. Growth rate, presented by population doubling time (PDT; b) and pluripotency presented by population percentage positive for SSEA4 marker (c), were found to be stable along 65 population doubling levels (PDL). Average PDT was found to be ~24 h. The blue line represents the acceptance criteria of ≥80% cells positive for SSEA4. (d) Digital karyotyping shows genomic stability between the early and late passages.

Cell growth rate and stability

The use of bESCs is cost-effective, since one thawed vial of cells is sufficient for several production batches. In addition, compared to satellite and mesenchymal cells, due to their small size and tendency to crowd, bESCs can reach a high cell density in 2D culture (cells per cm²), simplifying scale-up procedures prior to cell seeding in bioreactors. In addition, bESCs can also grow in suspension as aggregates, or attached to edible microcarriers which enables 3D culture in bioreactors. We therefore tested and demonstrated the ability of our derived bESC lines to grow as aggregates in small-scale vessels and showed the growth of aggregate size over 4 days of culture, indicative of cell growth (Fig. 5a). We next tested the growth of our bESC lines in stirred tank bioreactors (STBs) and showed that viable cell concentration increased by an average of 50-fold in 7 days in STBs, demonstrating that extensive growth can be obtained (Fig. 5b).

Fig. 5.

Cell growth and stability. (a) Representative brightfield images showing bESCs cultured as aggregates grown in growth medium in suspension. Aggregate size increased during 4 days in culture, indicative of cell growth. (b) Viable cell number increased by an average of 50-fold in 7 days in an STB, tested in four different STB runs.