Cell lines

Introduction

The cell lines used in cultivated meat production ultimately determine many of the downstream variables to consider. As starting material, cells that can self-renew and differentiate into the cell types that make up meat tissue (i.e. myofibers, adipocytes, fibroblasts, chondrocytes, endothelial cells, etc) are most attractive. In other words, we need to begin with a stem cell. There are several different possibilities for the starting stem cell population, delineated by their potency, or ability to differentiate into a diversity of cell types. For instance, embryonic stem cells have the ability to differentiate into cells of all three developmental germ layers (i.e. ectoderm, mesoderm, and endoderm), while adult stem cell populations found throughout our body are typically more specialized and limited to creating cells of the same germ layer or organ type. The most likely starting cell types for use in cultivated meat production are outlined in Table 1 and discussed below.

While not discussed throughout, it is also possible to use primary cell lines from specific organs or tissues for the generation of other consumable products such as foie gras derived from hepatocytes, or other organ tissues such as fish maw (swim bladder). The growth of mammary epithelial cells that create the nutritional components of milk (i.e. lactate, casein, and whey) can also be used to create human or animal milk products. Non-meat products fall under a larger umbrella of cellular agriculture, which include agricultural products such as wood, leather, enzymes, animal proteins and other materials produced using animal, plant, or microbial cell cultures.

Pluripotent stem cells

In order to use embryonic stem cells as a source for cultivated meat production, one must first have access to an embryonic stem cell line. Embryonic stem cells are derived from the inner cell mass of an early embryonic structure called a blastocyst, which forms just a few days post-fertilization. The first embryonic stem cell lines were derived from mice in 1981 and were followed thereafter by a select few additional species including human, non-human primates, rat, chicken, and fish. However, derivation of stable embryonic stem cell lines is extremely challenging, as the embryonic material is difficult to obtain and work with, the cells are highly sensitive to their growth substrate, and they require different sets of growth factors or inhibitors from species to species to maintain proliferation without spontaneous differentiation. Indeed, the derivation of embryonic stem cell lines from agriculturally-relevant bovine species was only recently achieved in 2018 (Bogliotti et al. 2018). Therefore, if cultivated meat is to be available for every commonly-eaten animal species, there exists significant work in the establishment of bonafide embryonic stem cell lines from a diverse set of species.

As an alternative, scientists can utilize a technology called cellular reprogramming to obtain induced pluripotent stem cells (iPSCs) that maintain the desirable properties of embryonic stem cells without being derived from an embryo. Cellular reprogramming enables the direct conversion of one cell type into any other cell type based on the expression of a defined set of important genes of the final cell type (Rackham et al. 2016) , typically a set of transcription factors. Reprogramming can be performed via viral-mediated over-expression of transcription factors, either via permanent genome integration (e.g. through the use of lentivirus) or stochastic non-integrating expression (e.g. via Sendai Virus (Fujie et al. 2014)) based on the virus type. However, reprogramming can also be achieved by additional non-integrating methods such as episomal or mRNA gene delivery (Schlaeger et al. 2015), proteins (Cho et al. 2010), or small molecules (Zhang et al. 2012). The generation of iPSCs can be performed from virtually any adult somatic cell, including easily obtained cells such as white blood cells or skin fibroblasts, by over-expressing the canonical set of transcription factor genes Oct4, Klf4, c-Myc, and Sox2, referred to as Yamanaka factors (Takahashi and Yamanaka 2006). Because these genes are highly conserved, iPSCs have been generated from most agriculturally-relevant species, although examples of fish iPSC or other marine species derivation are sparse (Rosselló et al. 2013). Thus, iPSCs are typically easier to derive than embryonic stem cells while being generally equivalent in their functionality (Choi et al. 2015).

The use of reprogramming technically permits other starting cell types (e.g. fibroblasts) to be directly converted (i.e. transdifferentiated) into muscle (Ito et al. 2017), fat (Wu, Jin, and Gao 2017), or other cell types, bypassing the iPSC state altogether. This approach is likely what is being used by Israeli-based cultivated meat company Future Meat, which uses fibroblasts as their starting cell line. Transdifferentiation strategies are somewhat limited in that their conversion efficiencies can be variable and incomplete, and they result in a post-mitotic population that undergoes limited expansion (Prasad et al. 2017). Given that the production of cultivated meat will require excessively large numbers of cells, proliferation would have to occur prior to transdifferentiation and all non-converted cells would be wasted, impacting the overall efficiency of the bioprocess. Nevertheless, these reprogramming technologies may be pursued using non-integrating methods in order to avoid potential regulatory issues with transgenes.

Adult stem cells

Mesenchymal stem cells

Many tissues in the adult body contain a reservoir of stem cells needed to replenish cell populations due to injury, cell death, or normal cellular turnover. These are referred to as adult stem cells. One of the most studied adult stem cell types is the mesenchymal stem cell (MSC), sometimes also referred to as mesenchymal stromal cells. MSCs are most commonly obtained from purified cell populations originating from a bone marrow or adipose tissue biopsy, although other sources such as the placenta, dental pulp, or umbilical cord have also been cited. Indeed, the diverse set of tissue sources and resultant cell phenotypes has called into question how MSCs should be defined and whether they are stem cells at all (Sipp, Robey, and Turner 2018). While guidelines from the International Society for Cellular Therapy (Dominici et al. 2006) have aimed to better define MSCs, some criteria such as cell surface marker expression may not be applicable in species outside of humans and thus will need to be defined. Additionally, MSCs are typically defined in part by their ability to form osteoblasts, adipocytes, and chondrocytes, while MSC potency toward skeletal muscle cells is somewhat limited and can be dependent on tissue source. In general, the multipotent nature of MSCs can be harnessed by culturing cells in specific cell culture medium formulations in order to bias their differentiation pathways. Thus, MSCs can serve as a readily attainable source of starting cells capable of making the principal cellular components of meat.

Fibroblasts, myofibroblasts, and fibroadipogenic progenitor cells

Homeostasis of the skeletal muscle involves a variety of resident muscle cell types. Deposition of extracellular matrix components that make up the muscle connective tissue is a key feature in guiding homeostasis, repair, and regeneration. This extracellular matrix deposition can occur in muscle from cell types including fibroblasts, myofibroblasts, and fibroadipogenic progenitor cells. Fibroadipogenic progenitor cells are a special type of mesenchymal progenitor cell capable of differentiating into myofibroblasts, adipocytes, chondrocytes, or osteogenic cells, but not myoblasts (Biferali et al. 2019). Although not responsible for the production of skeletal myotubes themselves, fibroadipogenic progenitors can secrete a variety of growth factors and cytokines that increase or influence myogenesis and myogenic differentiation (Wosczyna and Rando 2018). These cell types are also known to influence the accumulation of fibroblasts and adipocytes within the connective tissue, especially in disease contexts. Given that the defining features of these fibroblast populations are still being delineated, it’s possible that those working with mesenchymal stem cell populations derived from the skeletal muscle tissue will actually be culturing, in part, heterogeneous fibroblast populations.

Myosatellite cells

The resident stem cell populations in adult skeletal muscle tissue are referred to as myosatellite or satellite cells ). Satellite cells lie alongside myofibers under the basal lamina of the muscle tissue where they remain quiescent until activated upon injury or stress. In mouse, there are roughly 550 satellite cells per 1 mg of muscle tissue (Bentzinger, Wang, and Rudnicki 2012), making them one of the most abundant tissue-specific stem cell populations in the body. Satellite cells can be obtained from a small muscle biopsy, under local anesthesia or from animals recently slaughtered, and purified in the lab based on an array of well-characterized cell surface markers (L. Liu et al. 2015). However, maintaining their proliferative capacity in vitro outside of the resident muscle niche has been challenging and is an area of active research. As a tissue-specific stem cell, activated satellite cells readily give rise to myoblasts, which eventually lead to the formation of myocytes, multinucleated myotubes, and myofibers, each delineated by the expression of key transcription factors (Chal and Pourquié 2017). There is some evidence that satellite cells can enter an alternative mesenchymal-like pathway, leading to the generation of other cell types such as adipocytes (Shefer, Wleklinski-Lee, and Yablonka-Reuveni 2004), however this has been refuted (Starkey et al. 2011). Therefore, satellite cells offer the most direct method for obtaining skeletal muscle tissue in vitro, but may not be an ideal starting cell type for the creation of other cellular components of meat.

Cell banking

A reproducible and consistent cell line is essential for any bioprocess. Due to the large number of cell population doublings needed to make cultivated meat at scale, there exists a sizable concern for genetic drift and cell line stability, which may lead to inconsistencies in downstream processing and final product. In essence, as cells continue to divide and replicate their DNA, the probability of an increased burden of genetic variation (single nucleotide polymorphisms, copy number variation, large insertions or deletions, epigenetic changes, or aneuploidy) also increases. In some cases, these variations can be harnessed for improved processing (e.g. adaptation to suspension culture or lower concentration of growth factors), however, in general, genetic stability is favorable for a reproducible process.

In order to mitigate the risk of genetic drift, cells will need to be initially expanded, validated via rigorous quality control, and cryopreserved as a master cell bank. Individual vials from the master bank can then be serially subcultured to produce working cell banks. Animal-origin free and chemically-defined cryopreservation techniques will need to be optimized for a diverse set of cell types from various species. This strategy can allow for batch processing of cultivated meat or continuous processing of progenitor stem cells, at least until sufficient differences are detected wherein the culture can then be restarted. Similar strategies are already employed in other biomedicine industries, such as vaccine production, and these may serve as a guide for the cultivated meat industry.

Currently, there exist few publicly available cell lines of the aforementioned cell types from agriculturally-relevant species for cell-based meat production. Thus, there is a great need for the creation of new cell lines that can be banked and distributed to researchers in academia and industry alike. These biorepositories can be set up similarly to those dedicated to housing tissues and cell lines for efforts related to endangered animal conservation or large scale distribution networks (e.g. American Type Culture Collection). Having many cell lines available from a diverse array of animal species will be one of the most important factors in bootstrapping the research required for the cultivated meat industry to thrive long-term. The Good Food Institute is funding a project, the “Frozen Farmyard,” to accomplish this goal, and has partnered with reagent provider Kerafast to house cell lines in a biorepository for cultivated meat. Please e-mail cells@gfi.org if you want to learn how to deposit your own cell line or have a suggestion for an existing cell line that you’d like to see housed in the repository.

Species differences

Much of the knowledge for culturing stem cell lines has come from the fields of cell-based therapy and regenerative medicine. While there are many fundamental similarities in laboratory techniques, protocols, and reagents that can be used in cell-based meat production, an important difference lies in growing cells from different animal species. The vast majority of published literature using the cell types previously described comes from studies in human and mouse. While there exist examples of studies using the stem cells described from bovine, porcine, ovine, avian, and piscine animals, the field as a whole lacks well-established protocols and the degree of rich scientific literature from which to draw upon. Despite this, a vast amount of information, mostly held privately by corporations, does exist on the cellular biology and genetics of livestock species, which may be leveraged for adapting livestock species for cell culture. Additionally, resources such as sophisticated genome annotations, validated antibodies, and other -omics datasets will need to be generated. Lastly, while key developmental processes are generally conserved by evolution, it remains unclear the extent to which species differences will affect the success of applying human- or mouse-based cell culture strategies to evolutionarily distant species such as crustaceans or fish. Thus, the overall bioprocess will likely be replicated from species to species, but key differences, either advantageous or disadvantageous, are to be expected due to inherent biological differences across an evolutionarily diverse set of species.

Cell line considerations

Other cell line selection strategies may entail generation of multiple cell lines from an individual animal, different biopsy locations within an animal, the age or sex of the animal, different individuals within the same species or breed, or clones of each derived cell line. For example, selective breeding has produced desirable traits in specific animal breeds and thus these same traits may be strategically recapitulated in vitro (e.g. using cattle from the Belgian Blue versus Holstein breed for skeletal muscle production). This has begun to occur in the cultivated meat industry, exemplified by the Argentinian-based company Cell Farm Food Tech, which aims to produce mesenchymal stem cell lines from unique Argentinian cattle breeds and US-based JUST, which has partnered with the Toriyama farm in Japan to reproduce their renowned Wagyu beef. When possible, cell lines should also be obtained from animals that are raised in closed flocks, herds, or colonies, and free of known specific pathogens to reduce the risk of adventitious agents being present.

The location of an initial cell biopsy may also have downstream effects. As previously discussed, this could influence the differentiation potential of a mesenchymal stem cell population. For myosatellite cells, a muscle biopsy from a region of fast-twitch muscle fiber will in turn bias production of fast-twitch muscle fibers, which may influence final taste, texture, and metabolic rates of the cells in culture (Y.-C. Huang, Dennis, and Baar 2006). Location variables are less likely to influence pluripotent stem cell-derived outcomes, however individual and clonal variation amongst pluripotent stem cell lines is a notoriously difficult problem to manage (Kyttälä et al. 2016). With these considerations in mind, it’s likely that many species, stem cell types, and cell line variables will be explored in order to determine the best cell lines for cultivated meat production. However, one of the inherent advantages of using cell culture to produce meat rather than an animal is the range of real-time testing, data analysis, and parameter iterations which can be made from biopsy to product packaging. These advantages can accelerate the rate of innovation to reach success and create new products versus traditional animal agriculture.

Cell proliferation and immortalization

In general, the process of cultivated meat production following cell line selection can be broken up into two phases: proliferation and differentiation. In the proliferation phase, stem cells divide repeatedly to generate a large number of cells until they are transferred to a new environment and triggered to differentiate into a mature cell type via changes in scaffolding, medium composition, or both.

One hurdle in obtaining a large number of cells is that the number of times a cell can divide is inherently limited based on the Hayflick Limit. The Hayflick Limit imposes a limitation on cell divisions due to degradation of end-capping chromosomal telomeres following each cell division. Once a certain number of cell divisions occurs (typically around 30-50 in vitro for human cells), the cells enter a state of senescence and stop dividing. Thus, the number of potential cells acquired from a single starting batch is biologically limited. Some cells, however, can bypass the Hayflick Limit and achieve cell immortality. Pluripotent stem cells achieve immortality in part by epigenetic changes (Hochedlinger and Jaenisch 2015) and up-regulation of the enzyme telomerase (Y. Huang et al. 2014), which prevents telomere degradation. This property makes pluripotent stem cells especially useful in initial scaling, although genetic drift during proliferation may also lead to cell senescence or apoptosis.

While some adult stem cells can retain some telomerase expression (Hiyama and Hiyama 2007), it is insufficient to acquire immortality. One alternative method is to rely on the accumulation of a sufficient number of mutations in vitro to bypass normal cellular checkpoints and achieve spontaneous immortalization. Many cell lines used in research were derived from spontaneously immortalized lines, however the mutational burden can also alter the biology of the cells in unpredictable ways, potentially limiting their utility. Additionally, biological variation between species or cell type can contribute to the probability of spontaneous immortalization or cell transformation. For instance, the naked mole rat, which is highly resistant to cancerous cell transformation, is hypersensitive to contact inhibition (Seluanov et al. 2009) and resistant to iPSC reprogramming (Tan et al. 2017). Other species such as lobsters and fish, which retain high telomerase expression (Klapper et al. 1998; Gomes, Shay, and Wright 2010), or elephants, which have multiple copies of the p53 tumor suppressor (Sulak et al. 2016), may thus be easier and harder, respectively, to achieve transformation, although in vivo telomerase expression levels do not necessarily correlate to those observed in vitro (Venkatesan and Price 1998). These and other unique animal properties, known or currently unknown, may be strategically harnessed for cultivated meat production.

Having multiple cell lines or clonal lines will also enable companies to choose lines that have the best downstream characteristics (e.g. proliferation rate, differentiation potential, etc), potentially avoiding labor-intensive strategies such as directed evolution to acquire the same characteristics. For example, cellular proliferation can be boosted by naturally-occurring variation over time whereby selective advantage is conferred with variants associated with cell growth. By sequencing large batches of human pluripotent stem cells, growth advantages were observed to be conferred by chromosomal aberrations (Amps et al. 2011) and the acquirement of dominant negative mutations in p53 (Merkle et al. 2017). While it remains to be seen if these specific aberrations will naturally occur in non-human stem cells, it is nevertheless the case that selected stem cell lines from clonal populations or multiple individuals will likely harbor unique variation that drives their utility in the bioprocess being built.

A targeted immortalization approach can be achieved by over-expression of exogenously added genes or viral proteins. In these cases, the over-expression of telomerase in combination with inhibition of cell cycle genes such as p16 or Rb (Tsutsui et al. 2002) or use of viral elements such as the SV40 Large T Antigen (Jha et al. 1998) or adenovirus type 5 E1 gene (Sieber and Dobner 2007) have been utilized for cell immortalization. Precision gene editing (e.g. CRISPR) methods can be used in combination to insert these genes into safe harbor loci (Sadelain, Papapetrou, and Bushman 2011), minimizing the risk of alterations in global gene expression. Additionally, tandem use of genetic engineering strategies can create ON-OFF switches for immortalization, through use of Cre-lox or Flp-FRT recombination (Robin et al. 2015; Westerman and Leboulch 1996) or piggyBac transposon (Xie et al. 2016) systems. Thus, immortalization can be readily targeted or reversed, however, it is currently unclear how spontaneously immortalized or intentionally immortalized lines will be regulated, as significant genetic alterations and use of genetic engineering may warrant exclusionary regulatory standards.

Additional methods to improve proliferation rates or maintain proliferative capacity of non-immortalized stem cells also exist. For example, in myoblasts, use of small molecule compounds can assist in maintaining proliferative capacity via targeting of proliferation pathways (Bar-Nur et al. 2018) or inhibition of specific proteins such as STAT3 (Tierney et al. 2014), p38 (Ding et al. 2018), and Setd7 (Judson et al. 2018). Other strategies include mimicking an injured or regenerative state via growth in the presence of cytokines (Fu et al. 2015), addition of small peptides normally released following exercise (Vinel et al. 2018), or growth of cells in a hypoxic environment which may more closely mimic fetal oxygen levels (W. Liu et al. 2012). Similar strategies can tune biochemical pathways in other cell types such as adipocytes or chondrocytes. Many genetic engineering methods can also be employed to achieve similar results. These include gene over-expression via inducible or constitutively active systems and gene inhibition or knockout. In general, mimicking the native stem cell niche to retain stemness in vitro is an area of active research, and there exists a broad amount of possibilities for implementing these strategies for cultivated meat production.