In the bioreactor, stem cells undergo myogenesis - the differentiation into muscle tissue. Embryonic cells with high levels of twist and sloppy paired transcription factors code for zygotic genes which then activate the dorsal proteins within the cells. Dorsal proteins are inherited from the animal ’ s mother and encourage the cells to divide, morph and collectively form a monolayer over the ectoderm. The ectoderm is one of three embryonic germ layers which the new monolayer folds over in order to produce the mesoderm. The embryonic cells are now mesodermal cells. Because of “cross talk” between the ectodermal and mesodermal layers, ectodermal signaling molecules such as Wingless and Decapentaplegic induce patterning in mesodermal cells. In particular, they influence whether or not the mesodermal cells have cardiac or somatic fates. Signaling within the mesodermal layer (autonomous signaling) results in segmentation gene products called Sloppy Paired and Even Skipped. These segmentation gene products create larger patterning trends based on how much Twist transcription factor is expressed in a particular zone. For instance, Zones with low Twist and high Even Skipped eventually give rise to cells with cardiac and vascular lineage and cells with high Twist and high Sloppy Paired eventually give rise to cells with somatic muscle. 289x289px|Founder cells and fusion competent myoblasts connect to form multinucleated muscle fibres. This generates equivalence groups, or regions of cell with similar lineage potential. Notch Signalling will then act on these equivalence groups and encourage the mesodermal cells to divide into two identical daughter cells - one of which will become a myogenic progenitor and the other, a fusion-competent myoblast. Then, these myogenic progenitors divide asymmetrically. In the first case, the two daughter cells can become founder cells with low expressions of Twist. Founder cells are the basis for muscle tissue which other cells bind to in order to create fibers. Alternatively, one daughter cell can become a founder cell (with low Twist), and the other can become an adult muscle precursor (with high Twist). The adult muscle precursors (AMPs) are quiescent until the larval stage while the muscle precursors are not due to Numb Signalling. Notch Signalling combined with Twist expression discourages cell differentiation. Numb protein inhibits Notch and is mostly active around founder cell equivalence groups rather than AMPs. This results in differentiation being repressed in the AMPs while the founder cells begin to specialize. Temperature. Mammalian cells developing in vivo are incubated at 37 degrees celsius . Simulating such a warm climate in a bioreactor requires energy inputs which thus increase production costs. Insect tissue can be grown to scale at room temperature or colder with little to no hindrance in cell development. Culture Conditions. As mammalian cells digest and metabolize glucose, they produce byproducts such as lactic acid which accumulate and acidify the cell's environment. The ability with which cells are able to uptake nutrients depends on the pH of the environment - it must be within a certain window for optimal growth. Lactic acid accumulation leads to inferior growth conditions for the cell. As such the environment must be “rebalanced” which is typically accomplished by replacing the entire culture medium frequently as every 2–3 days. However, the saturated culture media may still contain viable nutrients which makes the practise wasteful and expensive. Insect cells in part circumvent lactate production but are also tolerant to more acidic environments. When insect cell growth was compared at a pH of 5.5, 6.5 and 7.5, negligible difference was noted. As a result, insect cultures can have their media replaced in as long as 90 days. This is compounded by the fact that insect cells do not deplete added nutrients as fast as mammalian cells. They consume triglycerides, glucose and proteins at a slower rate suggesting that they have more efficient metabolic pathways. Additionally, insect cell cultures are typically contaminated with lipid cells called trophocytes or vitellophages which are precursors to insect egg yolk cells. These cells are a natural source of fat which can be consumed by the other insect cells. Serum Free Culture Media. Culture media is an instrumental part of cellular agriculture and generating cultured tissue because it ’s effectively what allows scientists to begin with a relatively small sample of animal stem cells and end up with enough to constitute an entire tissue. In order to proliferate, a cell does not only require essential nutrients and macromolecules but also growth factors. When mammalian cells grow in vivo, these growth factors are supplied by the animal ’ s blood. In order to replicate this, the culture medium usually consists of a basal mixture supplemented with extra growth factors. The basal medium makes up the bulk of the culture and contains most of the nutrients while the growth factors are added in trace amounts. As a result, the natural starting point is combining Fetal Bovine Serum (FBS) into the culture media. FBS is somewhat controversial because it comes from the blood of a dairy cow fetus. The two issues with this is that it is a) reliant on animals, hence defeating the goal of cellular agriculture and b) expensive because it is so inaccessible. Additionally, from a scientific perspective, FBS is chemically undefined, meaning that its composition varies between animals. For the sake of research consistency, this is not desirable. The ideal culture medium is one that is simple, can stimulate proliferation, is unreliant on animals, is accessible and is cheap. However, because of the fact that mammalian cells rely on a complex array of growth factors, finding a culture medium that satisfies all 5 of these criteria is an ongoing challenge. Insect cells on the other hand, come from insects which are biologically simpler organisms than mammals. They contain a fluid called hemolymph rather than blood and so do not rely on all the same growth factors as mammalian cells. Instead, insect cell medium typically uses a basal medium (such as Eagle ’ s Medium, Grace ’ s Insect Medium or Schneider ’ s Drosophila Medium) which is supplemented with plant based additives such as yeastolate, primatone RL, hydrolysates, pluronic lipids and peptides.
In the bioreactor, stem cells undergo myogenesis - the differentiation into muscle tissue. Embryonic cells with high levels of twist and sloppy paired transcription factors code for zygotic genes which then activate the dorsal proteins within the cells. Dorsal proteins are inherited from the animal ' s mother and encourage the cells to divide, morph and collectively form a monolayer over the ectoderm. The ectoderm is one of three embryonic germ layers which the new monolayer folds over in order to produce the mesoderm. The embryonic cells are now mesodermal cells. Because of "cross talk" between the ectodermal and mesodermal layers, ectodermal signaling molecules such as Wingless and Decapentaplegic induce patterning in mesodermal cells. In particular, they influence whether or not the mesodermal cells have cardiac or somatic fates. Signaling within the mesodermal layer (autonomous signaling) results in segmentation gene products called Sloppy Paired and Even Skipped. These segmentation gene products create larger patterning trends based on how much Twist transcription factor is expressed in a particular zone. For instance, Zones with low Twist and high Even Skipped eventually give rise to cells with cardiac and vascular lineage and cells with high Twist and high Sloppy Paired eventually give rise to cells with somatic muscle. 289x289px|Founder cells and fusion competent myoblasts connect to form multinucleated muscle fibres. This generates equivalence groups, or regions of cell with similar lineage potential. Notch Signalling will then act on these equivalence groups and encourage the mesodermal cells to divide into two identical daughter cells - one of which will become a myogenic progenitor and the other, a fusion-competent myoblast. Then, these myogenic progenitors divide asymmetrically. In the first case, the two daughter cells can become founder cells with low expressions of Twist. Founder cells are the basis for muscle tissue which other cells bind to in order to create fibers. Alternatively, one daughter cell can become a founder cell (with low Twist), and the other can become an adult muscle precursor (with high Twist). The adult muscle precursors (AMPs) are quiescent until the larval stage while the muscle precursors are not due to Numb Signalling. Notch Signalling combined with Twist expression discourages cell differentiation. Numb protein inhibits Notch and is mostly active around founder cell equivalence groups rather than AMPs. This results in differentiation being repressed in the AMPs while the founder cells begin to specialize. Temperature. Mammalian cells developing in vivo are incubated at 37 degrees Celsius . Simulating such a warm climate in a bioreactor requires energy inputs which thus increase production costs. Insect tissue can be grown to scale at room temperature or colder with little to no hindrance in cell development. Culture Conditions. As mammalian cells digest and metabolize glucose, they produce byproducts such as lactic acid which accumulate and acidify the cell's environment. The ability with which cells are able to uptake nutrients depends on the pH of the environment - it must be within a certain window for optimal growth. Lactic acid accumulation leads to inferior growth conditions for the cell. As such the environment must be "rebalanced" which is typically accomplished by replacing the entire culture medium frequently as every 2–3 days. However, the saturated culture media may still contain viable nutrients which makes the practise wasteful and expensive. Insect cells in part circumvent lactate production but are also tolerant to more acidic environments. When insect cell growth was compared at a pH of 5.5, 6.5 and 7.5, negligible difference was noted. As a result, insect cultures can have their media replaced in as long as 90 days. This is compounded by the fact that insect cells do not deplete added nutrients as fast as mammalian cells. They consume triglycerides, glucose and proteins at a slower rate suggesting that they have more efficient metabolic pathways. Additionally, insect cell cultures are typically contaminated with lipid cells called trophocytes or vitellophages which are precursors to insect egg yolk cells. These cells are a natural source of fat which can be consumed by the other insect cells. Serum Free Culture Media. Culture media is an instrumental part of cellular agriculture and generating cultured tissue because it is effectively what allows scientists to begin with a relatively small sample of animal stem cells and end up with enough to constitute an entire tissue. In order to proliferate, a cell does not only require essential nutrients and macromolecules but also growth factors. When mammalian cells grow in vivo, these growth factors are supplied by the animal ' s blood. In order to replicate this, the culture medium usually consists of a basal mixture supplemented with extra growth factors. The basal medium makes up the bulk of the culture and contains most of the nutrients while the growth factors are added in trace amounts. As a result, the natural starting point is combining Fetal Bovine Serum (FBS) into the culture media. FBS is somewhat controversial because it comes from the blood of a dairy cow fetus. The two issues with this is that it is a) reliant on animals, hence defeating the goal of cellular agriculture and b) expensive because it is so inaccessible. Additionally, from a scientific perspective, FBS is chemically undefined, meaning that its composition varies between animals. For the sake of research consistency, this is not desirable. The ideal culture medium is one that is simple, can stimulate proliferation, is unreliant on animals, is accessible and is cheap. However, because of the fact that mammalian cells rely on a complex array of growth factors, finding a culture medium that satisfies all 5 of these criteria is an ongoing challenge. Insect cells on the other hand, come from insects which are biologically simpler organisms than mammals. They contain a fluid called hemolymph rather than blood and so do not rely on all the same growth factors as mammalian cells. Instead, insect cell medium typically uses a basal medium (such as Eagle ' s Medium, Grace ' s Insect Medium or Schneider ' s Drosophila Medium) which is supplemented with plant based additives such as yeastolate, primatone RL, hydrolysates, pluronic lipids and peptides.
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