Compositions and methods for increasing the culture density of a cellular biomass within a cultivation infrastructure
This patent application is Memphis Meats’ 3rd application that has been made public. It centers around the hippo signaling pathway, a pathway that regulates organ size during development. Memphis Meats discovered that by inhibiting this pathway, cells behave in a number of ways that are useful for bringing down the cost of large scale culture. The hippo pathway has been a focus of other research recently, but the novel part about this patent application is using the pathway as a tool for cell line and bioprocess development.
What is the hippo signaling pathway?
The hippo signaling pathway regulates “contact inhibition”–the tendency of cells to stop proliferating when they come into contact with other cells. During organ development, cells trigger this pathway as the organ grows and cell density increases. This is how the body regulates how large organs grow to be.
Memphis Meats realized that this pathway is triggered when cells come in close contact in cell cultures. Their insight was that by deactivating this pathway, cells can continue to proliferate even in high densities, creating a more efficient culturing process and ultimately a cheaper end product. In particular, Memphis Meats found that inhibiting this pathway causes:
increased cell density in both 2D and 3D cultures,
increased proliferation rates in both 2D and 3D cultures,
increased ability to proliferate without fetal bovine serum, and
decreased tendency for muscle stem cells to differentiate into mature muscle cells. This is important since some stem cells have a tendency to differentiate in high densities, but after doing so cannot continue proliferating. Cultures will often have expensive growth factors to make sure this doesn’t happen, but disabling the hippo pathway can be a cheaper way to achieve the same goal.
Memphis Meats also mentions how inhibiting the hippo pathway can induce anchorage independence , although the extent and exact meaning of Memphis Meats’ claims are unclear. There is some evidence in the literature that hippo inhibition can allow cells to grow without an anchoring substrate, i.e. in single-cell suspension . This may be what Memphis Meats is claiming. However, they could also be claiming that the lack of contact inhibition allows cells to grow in clumps in suspension, called “aggregates.” It’s unclear whether Memphis Meats experimentally confirmed either of these possibilities, or whether the discussion is purely theoretical.
Mechanics of the Pathway
The most important effect of the hippo signaling pathway is inhibiting the Yes-Associated Protein 1 (YAP1) . YAP1 is a transcriptional co-activator, meaning that it binds to a TEAD  transcription factor to trigger the transcription of a set of genes. These genes lead to proliferation, and the other qualities that Memphis Meats wants. YAP1 is active by default, meaning that it is bound to the TEAD transcription factor. When the hippo signaling pathway is active and contact inhibition is triggered, there is a signaling kinase cascade that eventually leads to the phosphorylation of YAP1. YAP1 then detaches from the TEAD protein, stopping gene transcription.
YAP1 can also be deactivated in response to mechanical signals. This is called mechanotransduction.
How to inhibit the hippo pathway
The patent application is agnostic as to how the hippo pathway is disabled. Memphis Meats discusses a number of ways to do it:
Target the YAP1 protein directly. Memphis Meats describes a modified version of YAP1 that is immune to phosphorylation. By either ectopically expressing this protein in the cells or adding it into the media, Memphis Meats can replace the natural YAP1 protein with their modified version, preventing the hippo pathway from disabling gene transcription. This solution also prevents deactivation of YAP1 by mechanotransduction. The downside is that if this protein is ectopically expressed, the meat would be classified as a GMO.
Target proteins that are necessary for the hippo signaling kinase cascade. For example, Memphis Meats uses CRISPR to knock out the LATS1 or LATS2 kinases, which disables the entire hippo signaling pathway. The downsides are that 1) completely knocking out a protein could have unintended side effects, and 2) disabling the hippo pathway doesn’t prevent mechanotransduction from deactivating YAP1. However, using CRISPR would not yield GMO meat based on US classifications.
Expose the cells to a chemical or protein that disables the hippo signaling pathway, such as lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin. The upside to this approach is that no cell modification is needed. However, it would again not prevent deactivation of YAP1 via mechanotransduction. Also, the media additive could end up in the end meat product, which might be undesirable.
In the experiments described in the patent application, Memphis Meats seems to favor either ectopic expression of the always-active modified YAP1, or exposure to LPA. However, it’s not clear which of the many options will be best at scale.
How useful is this technology?
In Memphis Meats’ experiments, disabling the hippo pathway had impressive results. In one experiment, they observed a 4-fold increase in cell density in 2D culture. In another, they observed a 216% increase in cell proliferation rates. Many of the benefits were validated in both 2D and 3D cultures, although not yet at the scales that Memphis Meats will eventually need. If these efficiency gains transfer to large scale culture, this technology could be a very useful tool for bringing down costs.
Thanks to Alene Anello and Parendi Birdie for reviewing drafts of this post.
 What is anchorage independence? Most mammalian cells grow within a structure in the body–typically, the extracellular matrix. This presents a challenge for the large scale production of cells, since it’s most efficient to scale by using large tanks. However, it’s difficult for some cells to grow in these tanks since their natural state is to be attached to a surface. Cells like this are considered anchorage dependent.
Anchorage independence is extremely important for hitting the low cost requirements for cell-based meat. The problem is that mature muscle cells are naturally anchorage dependent.
One solution is this problem is to modify muscle cells to become anchorage independent. This can be accomplished with either GM or non-GM techniques, but requires substantial R&D investment. Another solution is to use naturally anchorage independent stem cells for the proliferation phase. This solves the problem of anchorage independence, but presents other difficulties, such as lower cell densities and need for expensive growth factors to keep cells dedifferentiated.
 For example, see http://genesdev.cshlp.org/content/26/1/54.full. In this study, hippo inhibition is shown to suppresses anoikis (cell death due to lack of an anchoring structure).
 And the paralog TAZ.
 For example, https://en.wikipedia.org/wiki/TEAD1.