Patent Analysis: Modern Meadow's Bioprinting Patents
Modern Meadow was one of the first startups to explore cell-based meat technology. In their early days, they filed a number of patent applications around meat bioprinting. Their first patent, Engineered comestible meat, describes the basic bioprinting technology, and each further patent application describes improvements to this technology.
Engineered comestible meat (2011, Granted)
This patent describes a method of bioprinting in which non-human myocytes are aggregated into successively larger units until they form a full tissue. At first, individual cells are combined into “multicellular aggregates,” small spherical or cylindrical collections of cells that adhere to one another and form the base unit of the bio-ink. These aggregates are printed into a layer, then allowed to fuse together. These layers are then stacked on top of one another, and are themselves allowed to fuse together to form a complete tissue.
The multicellular aggregates can be structured a number of different ways. They generally have a diameter of between 100 and 500 microns, and sometimes contain endogenous extracellular-matrix for stability. One method of creating multicellular aggregates involves (1) centrifuging a suspension culture, (2) extruding the resulting cell pellet into small spheres or cylinders, and (3) incubating the aggregates until the cells adhere to one another. This isn’t the only way to make multicellular aggregates though–other methods are described in Modern Meadow’s later patent applications.
A major problem in conventional tissue engineering is that once the tissue becomes thick enough, oxygen and nutrients cannot reach cells in the center of the tissue. Modern Meadow’s technique mitigates this problem by first creating many thin layers of multicellular aggregates in 2D culture. After the layers are completed, they are stacked on top of each other to create a 3D tissue. Importantly, layers fuse together into a finished product faster than the cells in the center of the tissues die.
Another major advantage of Modern Meadow’s method is that it creates structured tissue without scaffolding, a major driver of cost and complexity in 3D tissue creation.
At the end of the patent, Modern Meadow fleshes out a particular example of this technology. They use primary pig aortic smooth muscle cells co-cultured with primary pig coronary artery endothelial cells. They create cylindrical multicellular aggregates via the method described above, then pack the aggregates into a bioprinter cartridge. They then deposit the aggregates onto an agarose-based gel inside a petri dish using a digitally controlled bioprinting head. The printer creates a 10 cm x 10 cm layer of aggregates. They pour media over the layer and leave it for 12 hours, in which time the aggregates partially fuse with one another. The agarose is semi-permeable so that the media can reach cells at the bottom of the layer. Once the aggregates fuse, the entire layer can be peeled off. Modern Meadow repeats this process 65 times to create 65 separate cell sheets. To form a tissue, they stack the sheets such that the direction of the cylindrical aggregates in each layer is perpendicular to the ones in adjacent layers. They submerge the tissue in media while the layers fuse together. Eventually, the cells in the center begin to die due to oxygen deprivation, but the layers fuse together at a faster pace than the cells die. The result is a 2 cm thick slab of meat with the consistency of a hamburger patty.
Claims: The claims of the patent are much broader than the particular example discussed. The highest level claim covers any method of making meat that involves creating multicellular aggregates from non-human myocytes, fusing these aggregates into layers, then fusing these layers into tissue.
Spherical multicellular aggregates with endogenous extracellular matrix (2012, Patent Pending)
This patent application describes another way to create multicellular aggregates. Instead of centrifuging a suspension culture and then extruding the resulting pellet, Modern Meadow uses a 2D culture to create a layer of cells, then lets the layer naturally ball up. The resulting ball of cells can then be used as a spherical multicellular aggregate.
In order to get the layer of cells to ball up, Modern Meadow uses media with high ascorbic acid content. Ascorbic acid is known to increase endogenous expression of extracellular matrix (ECM) proteins, leading to the balling behavior. After the cells are cultured to confluence on a 2D plate, the edges of the cell layer naturally curl. The layer is then peeled off the initial culture plate and placed on a non-adherent surface (made from e.g. agarose). The new surface allows the layer to continue to ball up due to the higher ECM content. This last step can be automated to help scaling. By lowering the temperature of the initial culture plate, the cell layer will naturally detach, allowing Modern Meadow to simply turn the plate upside down to drop the sheet onto the non-adherent surface.
The major advantage of this method over the original method of making multicellular aggregates is that it doesn’t require centrifugation, meaning that the cells are less tightly packed. This allows media to perfuse further through the aggregates and the resulting tissue, mitigating the problem where cells in the center of the tissue die due to oxygen deprivation. Another benefit is that Modern Meadow doesn’t need to use extra enzymes to detach cells from their growth substrate.
Claims: This patent application aims to protect any method of making multicellular aggregates that involves forming a confluent layer of cells on an adherent substrate, then moving the layer onto a non-adherent substrate so the layer balls up.
Methods and devices for preparing and continuously printing multicellular cylinders onto biocompatible substrates (2013, Patent Pending)
This patent application describes a bioprinting nozzle that can continually extrude cylindrical multicellular aggregates. The nozzle is filled with a suspension culture then centrifuged so that the cellular material collects near the tip of the nozzle. Performing centrifugation and extrusion within one device simplifies the entire bioprinting process.
The nozzle is made up of four parts. At one end is the extrusion tip, where cellular material is extruded from. Next is the internal cavity, where the cell suspension is deposited, and where the centrifuged cell pellet is eventually held. After that is the venting piston, which exerts force on the cell pellet to extrude it. This piston has a pluggable hole so that after centrifugation it can move through the supernatant. Finally, there is a positive displacement pump that generates force to extrude the pellet.
To bioprint meat, the extrusion tip is first blocked with a short wire plug, and the internal cavity is filled with a cell culture suspension. Before centrifugation, the venting piston is secured in place so that it doesn’t apply pressure to the suspension during centrifugation. Next, the entire nozzle is centrifuged so that a cell pellet forms next to the extrusion tip. The piston is then pushed down the internal cavity through the supernatant liquid so that it comes in contact with the pellet. The supernatant is then removed. The hole in the venting piston is plugged so that the pellet doesn’t flow through the hole during extrusion, and the extrusion tip is unblocked. Finally, the nozzle is attached to a bioprinter and the pellet is extruded through the extrusion tip.
The extrusion is continuous as long as there is enough cellular material, but it can be cut at constant lengths while it is being bioprinted. This results in cylindrical multicellular aggregates compatible with the process in Engineered comestible meat.
Interestingly, since this process explicitly requires centrifugation, it is incompatible with the non-centrifuged multicellular aggregates described in Spherical multicellular aggregates with endogenous extracellular matrix. The two patents describe mutually exclusive improvements to Engineered comestible meat.
Claims: This patent application attempts to protect any method for continuously extruding cellular material with a nozzle that includes all of the following steps:
Blocking the extrusion tip of the nozzle,
filling the internal cavity with a cellular suspension,
securing the venting piston so that it doesn’t move during centrifugation,
centrifuging the the nozzle to form a cellular pellet,
moving the venting piston to come in contact with the pellet,
removing the supernatant from the internal cavity,
closing the hole in the venting piston,
unblocking the extrusion tip, and
extruding the cellular material through the extrusion tip.
Edible and animal-product-free microcarriers for engineered meat (2013, Granted)
This patent describes a method to make edible animal-free microcarriers. The microcarriers can be seeded with cells to make spherical multicellular aggregates compatible with the process in Engineered comestible meat, and can go inside the bioprinting nozzle described in Methods and devices for preparing and continuously printing multicellular cylinders onto biocompatible substrates.
Cells like muscle need to grow on a surface in order to survive. Microcarriers are small beads that provide surface area for such cells to grow in 3D culture. Historically, microcarriers were made out of a bio-incompatible material and either separated from the cells after the culture, or discarded along with the entire culture, depending on the application. Modern Meadow’s microcarriers are edible and animal-free so they can be left in the culture and included in the end meat product.
The microcarriers have two components. The first is pectin, a common polysaccharide found in the cell wall of plants. Pectin makes up the majority of the microcarrier by volume, and is used to give the microcarriers structure. The second component is cardosin, a polypeptide found in artichokes. Cardosin contains the amino acid sequence of Arginine, Glycine, and Aspartate (RGD), a motif that can bind to cells so they grow on the microcarriers. Both components are chemically modified so they can be cross linked via disulfide bond. The pectin is derivatized with cystamine to make pectin-thiopropionylamide (PTP), and the cardosin is thiolized. The patent doesn’t go into detail about how these modifications are made, but the result is a PTP-cardosin hydrogel. The hydrogel is separated into microbeads with a coaxial airflow bead generator [1] and freeze dried to make the microcarriers.
When the microcarriers are used in cell culture, muscle cells bind to the cardosins and then proliferate in and around the bead. Once the microcarriers are cellularized, they can be bioprinted using the nozzle in Methods and devices for preparing and continuously printing multicellular cylinders onto biocompatible substrates. The cells covering the outside of each microcarrier allow the microcarriers to fuse together to form layers.
The major benefit of these microcarriers is that they allow adherent cells like muscle to be cultured in 3D suspension, and they don’t need to be separated from the cells since they can be included in the end meat product.
Claims: This patent protects any method to create an edible animal-free microcarrier for cell-based meat by cross linking pectin with an RGD-containing polypeptide (e.g. a cardosin), then forming microbeads from this polymer.
Dried food products formed from cultured muscle cells (2012, Granted)
The first meat product that Modern Meadow created was a cultured “meat chip” [2], which is described in this patent. First, cultured cells are mixed with a plant-derived hydrogel (e.g. made from pectin). Rather than culturing cells inside the hydrogel, the hydrogel is mixed in after culturing to add texture and structure to the product. Flavor components can also be added. The mixture is spread into a sheet and dehydrated, resulting in a flat meat chip product. The chip is similar to jerky, but is more tender. Modern Meadow discusses optionally culturing the cells with the edible microcarriers from Edible and animal-product-free microcarriers for engineered meat.
Claims: This patent protects any method for creating a food product by culturing muscle on a plant-derived hydrogel (e.g. made from pectin), spreading the mixture into a sheet, and dehydrating the mixture.
After their initial work on cell-based meat, Modern Meadow adapted their bioprinting technology for making leather. Instead of using myocytes to create multicellular aggregates, they used collagen-secreting cells. Their technology was well suited for leather since leather requires a consistent texture, and is generally thinner than meat. Modern Meadow also has a number of patents in this area not discussed here.
Let me know what you think in the comments! Part 4 will be the final installment until more patent applications are made public. I’ll discuss Integriculture’s feeder cell bioreactor design, as well a couple patents that aren’t associated with any company. I’ll also provide some analysis on the landscape as a whole.
Footnotes
[1] See https://www.ncbi.nlm.nih.gov/pubmed/28040932 for an explanation of how to use coaxial air flow to make microbeads. Basically, liquid is dropped from a nozzle, and a downward airflow controls drop size.