Source: Modern Meadow submitted to
ENGINEERED COMESTIBLE MEAT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0228895
Grant No.
2012-33610-19537
Project No.
MOW-2012-00097
Proposal No.
2012-00097
Multistate No.
(N/A)
Program Code
8.5
Project Start Date
Jun 1, 2012
Project End Date
Oct 31, 2013
Grant Year
2014
Project Director
Marga, F. S.
Recipient Organization
Modern Meadow
1601 S. Providence Road
Columbia,MO 65211
Performing Department
(N/A)
Non Technical Summary
Present farm and industrial meat production methods and technologies have a number of associated problems including health risks (infectious animal diseases, nutrition-related diseases), resource intensity (land, water, energy), damage to environment (green house gas emission, erosion, biodiversity loss) and ethical challenges (animal welfare). With increasing worldwide demand for meat, it is expected that some of these problems will become critical. The objective of this proposal is to develop a fundamentally new approach to edible meat production. The approach is based on bio-printing, a novel tissue engineering technology. In this technology, conveniently prepared multicellular aggregates (the bio-ink particles) are delivered into a biocompatible support structure according to a design template (compatible with the shape of the desired biological construct) by a computer-controlled delivery device (the bio-printer). Biological assemblies form after deposition of the discrete bio-ink particles, through morphogenetic processes akin to those evident in early embryonic development, such as cell sorting and tissue fusion. The resulting construct is transferred to special purpose bioreactor for further maintenance and maturation to make it suitable for use (e.g. implantation in medical applications). So far, bio-printing has been applied to build three-dimensional tissues and organ structures of specific architecture and functionality for purposes of regenerative medicine. Here we propose to adapt this technology to building meat products for consumption. The technology has several advantages in comparison to earlier attempts to engineer meat in vitro. The bio-ink particles can be reproducibly prepared with mixtures of cells of different type. This allows for control in composition that enables the engineering of healthy products of great variety. Printing ensures consistent shape, while post-printing structure formation and maturation in the bioreactor facilitates conditioning. As meat is a post mortem tissue, the vascularization of the final product is less critical than in medical applications (although important for taste an objective to be further pursued in Phase II). Overall, this process allows for greater structural precision than other approaches and higher throughput for eventual scaling to industrial production. We anticipate that this Phase I application will result in a macroscopic size (~2 cm x 1 cm x 0.5 mm) edible prototype and will demonstrate that bio-printing-based in vitro meat production is feasible, economically viable and environmentally practical. Successful in vitro meat engineering addresses a number of societal needs, thus the commercialization of the method has high market potential. The consumer acceptance of such products may not be without challenges. We expect it will first appeal to culinary early-adopter consumers and the segment of the vegetarian community that rejects meat for ethical reasons. With reduction in price, it can reach the masses with religious restrictions on meat consumption (people restricted to Hindu, Kosher, Halal diets) and finally populations with limited access to safe meat production.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
51135201030100%
Goals / Objectives
The objective of this proposal is to construct muscle tissues by a novel and versatile tissue engineering technology and to assess their texture and composition for use as minced meat. The patented "print-based" technology has several distinguishing features. It is scaffold-free - it does not rely on any artificial material to form the desired structure. The process to build a tissue construct utilizes the automated deposition of convenient multicellular units, suitable for rapid prototyping and high-throughput production. The method has solid scientific underpinning based on tissue self-assembly processes akin to those evident in early morphogenesis (i.e. tissue fusion, engulfment and cell sorting). The ultimate product that will be developed based on the proposed studies is an animal muscle strip that can be used as minced meat for the preparation of sausages, patties and nuggets. The two aims that will be pursued in this Phase I application are 1) to fabricate 3D cellular sheets composed of porcine cells and 2) to mature the cellular sheets into muscle tissue and measure its meat characteristics. The related technical objectives are 1) to determine the optimal cellular composition (type and ratio of muscle cells, fibroblasts and adipocytes) to produce "easy-to-handle" cellular sheets and 2) to find the most efficient stimulation method (mechanical, electrical or a combination of both) to achieve muscle formation with similar mechanical and biochemical properties to meat. The successful completion of the proposed project will provide the optimal parameters and conditions for engineering strips of mammalian muscle tissue ((2 x 1 x 0.5) cm3) with appropriate mechanical and biochemical properties to be used as minced lean meat. The building of larger pieces, that may need to be perfused and engineered around bio-printed blood vessels, would be carried out in Phase II.
Project Methods
The objective of this proposal, pursued through two Specific Aims is to construct a strip of edible porcine tissue using print-based tissue engineering approach, a viable technology for building biological structures of desired shape and functionality. Convenient bio-ink units composed of 3 cell types (muscle cells, fibroblasts, adipocytes) will be prepared and deposited in three-dimensional (3D) sheet arrangement, using agarose as support material (but not part of the final construct). Solid sheets will form during the post-printing fusion of the bio-ink units. We will systematically vary the ratios of the three cell types to optimize the quantity of fibroblasts necessary for sufficient extracellular matrix (ECM) production for easy-to-handle sheet formation. Easy handling will be defined by our ability to manipulate these constructs (i.e. lift, transfer to the bioreactor, etc.). Cylindrical units (that can have similar or different composition) of 2 cm length and 500 micrometer in diameter will be printed side by side into an agarose mold using the NovoGen MMX Bioprinter. Upon post-printing fusion of the cylindrical bio-ink units, sheets will be transferred into the bioreactor and subjected to chronic (i.e. persistent) low frequency stimulation. Electrical stimulations will be performed with an in-house built apparatus. The amplitude and frequency of the simulation will be varied to determine which parameters produce mature aligned muscle fibers. Electric stimulation will be evaluated by its effect on the biophysical (Water holding capacity (WHC), pH, tenderness) and biochemical (proximate analysis) properties of the bioprinted strip. These properties will be measured and compare to values obtained for meat. The method to measure WHC is based on the filter paper press. A small (300 mg) piece of the construct will be pressed onto an oven-dried Whatman 125-mm filter paper at 3000 psi for 3 minutes. The WHC values are calculated as the ratio of the area of expressed water to the area of the pressed meat sample. pH measurements are important for detecting meat quality traits (Normal, Pale, Soft and Exudative (PSE) or Dark, Firm and Dry (DFD)). Specific instruments have been developed to perform the pH analysis of meat products. Measurements are performed by directly inserting the device electrode into a meat sample with the resulting pH values appearing on the device display. Tenderness will be measured using the 'Warner-Bratzler shear force test' according to standardized protocol. Proximate compositional analysis will be performed using a SMART Trac System that uses a unique combination of microwave and NMR technology in one analyzer to directly measure fat and moisture quickly and accurately.

Progress 06/01/12 to 10/31/13

Outputs
Target Audience: Modern Meadow's target audience is the scientific community, the industry as well as the public. Several avenues have been used to communicate the company's objectives, such as public talks to raise awareness on industrial meat production and alternatives to it, invitational talks at academic institutions and conferences and participation at forums organized by federal agencies (e.g. NASA) or private foundations. The company also uses its website to communicate with the public. Finally, Modern Meadow has been featured in a number of media write-ups and the websites of organizations focusing on issues of high public interest (e.g. New Harvest, Humane Society). Changes/Problems: The major problem, described above, we encountered was working with satellite cells. As mentioned, we feel we are on the right track to solve this problem with the newly established collaboration with the Department of Animal and Poultry Sciences of Virginia Tech. So far we have not been able to locate a commercial source of porcine smooth muscle cells. Therefore we used bovine smooth muscle cells available from the Coriell Institute. However this factor does not affect any of the major goals and objectives of the project. TheDepartment of Animal and Poultry Sciences of Virginia Tech has access to pigs and cattles that are raised on campus. Established protocols for satellite cells from both species are available. One of their team members has developed biopsy methods for collecting tissue samples suitable for isolation of satellite cells. As Modern Meadow develops, a priority will be to establish primary cell lines from different species (bovine, porcine, ovine, etc.) directly from our own animal sources. Collaboration has been established with Dr. Carol Lorenzen (Animal Research Science Center, University of Missouri – Columbia) a Scientific Advisory Board member to the company, to obtain tissue samples. Her department will provide Modern Meadow with bovine tissues (skin, muscle, etc.). Cells will be isolated in Modern Meadow’s laboratory according to standard protocols (or protocols developed in collaboration with the the Department of Animal and Poultry Sciences of Virginia Tech) and cell lines will be established. Ultimately, cells will be isolated from biopsies. What opportunities for training and professional development has the project provided? From its inception, Modern Meadow has provided training opportunity for undergraduate students to participate in the company's activities. The company typically hires students with at least minimal experience in cell culture techniques and then trains them to perfect these skills and learn a number of sophisticated techniques to build extended biological structures, such as tissues using modern equipment, notably 3 dimensional bioprinters. As the company has special needs, team members are skilled in designing and building special purpose devices in house. Students also participate in this activity and become "tinkerers". In this Phase I SBIR project we provided training opportunity for two undergraduate students, Amanda Prescott and Madeline Komes, both undergraduates at the University of Missouri- Columbia. How have the results been disseminated to communities of interest? The salient features of the project resonate strongly with large segments of the population. Public awareness in the adverse impact of the meat industry on the environment and the well being of animals is growing. Modern Meadow has disseminated its objectives, approaches and consequences of its activity at various forums. Company members have been invited speakers during this project's period at a number of scientific conferences (detailed above) and public forums. The company has also been featured in numerous media write-ups and uses its own website to bring across its message. Presentations by Modern Meadow team members at public forums The future of meat, 2b AHEAD German Innovation Society Conference, June 19-20, 2012, Wolfsburg, Germany. The future of agriculture, Google Sci Foo Camp, Aug. 3-4, 2012, Google Campus Mountain View, CA On sustainable, scalable meat, Solve for X Google Symposium, Feb. 11, 2013 Mountain View, CA The future of industrial meat production, Future Tense New America Foundation Symposium, April 12, 2013, Washington DC Leveraging Science to Meet the World’s Burgeoning Demand for Animal Products, Chicago Council Global Food Security Symposium, May 21, 2013, Washington DC Tissue engineering meat, IdeaCity, June 19-21, 2013, Toronto, Canada Media/News Lab grown meat gives food for thought, Aug. 13, 2012, CNN, http://www.cnn.com/2012/08/13/tech/innovation/lab-grown-meat/index.html 3D printed meat: It's what's for dinner, Aug. 15, 2012 CNET http://news.cnet.com/8301-11386_3-57493377-76/3d-printed-meat-its-whats-for-dinner/ Peter Thiel’s Breakout Labs Funds Tissue Engineering, Aug. 15, 2012, Techcrunch http://techcrunch.com/2012/08/15/breakout-labs-grantees/ Peter Thiel Bets Big On 3-D Printed Meat, Aug. 15, 2012, FastCompany http://www.fastcoexist.com/1680383/peter-thiel-bets-big-on-3-d-printed-meat Peter Thiel Backs 3D Bioprinted Meat Start-Up Modern Meadow, Aug. 16, 2012, The Huffington Post http://www.huffingtonpost.com/2012/08/16/peter-thiels-3d-meat_n_1789665.html?utm_hp_ref=technology&ir=Technology#slide=1108839 Billionaire Peter Thiel’s Latest Investment: 3D-Printed Meat, Aug. 16, 2012, TIME http://techland.time.com/2012/08/16/billionaire-peter-thiels-latest-investment-3d-printed-meat/ A Pork Chop to Change the World (Yes, Really), Nov. 1, 2012, Inc. http://www.inc.com/john-mcdermott/big-ideas/modern-meadow-in-vitro-meat.html Modern Meadow aims to print raw meat using bioprinter, Jan. 21, 2013, BBC http://www.bbc.co.uk/news/technology-20972018 Finding ‘meatless’ meat for a world of cities, Feb. 25, 2013, The Seattle Times http://seattletimes.com/html/opinion/2020433414_peircecolumnmeatlesscitiesxml.html News 2050: All meat sales banned, 26 March, 2013, BBC http://www.bbc.com/future/story/20130326-news-2050-all-meat-sales-banned 100 Brilliant Companies: The Company Leading the Future of Farming, May 24, 2013 Entrepreneur http://www.entrepreneur.com/article/226697 Modern Meadow Makes Leather and Meat Without Killing Animals, June 6, 2013, BloombergBusinessweek http://www.businessweek.com/articles/2013-06-06/modern-meadow-makes-leather-and-meat-without-killing-animals Company Website www.modernmeadow.com What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact Statement This Phase I SBIR project addressed issues facing present day industrial meat production practices, which are predicted to eventually become unsustainable. The project focused on biofabricating comestible animal protein products using tissue engineering technologies developed by some of Modern Meadow’s team members. The work, carried out jointly by several team members and utilizing the collaborative support of external experts, led to major progress towards the laboratory scale production of edible animal protein sources. In particular, expertise was developed to source and culture satellite cells with the help of animal scientists at Virginia Tech. To reach proof of concept animal protein rich product by the end of the project, along with skeletal muscle, work was also carried out using bovine smooth muscle cells. The latter also allowed to experiment with multiple approaches and introduce innovative elements that would have been challenging with satellite cells. Constructs composed of smooth muscle cells were prepared the same way as originally proposed and retained the desirable characteristics stemming from the preparation process. (i.e. the final product is still kill-free, rich in animal protein and can be processed by our expert chef collaborator). The outcome of the project will lead to pilot and eventually large-scale production with reduced need for resources, with decrease in the release of toxic chemicals and green house gases and without harm to animals. Economic studies indicate, while higher initially, cultured meat will become price-competitive with presently available meat products. Importantly, cultured meat production would bring healthy animal protein products to segments of the world populations that have limited or no access to it at present. Specific Accomplishments The original objective of this Phase I SBIR project was to prepare cellular sheets and eventually skeletal muscle tissue constructs. While this was pursued, due to the complexity of growing skeletal muscle from satellite cells we have pursued the same goals using bovine smooth muscle cells. Activity was organized around two aims and these were pursued through technical objectives and specific tasks. Aim 1: Fabricate 3D cellular sheets composed of muscle cells 1. Establish research scale cell culture technology To grow cells needed to fabricate 3D cellular sheets we first employed roller bottles resulting in a significant increase in our cell culture productivity. With this still research scale technology we were able to reliably produce 3cm x 7cm x 0.5 mm smooth muscle sheets. In anticipation of pilot scale production in Phase II, recently we started using CellStack Vessels, which allow to reach the equivalent of our roller bottle capacity with less space and cell culture material. We are able to reuse these vessels several times, leading to a significant reduction in cost and increase in efficiency. 2. Edible microcarriers For economically viable, larger scale production even the CellStack system will be insufficient. Therefore in collaboration with Glenn Prestwich at the university of Utah, we are developing edible microcarriers that will be used in 3D bioreactors. As a first step, we constructed microsponges composed of pectin-containing hydrogels and RGD peptides (for cell attachment) (RGD peptides will be replaced with cardosin A, a plant-based product naturally containing RGD peptides). This collaboration resulted in a provisional patent application. 3. Sourcing and culturing bovine satellite cells Satellite cells are challenging and our initial attempts to source, expand and differentiate them were unsatisfactory. As the skill for large-scale expansion of satellite cells is non-existent, with help from the Department of Animal and Poultry Sciences of Virginia Tech, we developed our own. To have reliable and continuous source of cells we established a relationship with an abattoir (R&R Processing, Auxvasse, MO). For the isolation of fresh tissue we are recruiting the help of veterinarians from the Quail Valley Veterinary Clinic in Jefferson City, MO. 4. Continuous printing Extended 3D cellular sheets have been constructed by several methods. One utilizes multicellular spheroids, which after maturation are deposited (either manually or with a use of a bioprinter) onto a substrate to allow to fuse into the continuous sheet. Albeit simple and cheap this bioassembly method is inconvenient. It is challenging to produce identical size spheroids and to package them into bio-ink cartridges. Therefore we resorted to our earlier technology of bioprinting with finite length cylindrical bioink particles (composed of cellular paste). We then developed a method to continuous print the cellular paste (resulting in a utility patent application), to achieve faster, easier, more reproducible deposition and aesthetically more appealing sheet formation. Aim 2: Mature the cellular sheets into muscle tissue and measure its meat characteristics. 1. Assembly and post assembly maturation Multilayered printed sheet constructs are incubated to promote fusion of the cylinders. After fusion, the construct is rolled and further incubated to allow the rolled layers to fuse. This process results in a sausage-like product, whose casing is formed by the collagen support layer that is secreted by the cells themselves. The diameter of the sausage depends on the size and number of sheets rolled up and fused together. Sausages are removed from the culture medium, rinsed and stored in a fridge. 2. Testing the final tissue constructs For analysis and testing sausages were weighted and ground up. Proximate analysis was performed on a ~4g sample and pH measurements on 5g samples. The proximate analysis results were compared to those for ground beef and tripe. The proximate analysis revealed that our sample has high moisture content (87.73%) compared to ground beef (59.6-72.9%) and comparable to the moisture content of raw white tripe (84.16%). It is leaner (fat~1.07%) than ground beef (fat~6.2-21.7%) and tripes (fat~3.69%). The protein content was 11.2% for our samples. Protein content varies from 18.1 to 20.7% for ground beef and is ~12.07% for tripe. (Tripe is composed mainly of smooth muscle cells and is rich in collagen as are our samples.) The pH of our (two) samples was 6.75 and 6.96. This correlates with the pH of animal flesh at slaughter (~ 7.1). After slaughter, some of the glycogen in the meat turns into lactic acid resulting in lower pH. Our samples were directly frozen and such degradation didn’t occur. Water holding capacity test was performed by filter paper press method. No distinct line separated the sample and the moisture. We hypothesize that this is due to the high collagen content of our sample. In summary, we have successfully produced macroscopic size edible prototypes with a composition close to tripe. These were optimized for structure, form and cohesion but not yet for appearance, flavor or mouth feel. Samples were lightly grilled in olive oil and tasted by Modern Meadow employees and found quite acceptable.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Tissue Engineering Based Approach to Meat Production, Biannual Conference of the Farm Foundation Roundtable: Future of Global Animal Protein Products, January 11-12, 2013, Point Clair, Alabama
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Meat production by methods of tissue engineering, The Reciprocal Meat Conference of the American Meat Science Association, June 16-19, 2013, Auburn, Alabama


Progress 06/01/12 to 05/31/13

Outputs
Target Audience: Modern Meadow’s target audience is the scientific community, the industry as well as the public. Several avenues have been used to communicate the company’s objectives, such as public talks to raise awareness on industrial meat production and alternatives to it, invitational talks at academic institutions and conferences and participation at forums organized by federal agencies (US Army, the Navy, NIH and NASA), as detailed below. The company also uses its website to communicate with the public. Finally, Modern Meadow has been featured in a number of media write-ups (detailed below) and the websites of organizations dealing with issues of high public interest (e.g. the non-profit company New Harvest, http://www.new-harvest.org/). Changes/Problems: The major problem, described above, we encountered was working with satellite cells. As mentioned, we feel we are on the right track to solve this problem with the newly established collaboration with the group of Prof. J. Yoo at IRMWFU. So far we have not been able to locate a commercial source or porcine smooth muscle cells. Therefore we used bovine smooth muscle cells available from the Coriell Institute. However his factor does not affect any of the major goals and objectives of the project. The IRMWFU group is working with porcine satellite cells sourced (via biopsy) from laboratory pigs. As Modern Meadow develops, a priority will be to establish primary cell lines from different species (bovine, porcine, ovine, etc.) directly from our own animal sources. Collaboration has been established with Dr. Carol Lorenzen (Animal Research Science Center, University of Missouri – Columbia) a Scientific Advisory Board member to the company, to obtain tissue samples. Her department will provide Modern Meadow with bovine tissues (skin, muscle, etc.). Cells will be isolated in Modern Meadow’s laboratory according to standard protocols (or protocols developed in collaboration with the group of Prof. J. Yoo) and cell lines will be established. Ultimately, cells will be isolated from biopsies. What opportunities for training and professional development has the project provided? From its inception, Modern Meadow has provided training opportunity for undergraduate students to participate in the company's activities. The company typically hires students with at least minimal experience in cell culture techniques and then trains them to perfect these skills and learn a number of sophisticated techniques to build extended biological structures, such as tissues using modern equipment, notably 3 dimensional bioprinters. As the company has special needs, team members are skilled in designing and building special purpose devices in house. Students also participate in this activity and become "tinkerers". In this Phase I SBIR project we provided training opportunity for two undergraduate students, Amanda Prescott and Madeleine Komes, both undergraduates at the University of Missouri- Columbia. How have the results been disseminated to communities of interest? The salient features of the project resonate strongly with large segments of the population. Public awareness in the adverse impact of the meat industry on the environment and the well- being of animals is growing. Modern Meadow has disseminated its objectives, approaches and consequences of its activity at various forums. Company members have been invited speakers during this project's period at a number of scientific conferences (detailed above) and public forums. The company has also been featured in numerous media write-ups and uses its own website to bring across its message. Presentations by Modern Meadow team members at public forums The future of meat, 2b AHEAD German Innovation Society Conference, June 19-20, 2012, Wolfsburg, Germany. The future of agriculture, Google Sci Foo Camp, Aug. 3-4, 2012, Google Campus Mountain View, CA On sustainable, scalable meat, Solve for X Google Symposium, Feb. 11, 2013 Mountain View, CA The future of industrial meat production, Future Tense New America Foundation Symposium, April 12, 2013, Washington DC Leveraging Science to Meet the World’s Burgeoning Demand for Animal Products, Chicago Council Global Food Security Symposium, May 21, 2013, Washington DC Tissue engineering meat, IdeaCity, June 19-21, 2013, Toronto, Canada Media/News Lab grown meat gives food for thought, Aug. 13, 2012, CNN, http://www.cnn.com/2012/08/13/tech/innovation/lab-grown-meat/index.html 3D printed meat: It's what's for dinner, Aug. 15, 2012 CNET http://news.cnet.com/8301-11386_3-57493377-76/3d-printed-meat-its-whats-for-dinner/ Peter Thiel’s Breakout Labs Funds Tissue Engineering, Aug. 15, 2012, Techcrunch http://techcrunch.com/2012/08/15/breakout-labs-grantees/ Peter Thiel Bets Big On 3-D Printed Meat, Aug. 15, 2015, FastCompany http://www.fastcoexist.com/1680383/peter-thiel-bets-big-on-3-d-printed-meat Peter Thiel Backs 3D Bioprinted Meat Start-Up Modern Meadow, Aug. 16, 2012, The Huffington Post http://www.huffingtonpost.com/2012/08/16/peter-thiels-3d-meat_n_1789665.html?utm_hp_ref=technology&ir=Technology#slide=1108839 Billionaire Peter Thiel’s Latest Investment: 3D-Printed Meat, Aug. 16, 2012, TIME http://techland.time.com/2012/08/16/billionaire-peter-thiels-latest-investment-3d-printed-meat/ A Pork Chop to Change the World (Yes, Really), Nov. 1, 2012, Inc. http://www.inc.com/john-mcdermott/big-ideas/modern-meadow-in-vitro-meat.html Modern Meadow aims to print raw meat using bioprinter, Jan. 21, 2013, BBC http://www.bbc.co.uk/news/technology-20972018 Finding ‘meatless’ meat for a world of cities, Feb. 25, 2013, The Seattle Times http://seattletimes.com/html/opinion/2020433414_peircecolumnmeatlesscitiesxml.html News 2050: All meat sales banned, 26 March, 2013, BBC http://www.bbc.com/future/story/20130326-news-2050-all-meat-sales-banned 100 Brilliant Companies: The Company Leading the Future of Farming, May 24, 2013 Entrepreneur http://www.entrepreneur.com/article/226697 Modern Meadow Makes Leather and Meat Without Killing Animals, June 6, 2013, BloombergBusinessweek http://www.businessweek.com/articles/2013-06-06/modern-meadow-makes-leather-and-meat-without-killing-animals Company Website www.modernmeadow.com What do you plan to do during the next reporting period to accomplish the goals? The work so far performed led to several instructive consequences, which will be further explored in the remaining time. First, given that our constructs are to be prepared in planar geometry (even if the sheets are subsequently rolled into a sausage), we realized we can adhere to simpler and faster bio-assembly methods. Specifically, we can prepare multicellular aggregates (we have extensive experience in the preparation of such aggregates) and instead of using a bioprinter, simply either “pour” them on an appropriate substrate and let them fuse, or grind them collectively to arrive at a hamburger-like product. This approach is already being pursued and possibly will gradually become the assembly method of choice. Even though our cell culture methods are appropriate for the construction of laboratory size samples, in preparation for Phase II activity we have started pursuing a novel cell culture method specifically applicable to our project. Novel cell culture technology is based on single use bioreactors, which utilize disposable “bags”. This approach allows to use volume (instead of surface) to grow cells. However, in case of adherent cells (as ours) exploiting the volume necessitates the use of microcarriers. Most applications utilizing microcarriers aim at using not the cells but substances secreted by the cells. Thus separation of cells from microcarriers is not an issue. In our case such separation would be necessary making the approach tedious and expensive. We thus have started developing edible microcarriers that would not require cell separation and could serve as additional components for adjusting taste and texture. As mentioned, we have so far postponed working with skeletal muscle. We have however now established collaboration with the Institute of Regenerative Medicine at Wake Forest University (IRMWFU), one of the most eminent institutions of its kind. One project actively pursued at IRMWFU is engineering skeletal muscle tissue. Our collaborators (the group of Prof. J. Yoo) have developed robust techniques for working with human satellite cells (isolation, expansion, differentiation, maturation) and have started working with porcine satellite cells. As we have by now developed all the steps needed to build the extended “meat” construct, we anticipate that once satellite cells-derived myoblasts become available the appropriate construct formation and testing will be accomplished before the end of the Phase I project, by which we plan to build larger pieces, suitable for further processing by our professional chef-advisor.

Impacts
What was accomplished under these goals? The two aims were pursued through technical objectives. To reach these objectives several specific tasks had to be set up. 1. Establish research scale cell culture technology To fabricate 3D cellular sheets, representing extended biological constructs, large numbers of cells are needed. To be able to grow the required numbers of cells, since June 2012, we have acquired three roller bottle incubator systems resulting in a significantly increase in our cell culture productivity. One roller bottle system, when at full capacity, can hold 104 roller bottles. Each roller bottle has the equivalent capacity of 10 (15cm diameter) Petri dishes. This allows to grow the equivalent of 1040 petri dishes in one incubator. The roller bottle system has also dramatically improved efficiency. Processing two roller bottles takes approximately 10 minutes, while the equivalent petri dish capacity takes about 60-90 minutes. With this research scale cell culture technology we are able to reliably reproduce the needed cell capacity to produce 3cm x 7cm x 0.5 mm sheets; with one sheet needing approximately 7 roller bottles. 2. Continuous printing A new method based on the continuous printing of a cellular paste has been developed to achieve faster, easier, more reproducible deposition and aesthetically more appealing sheet formation than with individual multicellular cylindrical units. After culture in the roller bottles, the cells are detached and centrifuged. The concentrated cellular pellet is transferred in a specifically designed syringe that will serve as the head of a printer. The syringe has been engineered such that 1) its piston is immobilized during centrifugation 2) the supernatant removal is achieved through a hole into the piston (this step brings the piston in contact with the pelleted cells) and 3) the piston can be sealed before printing. The syringe is attached to the 3D printer. As the paste is extruded under medium, a cylindrical rod forms and the deposition can be pursued without interruption as long as cellular paste is available. 3. Assembly and post assembly maturation of the tissue constructs The continuously extruded cellular cylinder has lower cohesivity than individual multicellular units (aggregates or cylinders) since the cells have been in contact for a short time (only while in the syringe). This difference required printing in medium and providing a support to which the cells could attach. We chose to print on a collagen sheet produced by the cells themselves. Both fibroblasts and smooth muscle cells are able to produce such sheets in about 10 days when ascorbic acid is added to the medium to induce collagen production. The cells remain alive in the collagen sheet. The sheet assembly is performed by printing layers of cellular paste (in X and in Y direction) on top of the collagen sheet as shown in Figure 3 Left (the limiting factor for the number of sheets printed on top of each other is the amount of cellular paste in the syringe and the linear size of a sheet). The diameter of the cylinder and spacing between the lines can be varied and will affect the final thickness of the construct. The printed sheets are incubated for 2 days at 37oC and 5% CO2 to promote the fusion of the cylinders with each other and to the collagen sheet by migration of the cells. After fusion, the printed sheets are rolled on the top of each other using an in-house designed and fabricated rolling device and incubated an additional 2 days to allow the rolled layers to fuse. This process results in a sausage-like product, whose casing is formed by the collagen support layer. The diameter of the sausage depends on the size and number of sheets rolled up together. Once formed the sausages are removed from the culture medium, rinsed and then stored at -20oC. As we are currently using smooth muscle cells mechanical or electrical stimulation of the constructs is not necessary. 4. Testing the final tissue constructs for histology, biophysical and biochemical properties Before biochemical analysis, the sausages were weighed (their weight varied from 2 to 4.55 g depending on the number of sheets rolled together) and ground up in a meat grinder. A proximal analysis was performed on a ~4 g sample and pH measurements on 5 g (2 repeats). The proximal analysis results of our sample was compared to ground beef (Isaksson T et al. On-line, proximate analysis of ground beef directly at a meat grinder outlet. 1996 Meat Sci. 43, 245-53.) and 2 different tripes (Composition of green tripe, www.greentripes.com ; Composition of raw white tripe published at http://en.wikipedia.org/wiki/Tripe). The proximal analysis reveals that our sample has a high moisture content (87.73%) compared to ground beef (59.6-72.9%). It is comparable to the moisture content of raw white tripe samples (72.24 and 84.16% ). It is leaner (fat = 1.07%) than ground beef ( Fat ~ 6.2-21.7%) and tripes (Fat ~12.75 and 3.69%) (not surprisingly, as it does not contains fat cells) . The protein content was 11.2% for our sample. Protein content varies from 18.1 to 20.7% for ground beef and of 13.33 and 12.07% for the tripes. Tripe is mainly composed of smooth muscle cells and is rich in collagen as are our samples. The pH of our sample was 6.75 and 6.96. It correlates with the pH of the flesh of animals at slaughter (pH value of 7.1). After slaughtering, some of the glycogen in the meat turns into lactic acid. As a result, the pH value is lowered. Our samples were directly frozen and such degradation didn’t occur. A new series of measurement will be performed to follow the pH over time at 4oC. A water holding capacity test was performed by filter paper press method. No distinct line separates the sample and the moisture. Our hypothesis is that collagen present in high proportion in our sample may be responsible for this outcome since it is known to have high water retention. A rolled sausage has been processed for histology. Picrosirius red (PSR)-stained transversal histological sections of a sausage showing the presence of fibrous collagen content. The sample is heterogeneous with areas of various cell density. During this report period, we have successfully produced macroscopic size edible prototypes with a composition closed to tripe. Our products are a series of small “sausages” made from smooth muscle. These were optimized for structure, form and cohesion – not yet for appearance, flavor or mouth feel. Samples were tasted by Modern Meadow employees, without any salt/pepper/spices and only lightly grilled in olive oil. Not surprisingly the cooked samples had neutral taste, but otherwise were not distasteful at all.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: Cultured meat by self-assembly, Abstract for the 3rd TERMIS (Tissue Engineering and Regenerative Medicine) World Conference, Sept. 5-8, 2012, Vienna, Austria
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Tissue engineering based approach to meat production, Abstract for the Farm Foundation Round Table Meeting, Jan. 10-12, 2013, Point Clear, AL
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Building tissues and organs, NextMed Conference, Feb. 20-23, 2013, San Diego, CA
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Meat production by methods of tissue engineering, Conference Proceedings, Reciprocal Meat Conference, June 16-19, 2013, Auburn AL
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: Livestock-free production of edible animal protein, NASA Synthetic Biology Workshop on Food Production in Space, Sept. 19-20, 2012, NASA Ames