INTEGRATED APPROACHES TO ENHANCE SUSTAINABILITY, RESILIENCY AND ROBUSTNESS IN US AGRI-FOOD SYSTEMS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: EXTENDED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1027620
• Grant No.: 2021-69012-35978
• Project No.: MASW-2021-05678
• Proposal No.: 2021-05678
• Program Code: A9201
• Project Start Date: Sep 1, 2021
• Project End Date: Aug 31, 2026
• Grant Year: 2022

Project Director
Kaplan, D.

Recipient Organization
TUFTS UNIVERSITY
28 SAWYER AVE
MEDFORD,MA 02155-5811

Performing Department
Biomedical Engineering

Non Technical Summary
As the world population increases to 10 billion by 2050, total food and meat production must rise by 70 and 100%, respectively, to satisfy global demand. The US food production system faces several issues in meeting this demand due to limited available agricultural water and land and increased greenhouse gas emissions. Increasing water scarcity in major production regions and increasing vulnerability to disruptions from natural disasters due to climate change are just some of the growing issues that prompt the need for new technologies in meat production. Also, a critical challenge in food supply chains is food loss issues that present significant sustainability and security challenges, with 60 percent of meat becoming processing waste (1.4 billion tons for livestock; 800 million tons for seafood). New sources of sustainable protein would help alleviate these concerns and are the focus of the present proposal. Cultivated meat production is emerging as a feasible solution to address immediate societal problems by developing new sustainable agri-food systems to feed a rapidly growing global population. This industry will provide nutritious and safe foods while reducing environmental impact and resource usage (78-96% fewer greenhouse gas emissions, 99% less land use, and 82-96% less water use). This project aims to innovate the food supply chain from cell to fork and enhance food sustainability, nutrition, and food security by developing a cell-based meat platform based on the integration of physical, biological, and social sciences. Cultivated-meat production is emerging as an alternative source of sustainable protein to help address nutrition and food safety for consumer choices. The development of cultivated-meat faces many obstacles on an industrial scale: (a) questions related to consumer acceptance, perceptions and expectations; (b) technical sound life cycle and techno-economic analyses; (c) limited access to low-cost media and suitable cell lines impacting scalability; (b) lack of available sustainable biomaterials to achieve nutritious, safe, and organoleptically accurate cultivated-meat; (c) lack of systematic approaches for training the next generation of professionals. Our central hypothesis is that a sustainable, cost-effective, and scalable cultivated-meat platform will increase food availability options for consumers, while decreasing environmental impact. This proposed work aims to develop new adoptable techno-economically viable cultivated-meat systems and develop new educational platforms for training future professionals through specific aims: 1. Evaluate consumer acceptance and consumer willingness-to-pay for cultivated meats, as well as flavor profiles; 2. Analyze the environmental performance of cultivated meat products in the US; 3. Outreach, extension, and educating the next generation of professionals for workforce development; 4. Develop a sustainable pluripotent stem-cell line platform; 5. Develop economically viable serum-free media; 6. Develop sustainable biomaterials scaffolds, and tissue engineering strategies, to support meat quality; 7. Optimize the processes and biomaterials integration to enhance nutritional value, quality, and safety.

Animal Health Component

0%

Research Effort Categories

Basic

25%

Applied

50%

Developmental

25%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
501	7010	1030	20%
502	7220	1000	15%
701	5010	2020	15%
803	3260	2000	10%
903	3799	3020	20%
402	7410	3010	20%

Knowledge Area
803 - Sociological and Technological Change Affecting Individuals, Families, and Communities; 501 - New and Improved Food Processing Technologies; 903 - Communication, Education, and Information Delivery; 502 - New and Improved Food Products; 701 - Nutrient Composition of Food; 402 - Engineering Systems and Equipment;

Subject Of Investigation
7010 - Biological Cell Systems; 3260 - Poultry meat; 7220 - Sensory evaluation/analysis methods (human senses); 3799 - Cultured aquatic animals, general/other; 7410 - General technology; 5010 - Food;

Field Of Science
2020 - Engineering; 1030 - Cellular biology; 3020 - Education; 1000 - Biochemistry and biophysics; 2000 - Chemistry; 3010 - Economics;

Keywords

3d tissues

alternative meat

animal agriculture

cell culture

novel foods

nutrition

scaffolds

Goals / Objectives
The long-term goal of this project is to develop novel food production systems using transdisciplinary approaches to achieve 40% increase in American agriculture production, with a reduction in environmental footprint by 50%. This project aims to develop new adoptable techno-economically viable cultivated meat systems and develop new educational platforms for training future professionals. To achieve these goals, our interdisciplinary team consists of molecular biologists, biomedical engineers, food engineers, biosystems engineers, data scientists, protein chemists, flavor chemists, sensory experts, food safety specialists, environmental scientists, and consumer specialists and stakeholders from all around the US will be working together on following objectives: 1. Evaluate consumer acceptance, consumer willingness-to-pay and flavor profile of novel meat via cellular agriculture, as both a stand-alone product and an ingredient in prepared dishes; 2. Analyze the environmental performance of cultivated meat products in the US; 3. Develop outreach, extension, and education for the next generation of professionals for workforce development and as technology leaders; 4. Develop sustainable pluripotent stem-cell line platform with the robust, scalable proliferation and differentiation potential for broad utility in the field; as a source of cultivars; 5. Develop economically viable serum-free, value-added media, and media recycling to support cell proliferation and differentiation needs and reduce system waste, by integrating molecular modeling, coarse-graining and long-time scale umbrella sampling methods, artificial intelligence, and high throughput screening to optimize for advanced functionality; 6. Develop sustainable biomaterials scaffolds, tissue engineering strategies, and fermentation technologies to support meat structure, color, and flavor development; 7. Optimize the processes and biomaterials integration to enhance nutritional value, quality, and safety.

Project Methods
This project consists of several integrated research, education, and extension activities to develop innovative technologies, train the next generation professionals and improve outreach and extension activities in cellular agriculture. Aim 1 will evaluate consumer acceptance, consumer willingness-to-pay and flavor profile of novel meat via cellular agriculture, as both a stand-alone product and an ingredient in prepared dishes. We will evaluate the consumer willingness-to-pay for, and drivers of preference of the novel meat using Qualtrics surveys and Real Choice Experiment approach for eliciting preferences and responses to information treatments. We will supplement RCEs with a multi-year online panel. All consumer-facing work will be subject to approval by the Social, Behavioral and Educational Research IRB. We will develop a lexicon for cultivated meat by evaluating the flavor, and sensory attributes, and by applying AI and machine learning for predicting the sensory properties of the products. Aim 2 will analyze the environmental performance of cultivated meat products in the US. We will analyze the life cycle environmental impacts of the cultivated meats using ISO 14044-2006 compliant life cycle assessments. Aim 3. will develop outreach, extension, and education for the next generation of professionals for workforce development and as technology leaders. We will establish a Center of Excellence as a national institute for cellular agriculture (NICA) that will coordinate outcomes of this project and collaborate with other agencies and stakeholders focused on various aspects of cultivated meat. This will include website development, COE extension and training plans, education of future professionals and consumer education. Aim 4. Will focus on developing sustainable pluripotent stem-cell line platform with the robust, scalable proliferation and differentiation potential for broad utility in the field. We will develop pluripotent stem-cell lines from different species and evaluate the proliferation and differentiation potential for developed cell lines. We will characterize the cells such as transfection efficiency, proliferation efficiency, cryopreservation, karyotype, and gene expression analysis. We will also establish myogenic and pre-adipogenic cell lines. Aim 5. Will develop economically viable serum-free, value-added media, and media recycling to support cell proliferation and differentiation needs and reduce system waste, by integrating molecular modeling, coarse-graining and long-time scale umbrella sampling methods, artificial intelligence, and high throughput screening to optimize for advanced functionality. This will be achieved by integrating molecular dynamics, coarse-graining, long-time scale sampling, umbrella sampling methods, artificial intelligence, and high throughput screening to optimize for advanced functionality, utilizing data from different protein sources and bioprocessing methods. We will also apply machine learning techniques to develop and optimize low-cost and effective serum-free growth media for cell-lines. We will also use recirculating cell culture systems for growth media re-use through bio-inspired approaches. Aim 6. Will develop sustainable biomaterials scaffolds, tissue engineering strategies, and fermentation technologies to support meat structure, color, and flavor development. We will optimize the processes and biomaterials integration to enhance nutritional value, quality, and safety. We will explore edible food-grade scaffolding from plant- and microbial-based polymers, we will use different processing technologies including porous 3D printed scaffolds. We will characterize the physicochemical properties of scaffolds. Aim 7. Will optimize the processes and biomaterials integration to enhance nutritional value, quality, and safety. We will assess chemical composition (protein, fat, ash, moisture), amino acids and fatty acid profiles, minerals, vitamins, and chemical score; texture, color, water and oil holding capacity, rheological microstructural and gelation properties, protein solubility, and isoelectric points; microbial and chemical food safety assessments are included, as are shelf-life studies and assessments of thermal stability, protein secondary structure, and kinetics of quality changes in thermally processed cultivated meats.

Progress 09/01/21 to 08/31/22

Outputs
Target Audience: We have connected with various contingencies in industry, government, nonprofits and other academic centers. For industry, this has included a range of industries from start ups in cellular agriculture to larger traditional food producers. For government, this has included the USDA and DoD as examples. For Nonprofits, this has included New Harvest, the Good Food Institute and others. For other academic centers, this has included a range of universities and colleges, from Penn State, U. Wisconsin and Duke University, to others. Audiences have included students at universities via the list above as well a broader audience of users, students and industries via seminars (e.g., U. Wisconsin food consortium). We have hosted students and faculty, such as from other universities and countries to work with us on the various activities. We have also continued formal classroom options to develop academic paths in cellular agriculture, such as a lecture based cellular agriculture course in the fall semester, 2021 and a spring laboratory course this year. We continue to develop a new graduate Certificate program in Cell Ag, a four course cluster to provide a focused learning experience in the field. Changes/Problems:No major changes to date, no major problems to date.With Covid, zoom connections required different approaches, but the goals and activities have remained the same. What opportunities for training and professional development has the project provided? Initiated development of learning materials and teaching resources to support understanding and interest in cellular agriculture. Initiated plans for public resources to help the broader community understand and interact with cellular agriculture. At the university-level - development of the cellular agriculture courses and certificate program underway, with approval for 2 courses in place, a third course in preparation and approval for certificate program on Cellular Agriculture. Facilitating Alt-Protein Course for Industry in collaborating with Cambridge University and Good Food Institute- 8-week Program. Pre-College - Adaption of Shojin Meat set-up for outreach activities University- Level - Graduate certificate approved at Tufts for Cell Ag; Undergraduate minor for Cell Ag submitted for approval at Tufts; Cell-Based Seafood course is under development with Indian Researchers; Materials shared with partner organizations Student Club - established - planning speakers and discussions; GFI funded Virginia Alt Protein and Tufts University Projects. Two VT students (Amiti Banavar and Palak Gard) and two Tufts University students (Olivia Caulkins, Adham Ali) working on the project. How have the results been disseminated to communities of interest? Online webinars and related output; During year 1, more than 30 online webinars, new articles and related public affairs outreach provided. Conferences: attended several conferences and provided oral presentations, held several sessions on alternative proteins and cellular agriculture. Interviews with journals: interviewed by many journals including scientific notes, and local news papers on the topic of cellular agriculture. working with non-scientific journals for discussing Cell based meat and their impact on industry. Initiated student run seminar series - CULTIVATE -Monthly seminar series from global cellular agriculture leaders. Hosted by one of the six USDA-funded institutions each month.To introduce and create connections with cultivated meat industry and non-profit leaders/experts, to learn of the motivations behind the creation of cell ag companies and organizations, and to understand the current state of the industry. CULTIVATE YouTube (2022) - A platform aimed to introduce and educate viewers from any background and location to cellular agriculture, research, and lab basics. Cellular Agriculture 101 Playlist established and Research Lab Introductions Playlist What do you plan to do during the next reporting period to accomplish the goals?We plan to continue to progress on each of our 7 aims in the program, including: Pursuit of ongoing collaborative projects among the teams. Continuation of specific research projects for each aim of the program. Continue with regular zoom meetings to facilitate progress and synergy among the team and hold in person meetings when feasible based on Covid. Grow the student interactions among the teams to catalyze research activities. Grow the plans for the student team and industrial advisory board members. Further discussions with stakeholders for engagement and partnerships. Continue to develop training and course work plans to build educational outreach. Attend professional meetings to engage stakeholders and communicate the program Hold our first board meeting and program review (Sept 26) Initiate our new project proposal process

Impacts
What was accomplished under these goals? Overview: Established formal organizational structure and working teams for the 7 project aims Initiated collaborative projects among the teams Initiated specific research projects for each aim of the program Established regular monthly zoom meetings to facilitate progress and synergy among the team Initiated student leadership and interactions among the teams to catalyze research and outreach activities Established student leadership team and industrial advisory board Initiated discussions with stakeholders for engagement and partnerships Initiated training and course work plans to build educational outreach Established website for team publications, webinars and related materials for the project Initiated plans for data storage and preservation Actively participating in many professional and outreach venues to engage the community, stakeholders Planned for our first year hybrid (in-person and on-line) program review (September 26) Specifically: Aim 1 Designed and received IRB approval for survey and experiment on consumer response to cellular agricultural product nomenclature. Began collecting participant responses in US. Completing literature review on consumer perception of cultivated meat and product nomenclature Reviewing literature and methods (Deliberative Multicriteria Evaluation (DMCE)) on consumer acceptance of lab-based meat and seafood. Commenced online data collection with 158 respondents. Designed participatory workshop based on DMCE and other methods Presented project at the UMass Boston School for the Environment Earth Day Symposium Presented discussion of topic and survey to 150 students in undergraduate class Recruited collaborators in the US (Dr. Norbert Wilson, Duke) and Germany (Dr. Larissa Drescher, Weihenstephan-Triesdorf University of Applied Sciences) Meta-analysis of sensory properties of plant-based meat, conventional meat, and cultivated meat has been initiated at VT. Aim 2 Developed and initiated primary data collection systems for labs (Task 1) Developed product system model templates for lab-scale LCAs (Task 1) Defined goal and scope for lab-scale LCAs (Task 1) Collected primary data and completed life cycle inventory for first lab scale LCA, focused on recombinant growth factor production for cellular agriculture (Task 1) Submitted publication on LCA comparing insect and mammalian cell agriculture processes (Task 1) Completed interdisciplinary review of methods for simulating commercial scale cell ag systems (Task 2) Began modeling scaled up growth factor production system (Task 2) Completed review of available life cycle inventory data for comparison systems (Task 2) Defined goal and scope for commercial scale comparative LCA in Y2 (Task 2) Graduate Research Assistants working on the LCAs won Tufts Institute of the Environment Fellowships to extend their work Aim 3 - summarized later under outreach, education, broader impact Aim 4 Bovine satellite cells, chicken cells, porcine cells, and fish cells were isolated for propagation, to assess initial doubling times and survival with passaging; subsets of these cells will be further characterized for immortalization Primary cultures of oyster muscle cells generated from Crassostrea virginica, an economically important oyster specie in Eastern US that currently has no stable cell line available for cultivated meat production The team has moved through the assessment of various disinfectants, media combinations, and plate coatings to obtain conditions to reduce contamination during primary isolations, which is a particular challenge for marine species Studies are continuing on immortalization and subculture of the various cell lines. Directed differentiation of zebrafish embryonic stem cells (ZEM2S) into myogenic cell lines is underway, with the testing of growth factor cocktails and serum levels to monitor myogenic differentiation Several species including blue crab, cray fish, oyster, clam, Pompano, American Eel, and Flounder are under investigation Aim 5 Initiated hydrolysates from agriculture sources - specifically - marine byproducts, plants, and agri-wastes - enzymatically hydrolyzed. Identified fungal mycelia as sources of low-cost raw material for hydrolysis following similar protocols as for marine and agri-wastes. Established cell culture protocols to assess hydrolysates, including cell propagation and differentiation for muscle cells and tissue formation. Developed two modeling approaches to pursue in order to generate databases for the cell ag field with sequence data from ag waste protein sources correlated to cell receptors and growth factor mechanisms of interactions - to enable predictable outcomes on cell functions. Initiated studies with cell growth (bovine muscle cells) on cell ag sourced materials related to serum-free needs. Developed a MOF based electrochemical sensor for head-space and in liquid measuring short chain fatty acids Protein hydrolysates were extracted from 9 different sustainable sources, and basic characterization of these hydrolysates was conducted, followed by freeze-drying for downstream applications. The nine hydrolysates in various concentrations of FBS were assessed with a zebrafish embryonic stem cell lines to determine the best concentrations in reduced serum. Two machine learning methods, Response Surface Methodology and Artificial Neural Networks, were applied to reduce experimental conditions in creating chemically defined media with known concentrations of growth factors, vitamins, and minerals. Four different serum-free media experiments were run with zebrafish cell lines and a reduced-serum media with only five additional components needed identified. DOE utilized to optimize media for fish muscle Developed low serum (<5% FBS) medium for the fish muscle cells. Initially focused on FBS, insulin-transferrin-selenium (ITS), and linoleic acid Aim 6 Developed overall processing plans to generate composite materials from a family of low cost and available biomaterials. Also developed the plans for the characterization of these materials in the context of scaffolding for foods. Establishing a database of meat-related mechanical and morphological features as a guide to the scaffold designs being developed. Developed formulas of polysaccharide hydrogel based bioinks that are suitable for extrusion-based 3D printing with sub-millimeter scale resolution. Developed a novel process negative pressure assisted infusion process to combine cells and proteins with 3D scaffolds compositions by decellularization of plant tissues Characterized the role of fungal mycelium in promoting binding of cells and the proliferation. This project was developed in collaboration with David Block's lab, who is supported by NSF project on Cell Ag. Initiated development of plant-based scaffolds from bamboo shoots, banana leaf, celery, carrots, aloe vera, and cactus via decellularization. Aim 7 Developed list of possible food safety concerns in cultivated meat Developed Good Manufacturing practices and Good Cell Culture Practices protocols. Developed Food Safety Plan for the Cultivated Meat Industry Initiated literature review on food safety and possible technologies for enhancing food safety in the cultivated meat industry Developed vibrational spectroscopy fingerprints for conventional meats to enable use to compare to cultivated meats in the planned studies Evaluated the impact of photosensitizers on cells in 2D environments Evaluated the impact of microplastics on cells in 2D environments Initiated the interaction with FAO for developing the first comprehensive document on food safety for cell-based seafood Developed synergistic processing technologies for the inactivation of mycoplasma in culture media to aid in reducing the loss in productivity of cell lines and promote re-use of spent media Completed a study characterizing the chemical and structural differences between real meat and plant based alternative meat products; drafting a manuscript

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Andrew J. Stout, David L. Kaplan, Joshua E. Flack, Cultured meat: creative solutions for a cell biological problem, Trends in Cell Biology, Volume 33, Issue 1, 2023, Pages 1-4, ISSN 0962-8924, https://doi.org/10.1016/j.tcb.2022.10.002.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Ashizawa R, Rubio N, Letcher S, Parkinson A, Dmitruczyk V, Kaplan DL. Entomoculture: A Preliminary Techno-Economic Assessment. Foods. 2022 Sep 30;11(19):3037. doi: 10.3390/foods11193037.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Batish I, Zarei M, Nitin N, Ovissipour R. Evaluating the Potential of Marine Invertebrate and Insect Protein Hydrolysates to Reduce Fetal Bovine Serum in Cell Culture Media for Cultivated Fish Production. Biomolecules. 2022; 12(11):1697. https://doi.org/10.3390/biom12111697
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Goswami M, Belathur Shambhugowda Y, Sathiyanarayanan A, Pinto N, Duscher A, Ovissipour R, Lakra WS, Chandragiri Nagarajarao R. Cellular Aquaculture: Prospects and Challenges. Micromachines. 2022; 13(6):828. https://doi.org/10.3390/mi13060828