Source: TUFTS UNIVERSITY submitted to
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
Reporting Frequency
Annual
Accession No.
1027620
Grant No.
2021-69012-35978
Project No.
MASW-2021-05678
Proposal No.
2021-05678
Multistate No.
(N/A)
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
5017010103020%
5027220100015%
7015010202015%
8033260200010%
9033799302020%
4027410301020%
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 09/01/22 to 08/31/23

Outputs
Target Audience:The National Institute has a wide-ranging target audience including consumers, policy makers, regulatory agencies, startup companies, researchers, extension agents, local Departments of Agriculture and Consumer Services, nutrition practitioners, college and high school students and institutions. The target audience can be categorized into five groups. Consumers and the public - Members of the public who wish to learn about cellular agriculture. Policy makers and regulatory agencies - National and state officials setting policy that impact the future of cellular agriculture. Business enterprises - Entities researching, designing, producing, and promoting cell cultivated products. Academic and industry researchers - Individuals researching cell agriculture from academic and private institutions. Students and educational institutions - Individuals enrolled in courses on cellular agriculture. Changes/Problems:Personnel Changes Reza Ovisspour moved his lab from VT to Texas A&M. This will expand our impact via resources and programs at Texas A&M Challenges Outreach efforts (Aim 3) have had challenges identifying individuals to engage in the development of resources who have both educational expertise and deep knowledge of cellular agriculture. This has delayed some of the deployment of videos and activities. However, there are additional team members that are part of the Tufts Center for Cellular Agriculture that have joined the project in teaching and communications that are helping to mitigate and advance those efforts. What opportunities for training and professional development has the project provided?Training High school students from VA Governor High School were exposed to cellular agriculture concepts, and were able to grow cells, count, transfer, change the media, work under aseptic conditions, and then harvest cell for scaffold adhesion. Across the greater Boston area pre-college and university-level students have been provided opportunities for understanding pathways through cellular agriculture, career opportunities and highlights of key issues in the field. Three students from the Ovissipour lab at Virgina Tech received specialized training on imaging from Imaging Center, raman spectroscopy from Thermo Fisher, Python training, and general cell culture practices. Three students in the Cash lab at Tufts have received specialized training in consumer research and preference elicitation through this grant. Job Placement Three students from the Ovissipour lab at Virginia Tech graduated and found jobs as researchers with Upside Foods, Wild Type. Two students and two postdocs have completed training in the Nitin lab at UC Davis. One of the students has joined Genentech and the other student has joined UCANR as a tenure track farm advisor. Both the postdocs have successful received faculty positions - one at Florida State University in the US and the other in S. Korea. Additionally, the project is supporting the ongoing training of 2 postdocs and one graduate student. Two students from the Cash lab at Tufts have completed their training and secured their next position. The first with the Coolfood project (climate-and-food-oriented database) at the World Resources Institute, and the second has taken a job as a Consumer Scientist I at Mondelez International. Professional development Dr. Amin Nikkhah (Blackstone Lab, Tufts) presented at the International Symposium for Sustainable Systems and Technologies, his first professional society presentation in the field of cultivated meat. The conference provided an opportunity for him to further develop his professional network. Dr. Nikkhah also participated in online courses in coding (using Python), response surface methodology (using Expert Design), and process simulation (with SuperPro Designer). Dr. Katherine Fuller (Cash Lab, Tufts) presented at the Agricultural and Applied Economics Association Annual Meeting which provided an opportunity to expand her professional network. Students from Tufts visited research and production facilities in the San Francisco Bay area. This provided an opportunity for students to interact with industry members, tour production facilities, taste cell-cultivated products, and meet with academic team members at UC Davis. How have the results been disseminated to communities of interest?Results have been disseminated through scientific publications, YouTube videos, social media, seminars, presentations, and interviews with local, national and international agencies. Consumers and the public In addition, public events at Yorktown Library to educate the public through face-to-face meetings. Panelist for a Movie Night at Richmond Library (VA) after showing "Meat the Future" movie. YouTube Channel @cellagtube Recorded seminars and interviews Instagram @cellaggram "Method Monday", channel takeovers, life a CellAg Ph.D. New and Press Celtics Dancer and Biomedical Engineering Student Strikes Balance On, Off Court. Tufts Now 2023 Cultivated meat in focus at Tufts cellular ag innovation day: From bioreactor bottlenecks to a "global battle cry' for government funding. Foodnavigator 2023 Cultured meat industry experts call for government funding during Tufts University Cellular Agriculture Innovation Day. FOOD MATTERS live. 2023 Leaders in Cellular Agriculture Gather at Tufts to Transform the Way We Eat Meat. Tufts Now. 2023 What is Cellular Agriculture. Tufts Now. 2023 What's Next for Cellular Agriculture: 5 Takeaways From Tufts Cellular Agriculture Innovation Day. The Spoon. 2023 Editor's blog: Tufts' CellAg Innovation Day looks ahead with energy. Alt-Meat. 2023 For one afternoon, lab-grown meat was the talk of the town. Boston Globe. 2023 After years of promise, cell-based meat is moving from the lab to dinner plates. Boston Globe. 2023 Policy makers and regulatory agencies Multiple webinars were hosted with specific presentations for educating Virginia state USDA agents, and inspectors about cellular agriculture. Meeting with Massachusetts State Senator to support their funding initiative for cell-cultivated technology across the greater Boston area. Webinars United States Oversight of Meat and Poulty Made Using Animal Cell Culture Technology - Philip Bronstein (USDA), Jeremiah Fasano (FDA) Regulatory & Food Safety Concerns for Cell-Cultured Meat and Seafood - Barbara Rasco (Univ. of Wyoming), Noreen Hobayan (BlueNalu) Business enterprises Discussion on the state of scientific evidence on sustainability and equity of cultivated meat with key cultivated meat industry and investor stakeholders through a panel discussion and full-day event on innovation in cellular agriculture in Boston (January 2023). Industry members of companies involved in cellular agriculture have participated in advising and planning for upcoming recruiting. Webinars United States Oversight of Meat and Poulty Made Using Animal Cell Culture Technology - Philip Bronstein (USDA), Jeremiah Fasano (FDA) Regulatory & Food Safety Concerns for Cell-Cultured Meat and Seafood - Barbara Rasco (Univ. of Wyoming), Noreen Hobayan (BlueNalu) Researchers Life cycle assessment workshop at the Tufts Nutrition Data Symposium Conference presentations and publications Students and educational institutions High School and Pre-college Students High School Students - High school students enrolled in Tufts pre-college summer programs were the target of direct testing and intervention with AIM 3 outreach tools that included ethical considerations, sensory evaluation and life-cycle analysis. 17% of the program receives scholarships and participants are from across the country as well as international locations. Pre-college Educational Providers - The 2022 activities were further refined and had educator guides developed that are freely available via downloadable PDF. The 2023 activities will go through a similar process in Year 3. Dissemination of Cell Ag Design Talk Activity (Pre-College Activity 1) Web - https://go.tufts.edu/cellagdiscussionguide1 Undergraduate Students, Graduate Students, Postdoctoral Scholars The courses developed at Tufts reach students across levels and majors (engineering, nutrition) Graduate students are involved with the design and dissemination of the Cellular Agriculture club across USDA grant participating institutions. Graduate students involved span biomedical engineering, nutrition and other affiliated disciplines. Training and development of Postdoctoral Scholars through Research Assistantships Webinar aimed to increase awareness of cell-agriculture What do you plan to do during the next reporting period to accomplish the goals?Aim 1 - Year 3 Plans: Scale up the study on consumer acceptance of cultivated meat and seafood by working with partnering institutions in Nigeria and China. Conduct and publish the content analysis study on the USDA label and sale approvals of cell-cultivated meat Conduct sensory analysis of commercially available cultivated meat and willingness to pay for retail products. Complete the impact of information and nudging on consumer preference for cultivated meat including finishing the data collection, data analysis and manuscript writing. Conduct an experiment to identify the role of trust when communicating cultivated meat scaffolding information to consumers. Aim 2 - Year 3 Plans: Complete modeling and validation of multi-objective optimization of media composition for differentiation phase with a fish cell line that considers cell performance, environmental impacts and cost. Collaboration between Blackstone and Ovissipour Labs. (Task 1) Transdifferentiate chicken fibroblast cells into muscle and/or fat phenotypes for parameter data collection and life cycle inventory development relevant to differentiation-based cultivated chicken LCA modeling. Collaboration between Blackstone and Kaplan Labs. (Task 2). Compare the nutritional and sensory attributes of undifferentiated cultivated chicken fibroblast cells against conventional chicken samples. Collaboration between Blackstone and Kaplan Labs. (Task 2). Complete analysis of first commercial scale LCA comparing cultivated chicken meat to its conventional alternative. Collaboration between Blackstone and Kaplan Labs. (Task 2). Exploratory analysis of commercial scale recombinant protein production (Task 2). Adapt adherent bovine cells to suspension culture in serum-supplemented and serum-free media conditions for parameter data collection and life cycle inventory development relevant to comparative cultivated beef LCA modeling. Collaboration between Blackstone and Kaplan Labs. (Task 2). Begin building commercial scale model of bovine cultivated meat in SuperPro Designer (Task 2). Aim 3 - Year 3 Plans: Pre-College Finalize and Disseminate Pre-College Activities 2 & 3 Webinar on Pre-College Activities University- level Development of infrastructure for sharing course modules across institutions New courses deployed during 2023/2024 academic year BME194 Value Creation in Cellular Agriculture (Fall 2023, Tufts University) Courses BME173/NUTR173 Biofabricated Foods and Cellular Agriculture Lecture (Fall 2023, Tufts University) BME174/NUTR174 Biofabricated Foods and Cellular Agriculture Lab (Fall 2023, Tufts University) Student Club As a part of the @cellaggram Instagram initiative, we plan to continue current efforts and develop a "Life in the [insert PI's name] Lab" series in which graduate and undergraduate students/post-doctoral candidates/lab technicians share their day-to-day activities and provide insights into what each lab is like. As a part of the @cellagtube YouTube initiative, we plan to continue current efforts and develop playlists of educational videos breaking down each phase of the cultured meat production process, consumer acceptance, life cycle assessment, and techno-economic assessment considerations. As a part of the newsletter initiative, we plan to design and release the Summer 2023, Fall 2023, Spring 2024, and Summer 2024 National Cellular Agriclub newsletters. As a part of the CULTIVATE Cellular Agriculture Seminar Series initiative, we plan to offer bimonthly seminars throughout the 23-24 academic year. Industry In-progress - Working to identify potential topics for 2023/2024 workshops Survey/Feedback Session with industry planned Cellular Agriculture Job Fair (January 2024) Communication Continue work across YouTube Hiring professional production team for Explainer videos Aim 4 - Year 3 Plans: Develop and characterize the non-immortalized and immortalized cell lines from different species. Develop genomics characterization methods for these cells and cell lines Develop 3D cell culture environments for differentiation studies (flasks, bioreactors) Continue to identify additional cells, cell sources, modes of isolation and modes of purification and propagation to the support the program Implement cell distribution plans Develop plans for safety evaluation of cells developed in the program, in conjunction with Aim 7 Aim 5 - Year 3 Plans: Develop new bioprocessing and pre-treatment methods to enhance the quality of peptides and reduce costs Screen different feedstocks to develop novel protein sources for growth factors Apply AI for differentiation and proliferation media Apply AI for characterizing and developing novel molecules that can mimic the natural growth factors but at lower cost Validate the efficacy of metabolic imaging to enable screening of serum replacement and optimizing growth factors in media Aim 6 - Year 3 Plans: Expand the current library of food grade, low-cost materials that support cell adhesion, proliferation and differentiation. Evaluate cell differentiation of cells on these composite materials to support development of cell based meat Develop a benchmarking framework based on a multi-institutional collaboration to guide the selection of scaffold compositions for cell-Ag applications. Develop novel protein-based scaffolds using top-down and bottom-up approaches. Evaluate differentiation of cells on the scaffolds to support development of cell-based meat. Develop novel protein nanofibers-based scaffold for supporting cells attachment, proliferation, and differentiation. Aim 7 - Year 3 Plans: Optimize the design of the developed light-induced antimicrobial matrix for enhancing the microbes inactivation rate. Develop novel matrices for removal of ammonia and lactic acid from the culture media to support the re-use of culture media. Develop novel matrices for capturing of the microplastics to prevent their release into the cultured meat bioreactors.

Impacts
What was accomplished under these goals? Accomplishment Highlights Published 13 peer-revied manuscripts Developed a continuous muscle cell line from Atlantic mackerel Used AI towards developing media, scaffolds, and other biomaterials Developed a novel process to infuse cells into fungal microcarriers Multiple student organized outreach events (seminars, YouTube, Instagram) Aim 1 - Accomplishments: Completed data collection, analysis, and drafted manuscript for the nomenclature study (815 participants in the US and 848 in Germany) Designed, received IRB approval, and currently pre-testing a survey experiment on the impact of information and nudging on consumer preferences for cultivated meat Designed and launched an internal survey of the USDA-funded research team on potential concerns current innovations in cultivated meat might raise for consumers Completed a pilot assessment of consumer acceptance of cultivated meat and seafood Designed a media content analysis on the USDA label and sales approvals of cell-cultivated meat Worked on recruiting commercial food samples for sensory testing (in progress) Screened articles for a scoping literature review on cultured meat sensory evaluation Aim 2 - Accomplishments Completed experiments, modeling, and validation of multi-objective optimization of media composition to maximize cell growth in a fish cell line (proliferation phase), while minimizing cost and carbon footprint. Collaboration between Blackstone and Ovissipour Labs. Developed a baseline commercial scale model for food-grade recombinant protein production, a key media input. Development of life cycle inventory and preliminary analysis of commercial scale Beefy-R serum free media. This will result in a publication in Y3 and will serve as an input to a comparative cultivated beef LCA. Developed future scenarios for conventional chicken and beef comparator systems based on literature and industry reports Adapted adherent chicken cells to suspension culture in serum-supplemented and serum-free media conditions Aim 3 - Accomplishments: Pre-College Testing of two new USDA Cellular Agriculture Activities with 80 High School Students Pre-College Activity 2: Sensory Evaluation - Students compare plant-based nuggets using established research methods and discuss the ways in which cellular agriculture will have to address appearance, textures, and smell Pre-College Activity 3: Life Cycle Analysis - Students learn about the principles of Life Cycle Analysis and conduct a simplified LCA Analysis of a food product. Students discuss how LCA is being conducted in cellular agriculture. University- Level Tufts University Cellular Agriculture Courses Fall 2022 - BME173 - Biofabricated Foods and Cellular Agriculture Lecture course Spring 2023: BME174 Biofabricated Foods and Cellular Agriculture Lab course. Cellular Agriculture Certificate Program - Fall 2024 Student Club Supported multiple events and built a thriving community across multiple institutions Launched the @cellaggram Instagram account featuring multiple educational series: "Methods Monday'' and "Feature Friday". Launched the @cellagtube YouTube channel featuring multiple filmed/edited "73 questions" style interviews with faculty across the institutions and recordings of the CULTIVATE Cellular Agriculture seminar series. Designed and disseminated the Winter 2022 and Spring 2023 National Cellular Agriclub newsletters featuring "Recent Publications", "Researcher Spotlights" "Upcoming Events", and "Job Openings" for each institution involved in the grant. Industry Initialed industry focused webinars Aim 4 -Accomplishments Initiated ESC and/or adult stem cell line cultures from different seafood species from different stages including embryonic, larvae, fingerling, and adults. Disinfectant methods were developed and optimized for different species. Initiated differentiation of seafood-related ESCs and/or adult stem cells into myogenic and pre-adipogenic cells. Bovine muscle satellite stem cells were successfully developed and characterized Porcine adipogenic cells were successfully isolated and characterized Crab cell lines were developed from Blue Crab embryonic stage Mackerel stem cell lines were successfully developed and characterized, and shared with cell banks such as Kerafast Plans to formally distribute cell lines developed in the program were established Aim 5 - Accomplishments Post-biotic materials were characterized in terms of nutritional value, peptide size, amino acids, molecular size, and degree of hydrolysis, and functional properties. AI models were successfully applied for cell differentiation and proliferation media formulation. Evaluated a metabolic imaging based molecular imaging approach to monitor cell proliferation in response to different formulations of culture media. Conducted computational and bench experiments evaluating the binding of different fibroblast growth factor 2 variants to receptors. Experimentally, different FGF2 variants, such as hyperstable and fish orthologs, were screened for short-term growth in immortalized bovine satellite cells. Molecular dynamics computations predicted goldfish FGF2's enhanced binding ability, as predicted in binding energy and receptor dimerization distances. Atlantic mackerel (Scomber scombrus) RNA sequencing was performed and successfully aligned and analyzed. An adaptable, easy to implement bioinformatic platform for understudied species was developed. Proteomics comparing intracellular protein profiles of iBSCs grown in serum-free and serum-containing media was successfully performed. Aim 6 - Accomplishments Utilized machine learning to predict the 3D printability of bio inks based on rheological properties. Developed a 3D printed scaffold for cellular attachment and proliferation. Based on the rheological, mechanical, and cell-based assays with two types of polysaccharides (pectin and alginate) and proteins-polysaccharide combination (soy protein isolate (SPI) and pea protein isolate (PPI) combined with pectin), the pectin and pectin-protein compositions presented mechanical and biochemical properties to support cell attachment and proliferation. Developed 3D foam-based scaffolds for cultivated meat using PPI and tannic acid to induce crosslinking within the scaffold matrix. The porous structure of the scaffold supported the growth of skeletal satellite cells. Applied a glucose-based Maillard reaction to further improve PPI scaffolds' mechanical and water resistance properties. This process promoted attachment and proliferation of diverse cell types (C2C12, iBSC). The initial studies also support the potential of this scaffold to promote differentiation of the cells. Furthermore, the scaffolds presented Maillard reaction-derived flavors, compared to the scaffold without glucose addition. Developed 3D oleogel-based scaffold compositions with high oil content to influence both the texture and other organoleptic properties for cultured meat. The scaffolds showed good C2C12 attachment and proliferation. Developed a patent pending process to develop hybrid composition combining fungal microcarriers with cells including yeast and mammalian cells. Aim 7 - Accomplishments Developed novel LED light induced photodynamic matrix for the rapid and effective inactivation of microbes in culture media to reduce the loss in productivity of cell lines and promote the re-use of spent media. Developed fungal pellet-based scaffolds for encapsulation of cells, nutrients and other bioactive. Developed models to predict the enhancement in nutrient content of cells and their release using novel encapsulation process. Determined the impact of microplastics on cell adhesion, cell proliferation and differentiation under the 2D environment. Developed the D-value and Z-value for different pathogenic bacteria in cell culture media containing different concentrations of serum.

Publications

  • 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) Nov 16, 12(11), 1697. https://doi.org/10.3390/biom12111697
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Goswami M, Pinto N, Yashwanth BS, Sathiyanarayanan A, Ovissipour R. Development of a cell line from skeletal trunk muscle of the fish Labeo rohita. Cytotechnology. (2023) Jun 1:1-3. https://doi.org/10.1007/s10616-023-00581-3
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Goswami, M., Belathur Shambhugowda, Y., Sathiyanarayanan, A., Pinto, N., Duscher, A., Ovissipour, R., Lakra, W.S., Chandragiri Nagarajarao, R. Cellular Aquaculture: Prospects and Challenges. Micomachines. (2022), 13, 828. DOI:/10.3390/mi13060828
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Hu, Y. & Buehler, M.J. Deep language models for interpretative and predictive materials science. APL Machine Learning. (2023) 1 Mar (1), 010901. DOI:/10.1063/5.0134317
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Hu, Y. & Buehler, M.J. End-to-End Protein Normal Mode Frequency Predictions Using Language and Graph Models and Application to Sonification. ACS Nano. (2022) 16, 12, 20656-20670. DOI:/10.1021/acsnano.2c07681
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Lara L�quez-Caravaca, L., Minami Ogawa, M., Rewa Rai, R., Nitin Nitin, N., Juan Moreno, J., Garc�a-Mart�nez, T., Juan Carlos Mauricio, J., Juan Carlos Jim�nez-Uceda, J. & Jaime Moreno-Garc�a, J. Yeast cell vacuum infusion into fungal pellets as a novel cell encapsulation methodology, Applied Microbiology and Biotechnology. (2023), 107, 5715  5762. DOI:/10.1007/s00253-023-12681-3
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Lu, R. Rai & N. Nitin. Image-based assessment and machine learning-enabled prediction of printability of polysaccharides-based food ink for 3D printing, Food Research Intl. (2023), Volume 173, Part 2, 1133842023. https://doi.org/10.1016/j.foodres.2023.113384
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Nikkhah, A., Rohani , A., Zarei, M., Kulkarni, A., Batarseh, F.A., Blackstone, N.T. & Ovissipour, R. (2023). Toward sustainable culture media: Using artificial intelligence to optimize reduced-serum formulations for cultivated meat. Science of the Total Environment. (2023), 894, 164988. DOI:/10.1016/j.scitotenv.2023.164988
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Portsmore, M. D., Pangan, T. J., & Starling, B. D. Cellular Agriculture: An activity guide to support an engineering ethics and impacts discussion in high school settings (Resource Exchange) Paper presented at 2023 ASEE Annual Conference & Exposition, Baltimore, Maryland. https://peer.asee.org/43142
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Renata, C., Cierra, J., Thet, A., OKeefe, S. Challenges for flavoring fish products from cellular agriculture. Current Opinion in Food Science. (2022), 47, DOI:/10.1016/j.cofs.2022.100902
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Saad, M.K., Yuen, J.S.K., Joyce, C.M., Li, X., Lim, T., Wolfson, T., Wu, J., Laird, J., Vissapragada, S., Calkins, O.P., Ali, A., & Kaplan, D. Continuous fish muscle cell line with capacity for myogenic and adipogenic-like phenotypes. Scientific Reports. (2023), 13, 5098. DOI:/10.1038/s41598-023-31822-2
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Shen, S.C., Khare, E., Lee, N.A., Saad, M.K., Kaplan, D.L., & Markus J. Buehler, M.J. Computational Design and Manufacturing of Sustainable Materials through First-Principles and Materiomics. Chemical Reviews. (2023) 123, 5, 2242-2275. DOI:/10.1021/acs.chemrev.2c00479
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Trinidad, K., Ashizawa, R., Nikkhah, A, Semper, C., Casolaro, C., Kaplan, D.L., & Blackstone, N.T. Environmental life cycle assessment of recombinant growth factor production for cellular agriculture applications. Journal of Cleaner Production. (2023), 138153. DOI:/10.1016/j.jclepro.2023.138153
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Cellular Aquaculture. Reza Ovissipour. PFT 2023 (Pacific Fisheries Technology)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Cultivating the Right Image: Consumer interest in naming conventions for Cellular Agriculture Products. Sean Cash. Morrison School of Agribusiness, WP Carey School of Business, Arizona State University, September 28, 2022
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Towards sustainable culture media: Using artificial intelligence to optimize reduced-serum formulations for cultivated meat. Amin Nikkhah. International Symposium on Sustainable Systems and Technology (ISSST) 2023 Conference
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Vegan cells: how environmentally sustainable is Beefy-R animal-free culture media? Amin Nikkhah. Tufts Food & Nutrition Innovation Institute, 1st Annual Research Poster Competition. April 24, 2023
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Cellular Aquaculture as an Alternative Food Production System. Michael Saad. Society for In Vitro Biology Conference 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Tensions and Tradeoffs in Diet Sustainability Analyses. Sean Cash, Nicole Blackstone. Tufts Nutrition Data Symposium
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: A Cross-Country Choice Experiment in Naming Effects for Cellular Agricultural Meat Products. Katherine Fuller. Agricultural and Applied Economics Association Annual Meeting, Washington DC, July 24, 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Cellular agriculture for alternative foods. David Kaplan. National Animal Nutrition Summit, Washington DC. 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Challenges and Opportunities for Future Foods  cells, biomaterials, scaling. David Kaplan. June 26 - Gordon Research Conference  Advanced Cell and Tissue Biomanufacturing, Newry, Maine. 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Consumer Preference for the Meat of the Future. Kathrine Fuller. Tufts - Friedman School Speaker Series 2022
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Foods of the Future via Cellular Agriculture. David Kaplan. IFT Annual Meeting. 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Opportunities and Challenges with Biomaterial Scaffolding for Cultivated Meat. David Kaplan. 5th Industrializing Cultivated Meats and Seafood Summit, Boston MA. 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Sustainable proteins to advance plant-based, cultivated and fermentation-made food. David Kaplan. GFI Europe and Cell Press Sustainable Proteins Forum, zoom event. 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Talk  Biopolymers and Sustainability  from food to materials. David Kaplan. TechConnect World 2023, Washington DC. 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Consumers' Willingness to Pay for Cellular Meat Products and Naming Preferences. Kathrine Fuller September 2022


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