A serum-free media formulation for cultured meat production supports bovine satellite cell differentiation in the absence of serum starvation

Nature Food ◽  
2022 ◽  
Author(s):  
Tobias Messmer ◽  
Iva Klevernic ◽  
Carolina Furquim ◽  
Ekaterina Ovchinnikova ◽  
Arin Dogan ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Satellite Cell ◽  
Serum Starvation ◽  
Meat Production ◽  
Serum Free ◽  
Media Formulation ◽  
Cultured Meat ◽  
Free Media

Truncation in CCND1 mRNA 3′ UTR Is Associated with An Aggressive Phenotype and Chemoresistance

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5287-5287
Author(s):  
Robert W Chen ◽  
Myo Htut ◽  
Britta Hoehn ◽  
Eamon Berge ◽  
William Robinson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Expression ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Serum Starvation ◽  
Cycle Progression ◽  
Serum Free ◽  
Aggressive Phenotype ◽  
Free Media

Abstract Mantle Cell Lymphoma (MCL) represents 5–10% of all non-Hodgkins lymphomas, making it an uncommon but difficult form of lymphoma to treat. It has the worst prognosis among the B cell lymphomas with median survival of three years. The genetic hallmark of MCL is the t(11,14)(q13;32) translocation causing amplification of cyclin D1 (CCND1). It is a well known cell cycle regulator. Multiple reports have shown a truncation in the cyclin D1 mRNA 3′ untranslated region. This truncation increases CCND1 protein expression by not only enhancing the half-life of CCND1 mRNA, but also evades microRNA regulation of mRNA translation. The dramatic overexpression of CCND1 mRNA and protein has been associated to poor clinical outcome in patients. We hypothesize that this truncation leads to a more aggressive phenotype and induces chemoresistance in MCL. We have identified 4 MCL cell lines (Granta-519, JVM-2, Jeko-1, and Z138) with different levels of the truncated CCND1 mRNA. We were able to show that Z138 and Jeko-1 have a much higher ratio of truncated CCND1 mRNA to the full length CCND1 mRNA as compared to Granta-519 and JVM-2. We were also able to show that this truncated mRNA leads to an increase in CCND1 protein expression. By using flow cytometry, we correlated the increase in CCND1 protein expression to faster cell cycle progression. We proposed that cell lines with increased CCND1 expression are phenotypically more aggressive, and would be able to continue cell cycle progression without serum support. We were able to arrest JVM-2 in G1 phase after 48 hours of serum starvation. However, we were not able to arrest cell cycle progression in Jeko-1 even after 96 hours of serum starvation. Western blot analysis shows that CCND1 protein expression is decreased in JVM-2 but remains unchanged in Jeko-1 with serum starvation. The same phenomenon was observed in Granta-519 and Z138. The MCL cell lines (Jeko-1 and Z-138) with more CCND1 protein expression were able to continue cell cycle progression in serum free media. The MCL cell lines (JVM-2 and Granta-519) with less CCND1 protein expression were not able to continue cell cycle progression in serum free media. This shows that CCND1 overexpression is associated with a more aggressive phenotype. We then treated the 4 MCL cell lines with varying concentrations of doxorubicin, a standard anthracycline chemotherapy used in the treatment of MCL patients. We used MTS assay to assess cell proliferation after treatment with doxorubicin. We found the IC 50 (inhibitory concentration 50%) of doxorubicin in these cell lines varied from 6nM to 600nM. The cell lines (Jeko-1 and Z-138) with more CCND1 protein expression have a much higher IC 50 as compared to the cell lines (JVM-2 and Granta-519) with less CCND1 protein expression. This demonstrates that CCND1 overexpression is associated with chemoresistance. We conclude truncation in CCND1 mRNA leads to increased CCND1 protein expression and faster cell cycle progression CCND1 overexpression is associated with an aggressive phenotype CCND1 overexpression is associated with chemoresistance.

scholarly journals Simple and effective serum-free medium for sustained expansion of bovine satellite cells for cell cultured meat

2021 ◽  
Author(s):  
Andrew Stout ◽  
Addison Mirliani ◽  
John Yuen ◽  
Eugene White ◽  
David L. Kaplan
Keyword(s):  
New Media ◽  
Satellite Cells ◽  
Food System ◽  
Low Cost ◽  
Population Doubling ◽  
Average Growth ◽  
Serum Free ◽  
Cultured Meat ◽  
Starting Point ◽  
Free Media

Cell-cultured meat offers the potential for a more sustainable, ethical, resilient, and healthy food system. However, research and development has been hindered by the lack of suitable serum-free media that enable the robust expansion of relevant cells (e.g., muscle satellite cells) over multiple passages. Recently, a low-cost serum-free media (B8) was described for induced pluripotent stem cells. Here, we adapt this media for bovine satellite cells and show that the addition of a single component, recombinant albumin, renders B8 suitable for the long-term expansion of cells without sacrificing myogenicity. We show that this new media (Beefy-9) maintains robust cell growth over the entire culture period tested (seven passages) with an average growth rate of 39 hours per population doubling). Along with demonstrated efficacy for bovine cells, this work provides a promising starting-point for developing serum-free media for cultures from other meat-relevant species. Ultimately, this work offers a promising foundation for escaping the reliance on serum in cultured meat research, thereby accelerating the field.

scholarly journals Bovine Satellite Cells Isolated after 2 and 5 Days of Tissue Storage Maintain the Proliferative and Myogenic Capacity Needed for Cultured Meat Production

2021 ◽  
Vol 22 (16) ◽  
pp. 8376
Author(s):  
Stig Skrivergaard ◽  
Martin Krøyer Rasmussen ◽  
Margrethe Therkildsen ◽  
Jette Feveile Young
Keyword(s):  
Satellite Cells ◽  
Myogenic Differentiation ◽  
Expression Patterns ◽  
Meat Production ◽  
Positive Trend ◽  
Tissue Storage ◽  
Myogenic Potential ◽  
Cultured Meat ◽  
Start Up ◽  
Myogenic Satellite Cells

Cultured meat is an emerging alternative food technology which aims to deliver a more ethical, sustainable, and healthy muscle-tissue-derived food item compared to conventional meat. As start-up companies are rapidly forming and accelerating this technology, many aspects of this multi-faceted science have still not been investigated in academia. In this study, we investigated if bovine satellite cells with the ability to proliferate and undergo myogenic differentiation could be isolated after extended tissue storage, for the purpose of increasing the practicality for cultured meat production. Proliferation of bovine satellite cells isolated on the day of arrival or after 2 and 5 days of tissue storage were analyzed by metabolic and DNA-based assays, while their myogenic characteristics were investigated using RT-qPCR and immunofluorescence. Extended tissue storage up to 5 days did not negatively affect proliferation nor the ability to undergo fusion and create myosin heavy chain-positive myotubes. The expression patterns of myogenic and muscle-specific genes were also not affected after tissue storage. In fact, the data indicated a positive trend in terms of myogenic potential after tissue storage, although it was non-significant. These results suggest that the timeframe of which viable myogenic satellite cells can be isolated and used for cultured meat production can be greatly extended by proper tissue storage.

scholarly journals Stated Preferences for Plant-Based and Cultured Meat: A Choice Experiment Study of Spanish Consumers

2021 ◽  
Vol 13 (15) ◽  
pp. 8235
Author(s):  
Alfredo J. Escribano ◽  
Maria Belen Peña ◽  
Carlos Díaz-Caro ◽  
Ahmed Elghannam ◽  
Eva Crespo-Cebada ◽  
...  
Keyword(s):  
Choice Experiment ◽  
Meat Products ◽  
Meat Production ◽  
Experiment Study ◽  
Negative Effects ◽  
High Quality ◽  
Cultured Meat ◽  
Production And Consumption ◽  
Alternative Sources ◽  
Negative Impacts

Meat production and consumption have been claimed to have negative impacts on the environment, and even on the consumer’s health. In this sense, alternative sources of protein, mainly meat substitutes and cultured meat, have emerged due to those perceived negative effects. Our paper carries out a choice experiment to analyze the preferences of 444 Spanish consumers and their willingness to pay for plant-based and cultured meats, as compared to conventional meat. Spain was considered of interest for this study due to its significant gastronomic culture, with high-quality meat products that make a great contribution to the economy, meaning that this could be a suitable and also challenging market in which to test alternative sources of protein. The findings show that consumers’ motivations and their interactions with these products are complex. Additionally, a cluster analysis allowed us to identify three types of consumers in terms of preference for these products: price-sensitive millennials, conscious/concerned consumers, and indifferent consumers. Only one group showed some level of acceptance of these alternative products meats.

scholarly journals Expansion and characterization of bone marrow derived human mesenchymal stromal cells in serum-free conditions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Samatha Bhat ◽  
Pachaiyappan Viswanathan ◽  
Shashank Chandanala ◽  
S. Jyothi Prasanna ◽  
Raviraja N. Seetharam
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Population Doubling ◽  
Seeding Density ◽  
Population Doubling Time ◽  
Differentiation Potential ◽  
Serum Free ◽  
Safety Issues ◽  
Free Media

AbstractBone marrow-derived mesenchymal stromal cells (BM-MSCs) are gaining increasing importance in the field of regenerative medicine. Although therapeutic value of MSCs is now being established through many clinical trials, issues have been raised regarding their expansion as per regulatory guidelines. Fetal bovine serum usage in cell therapy poses difficulties due to its less-defined, highly variable composition and safety issues. Hence, there is a need for transition from serum-based to serum-free media (SFM). Since SFM are cell type-specific, a precise analysis of the properties of MSCs cultured in SFM is required to determine the most suitable one. Six different commercially available low serum/SFM with two different seeding densities were evaluated to explore their ability to support the growth and expansion of BM-MSCs and assess the characteristics of BM-MSCs cultured in these media. Except for one of the SFM, all other media tested supported the growth of BM-MSCs at a low seeding density. No significant differences were observed in the expression of MSC specific markers among the various media tested. In contrary, the population doubling time, cell yield, potency, colony-forming ability, differentiation potential, and immunosuppressive properties of MSCs varied with one another. We show that SFM tested supports the growth and expansion of BM-MSCs even at low seeding density and may serve as possible replacement for animal-derived serum.

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