Optimizing Cultured Beef Production:
Development of Conditioned Media Enriched with Afamin and Wnt3a Proteins

Problem

Introduction

Natural beef and its production processes are causing more problems in society than providing humanity with a food supply. These various problems can be narrowed down into the following:

• Excessive carbon emissions
  • The total emissions from global livestock are about 7.1 gigatonnes of CO2-equivalent per year [9].
    • Beef alone is responsible for 65% of livestock emissions [9].
    • This accounts for approximately 14.5 percent of all anthropogenic greenhouse gas emissions [9].

• Water shortage
  • To produce one pound of beef, approximately 1,847 gallons of water (39 bathtubs) are required [7].

• Potential food scarcity
  • The global consumption of meat has increased since 1961 as the world urbanizes. It is estimated that we may need 70% more animal products by 2050 to feed the world [8].

• Violation of animal rights
  • The cruel raising methods and inhumane slaughtering procedures that farms use treat livestock as not living beings.

Problems of Current Cultured Meat

Apart from the environmental challenges and sustainable food supply, there are also problems within the cultured meat industry. The three major challenges can be classified into the following:

• High production costs [2]
• Technical hurdles [3]
• Scale-up challenges [10]

Our Approach

Overview

Our solution aims to address the aforementioned problems of conventional meat production. We hypothesized that Wnt3a and Afamin proteins could improve cell differentiation in the media, thus boosting bovine muscle cell growth. By utilizing recombinant proteins to replace costly growth-promoting supplements, our project aims to enhance muscle cell growth efficiency and resolve the cost issue.

We aim to make lab-grown meat more accessible through the following:

• Cost-effectiveness
• Sustainability
• Public awareness

In particular, we chose our project to materialize our passion for spreading public awareness surrounding cultured meat in South Korea. Due to the Livestock Products Sanitary Control Act, cultivated meat is not yet consumable in Korea. Through our project, we hope to promote a healthy, normalized perception of cultured meat that can make a change through widespread consumption.

About Conventional Serum

The overall process of cultivating meat involves taking a small sample of animal cells and multiplying them in a nutrient-rich medium in a bioreactor to form muscle tissue. This conditioned medium contains components secreted by cells that are required by another set of cells.

Conventional serum usually contains the following:

• Glucose
• Amino acids
• Inorganic salts
• Vitamins
• Buffers
• Fetal bovine serum

Conventionally, fetal bovine serum (FBS) is used as it provides nutrition and other factors that accelerate growth rates. However, it is extremely expensive and can be reduced with the introduction of these recombinant factors.

The current practice of fetal blood harvesting is also inhumane as it involves killing the mother cow during pregnancy and draining the unborn calf’s blood to produce the serum. Not only that, a serum-free growth medium reduces carcinogenic and zoonotic disease risk [6, 4].

Our Solution

Incubating the media with producer cells that secrete recombinant proteins—which regulate cell growth and division—allows for the cell culture to be more affordable by reducing the cost of producing purified recombinant proteins.

This also addresses the unethical practice of using FBS and its risks. Thus, significant cost reductions are possible without large technological leaps [11].

After the media has been conditioned with the growth factors, it can be stored or transferred immediately onto the cells of interest that require it [5]. To lower the cost of the cultured medium, we used the recombinant proteins Wnt3a and Afamin.

Utilizing Recombinant Proteins

Our first protein, Wnt3a, is a signaling protein that plays an important role in various cellular processes, including…

• Cell growth and differentiation
  • Process in which a stem cell changes from one type to another
• Cell migration
  • Directed movement of cells in response to signals
• Tissue development
  • Process by which a group of cells increases its size

Wnt3a also activates Wnt signaling, which can stimulate the proliferation of muscle stem cells, which are a critical component of cultured meat production [1].

Our second protein, Afamin, is a protein that is involved in various physiological functions, including:

• Transport of vitamin E
• Regulation of insulin secretion
• Modulation of cell proliferation and differentiation

Particularly, Afamin transports vitamin E into cultured cells, which in the context of cultured meat can help to improve the quality and stability of the resulting meat products. Supplementing the growth medium with vitamin E can produce the following benefits:

• Reduce lipid oxidation
• Improve color stability
• Extend the shelf life of cultured meat

Additionally, Afamin increases the stability of Wnt3a, with Afamin protecting Wnt3a from degradation by proteases or other cellular mechanisms [1].

Experimental Design

Methodology

• Step 1: Establishment of Afamin-Wnt3a Producer Cells
  • We used polymerase chain reaction (PCR) and gel electrophoresis to assess the presence of the proteins in the BII-CMV-AfmW3A vector with a circular DNA form consisting of 10,434 base pairs.

• Step 2: Generate the Afamin-Wnt3a Enriched Media
  • HEK293T producer cells (human embryonic kidney cells) are competent in producing a growth medium that does not contain serum.
  • The stability of the transfection process is improved with liposome assistance for DNA insertion.
  • Puromycin is used to eliminate non-transfected cells, leaving only viable ones.
  • Afamin and Wnt3a proteins are secreted in an equal 1:1 ratio when in media.
• Step 3: Analyze the Growth Rate of Bovine Muscle Cells for Meat Production
  • We used the Bradford assay to determine the abundance of targeted proteins by measuring the total protein concentration.
  • We used a cell proliferation assay to analyze the growth rate of bovine muscle cells and determine the number of live cells during growth and division.
  • We analyzed the expression levels of genes associated with muscle growth and differentiation.

Step 1: Establishment of Afamin-Wnt3a Producer Cells

The initial step entailed confirming the viability of the vectors designated for our project, namely the presence of Afamin and Wnt3a proteins in the DNA plasmid vector. This involved employing PCR and gel electrophoresis techniques to assess the presence of the proteins in the BII-CMV-AfmW3A vector with a circular DNA form consisting of 10,434 base pairs.

• Each gene in the vector (CMV promoter, Afamin, T2A sequence, Wnt3a, puromycin resistance gene, ampicillin resistance gene) has a specific role in making Afamin-Wnt3a conditioned media.

• To confirm this, 1x TAE buffer and agarose gel electrophoresis were used.

Step 2: Generate the Afamin-Wnt3a enriched conditioned media

Having confirmed the presence of Afamin and Wnt3a proteins, the next phase involved transfection of the vectors into HEK293T cells—human embryonic kidney cells—to integrate the vectors into the cell genome.

Step 3: Analyze the growth rate of bovine muscle cells for meat production

We employed the Bradford assay to measure total protein concentration to determine the abundance of targeted proteins, thereby ensuring equal protein amounts for subsequent western blotting.

• Bradford Assay: Measures absorbance of orange color from coomassie blue
  • Reliable measure
• Western Blot Analysis: Uses SDS-PAGE gel
  • Detects Afamin and Wnt3a proteins
  • Involves loading samples into gel and electrophoresis
• Cell Proliferation Assay: Uses bovine cultured cells (MyoB)
• Data Analysis: Final step after completing the procedures

Final Thoughts

In conclusion, our project—Conditioned Media Enriched with Afamin and Wnt3a Proteins—aims to address the environmental and ethical problems associated with current conventional and cultured meat production processes.

We hypothesized that incubating cell culture media with producer cells that secrete Afamin and Wnt3a recombinant proteins could improve cell differentiation and thus boost bovine muscle cell growth.

Our experiment results showed that a 10% concentration of Afamin-Wnt3a conditioned media significantly enhances cell proliferation, proving the media to be a valuable resource for cultivating bovine muscle cells.

Ultimately, with our project, Korea-HS aims to enhance muscle cell growth efficiency and reduce production costs.

References