Product Review

March 17, 2022

Transferrin is an essential component for cell survival and proliferation in culture, where it’s responsible for the uptake and transport of iron into the cell. What does that mean?

Most living things need iron, and Transferrin has the privilege of being best friends with iron.

Transferrin plays a central role in iron metabolism and ensures a vital iron pool is available for cells. This allows proper oxygen uptake and also stimulates growth-related enzyme activity.

Transferrin loves iron so much that it grabs up all the extra iron it can. In fact, it is so iron-rich, the color of recombinant Transferrin is a nice orangey-red color (that you can see in our Ento-Tf product). This also allows Transferrin to play a role in innate immunity by reducing bacterial survival through iron sequestration.

For the cellular agriculture industry, Transferrin is a key input for meat products such as steaks, hamburgers, or even brisket!

With all the amazing things Transferrin does, we are excited to publicly share performance data on our Bovine Transferrin — Ento-Tf.

Produced with our EntoEngine™ platform, Future Fields’ Transferrin provides consistent performance across multiple metrics — so you can get the most out of your cell cultures every time.

If you have any questions about anything in this article, please reach out to support@futurefields.io.

Advantages of producing Bovine Transferrin using EntoEngine™

The EntoEngine™ is a robust system where we use Drosophila melanogaster as part of our platform to produce high quality, precisely folded and post translationally modified proteins. This allows us to scale up production of higher quantities of proteins using proprietary genetic engineering and scaling techniques.

Keeping in mind safety and regulatory thresholds for our internal standards in extraction, purification and quality check assays, we have developed a fully characterized, affordable option for bovine Tf that consumes less energy, resources and labour than the conventional recombinant expression platforms. ‍

‍ Finally, we also vigorously test all individual batches of EntoEngine products in a predetermined panel of tests, as directed by the FDA and other regulatory agencies, that include endotoxins, allergens, pathogens, pesticides, heavy metals and others.

What are other methods of Transferrin production and their limitations?

Recombinant human/bovine Tf can be produced in bacterial-, yeast-, plant- and cell culture-based expression systems. Each of these comes with their own advantages and disadvantages. Even though functionality of the proteins have been reported for recombinant Tf sourced from any of the above expression systems, when it comes to cost, scalability and sustainability, these options can fall short.

Additionally, post translational modifications are once again a concern for recombinant mammalian proteins that are expressed in non-mammalian systems. This means that even if an expression system can produce a recombinant protein it might not work as expected when used in cell culture. For example, rice-derived recombinant human Tf is expressed at ~1% seed dry weight (10 g/kg); However, MALDI MS analysis of the rice-derived transferrin shows a predominant peak that is slightly higher than the expected size, indicating the presence of post translational modifications that are usually not seen in mammalian systems. In other words, it can be hard to get things like rice to make proteins that are normally made in animals!

Using the EntoEngine platform to produce Bovine-Tf

Using genetic engineering techniques, we have generated D. melanogaster stocks that robustly expresses Bovine Transferrin. This was confirmed using a western blot analysis in which Ento-Tf was run alongside positive control (purified Bovine-Tf) and negative control from WT D. melanogaster. We observe Ento-Tf and Bovine-Tf (positive control) migrate similarly in the gel indicating appropriate translation.

Figure 1. Western blot showing positive and negative control for bovine-Transferrin (bvTf) along with our bovine Ento-Tf product. Ento-Tf migrates similarly to purified bovine-Transferrin from a commercial source.

Performance Data

Validation of functionality of EntoEngine Bovine Ento-Tf

All Ento-products at Future Fields are tested for their ability to aid cell survival and proliferation using C2C12 cells. These are mouse derived myoblast cells that have been immortalized. We recognize that this may not accurately represent the cell lines that our customers use and are in the process of cross testing our products against primary bovine and porcine cells using species-specific growth factors and appropriate controls.

Population Doubling: The population doubling time or growth rate of a cell culture is the time required for a cell population to double its size, reaching exponential growth. Many factors contribute to growth rate such as cell density, availability of nutrients and presence of growth factors.

Morphology images

Figure 2. Serum-free adapted C2C12 cells were grown for 72 hours with medium containing Future Fields’ Ento-Tf bovine Transferrin, a competitor source of human Tf or medium lacking a source of transferrin.

EntoEngine™ derived Bovine Tf drives cell survival and proliferation

A proliferation assay using adapted C2C12 cells was performed to compare the efficiency of EntoEngine™ derived Bovine Tf to commercially available competitors. Here, adapted cells were treated with either of the above sources of Tf or no Tf (negative control), and proliferation was assessed by microscopy after 3 days of incubation. Both the commercially derived competitor and Ento-Tf were able to support cell survival and proliferation; Ento-Tf had better performance compared to the rice-derived competitor in this assay.

Figure 3. Serum-free adapted C2C12 cells were seeded at 6000 cells/cm2 and treated for 72 hours with medium containing a competitor source of human Tf or Future Fields’ Ento-Tf bovine Transferrin. The experiment was blinded and population doubling was measured by cell counting. Results are relative to a commercially available serum-free media. Statistical significance was determined by an unpaired t-test. The error bars represent the standard deviation. Biological replicates: Rice competitor n=24, Bovine Ento-Tf n=36.

Long term usage of Ento-Tf supports cell survival and growth

Serum-free adapted C2C12s were also tested for their ability to grow in the presence of Ento-Tf or rice competitor human-Tf as the sole source of Tf in the media. We found that both the positive control and Ento-Tf supported cell proliferation in a comparable manner. These results indicate that cells are able to utilize Ento-Tf as the source for iron metabolism for longer periods across different generations without any drop in proliferation or survival rates.

Figure 4. Serum-free adapted C2C12 cells were seeded at 10,000 cells/cm2 in flasks and treated with medium containing a competitor source of human Tf, Future Fields’ Ento-Tf bovine Transferrin, or medium without Transferrin (negative control). At about 70–80% confluency, population doubling was measured by cell counting, then cells were passages into new flasks. Results are relative to a competitor source of human Tf. The error bars represent the standard deviation.

EntoEngine™ derived FGF2 and Tf drive proliferation in C2C12 cells

Future Fields' EntoEngine™ is a flexible platform that can be used to produce numerous growth factors, cytokines and other media essentials that can find application for various cell lines of different species. In this particular experiment, we wanted to test the effect of combining Ento-F bovine FGF2 and Ento-Tf on C2C12 population doubling, in comparison to commercially available human Tf and FGF2. In addition to cell counts, we also assessed the metabolic activity of cells via resazurin conversion. We observed that Ento-F and Ento-Tf drives proliferation significantly better compared to commercially sourced FGF2/Tf, or commercial FGF2/Ento-Tf. The results indicate that EntoEngine proteins are compatible with other commercially available sources to meet the requirements of cell growth and survival. In combination, EntoEngine Ento-Tf and Ento-F consistently work synergically to increase proliferation with our model myoblasts.

Figure 5. Serum-free adapted C2C12 cells were seeded at 6000 cells/cm2 and treated for 72 hours with the medium containing a competitor source of human Tf (with a commercially available hFGF2), Future Fields’ Ento-Tf bovine Transferrin (with a commercially available hFGF2), a medium containing combined Ento-TF and Ento-F bovine FGF2 (as the only FGF2 source, or a medium without Transferrin (with a commercially available hFGF2). (A) The experiment was blinded and population doubling was measured by cell counting. (B) Treatment conditions were repeated and the final output measured the metabolic activity rate of cells using a resazurin based readout. Results are relative to a commercially available serum-free media (A, B). Statistical significance was determined by an unpaired t-test. The error bars represent the standard deviation. (A) Biological replicates: Rice competitor n=24, Bovine Ento-Tf n=36, Ento-F FGF2 + Ento-Tf n=28. (B) Biological replicates: n=10 for all treatments.

We perform rigorous quality assessment analyses on all our EntoEngine™ products and perform a battery of Food Safety tests that include bacterial/fungal contaminations, endotoxins, other toxins, heavy metals, pesticide/herbicide residues and allergens. Ento-Tf has successfully passed all these tests and are highly suitable for mammalian cell cultures systems.

Our Recombinant Protein Library

We have three products available on the market today:

Ento-F (bovine): Future Fields Ento-F FGF2 is an Enriched Growth Media Supplement can directly replace FGF2 needs in cell culture. Produced with the EntoEngine™ recombinant protein platform, the supplement comes in a 1000X concentrate, containing enough recombinant bovine FGF2 to sustain cell culture in two and three dimensions with cross species performance equivalent or better than expensive alternatives. Ento-F is the most cost effective and sustainable option for cell culture FGF2 needs on the market today.

FGF2 (bovine): Future Fields Recombinant Bovine FGF2 (> 95 % purity) produced with the EntoEngine™ recombinant protein platform. Fulfills the needs of FGF2 requirements in cell culture across a variety of land-dwelling and aquatic species.

Ento-Tf (bovine): Transferrin Enriched Growth Media Supplement. 1000X concentrate recombinant bovine Transferrin enriched growth media supplement produced with the EntoEngine™ recombinant protein platform. Ento-Tf fulfills the transferrin needs in cell culture and is a cost effective alternative to other recombinant Transferrin products on the market.

Don’t see what you need? We have additional products in our pipeline. Get notified for our future proteins here.

For more information, please email support@futurefields.io.