Increasing skeletal myoblasts culture density by an order of magnitude in novel scalable perfusion bioreactors

Background: Using stirred tank reactors (STRs) for scalable production of cells is common in industry [1]. However, their maximum capacity for growing adherent mammalian cells has not been reported more than 106 cells/mL which is very far from optimum cell density needed for economically-viable cultured meat [2]. This work shows that our previously introduced novel method for making 3D, vascularized scaffolds is able to grow C2C12s with densities up to 1.39×107 cells/mL in scalable perfusion bioreactors (PBR).

Objectives: To scale up our previously introduced novel PBR in terms of diameter and perfusion length. To determine the effects of scaling on cell growth and cellular density. To determine the maximum cell culture density.

Methods: Channelled polycaprolactone (PCL) sheets were produced in different sizes as stated in our previous work [3]. The smallest scaffold was a cylinder with diameter of 8 mm and height of 5 mm, while the largest was 24 mm in diameter and 20 mm in height. C2C12s were seeded on scaffolds at seeding density of 106 cells/mL. Proliferation was assessed in all scaffolds after 72 h and 120 h of culture by cell counting using enzymatic cell suspension and NucleoCounter®.

Results: Increasing the diameter by 3 times and increasing the perfusion length from 5mm to 20mm and then 40mm – a volumetric increase of 72x - showed no adverse effect on final cell density. Cell densities were comparable in all the scales (72 h). Densities as high as 1.39×107 cells/mL were seen after 120 hr of proliferation (reactors with 8 mm diameter/ 10 mm length).

Conclusions: The novel PBRs were demonstrated to be scalable, with comparable cell numbers and viability at all three sizes tested, suggests they can be scaled up further. The cell densities were an order of magnitude higher than those in STRs. Therefore, we believe our method offers a potential alternative to STRs for optimising cultured meat industrialization.
References:
1) Storm, M., et al., Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture. J. Vis. Exp. (111).
2) Allan, S., et al., Bioprocess Design Considerations for Cultured Meat Production with a Focus on the Expansion Bioreactor, 2019, Front. Sustain. Food Syst. 3:44.
3) MM Fakhar - 5th International Scientific Conference on Cultured Meat, 2019, Maastricht