Scale-Up Technologies for the Manufacture of Adherent Cells

Abstract

Great importance is being given to the impact our food supply chain and consumers' food habits are having on the environment, human health, and animal welfare. One of the latest developments aiming at positively changing the food ecosystem is represented by cultured meat. This form of cellular agriculture has the objective to generate slaughter-free meat products starting from the cultivation of few cells harvested from the animal tissue of interest. As a consequence, a large number of cells has to be generated at a reasonable cost. Just to give an idea of the scale, there were billions of cells just in a bite of the first cultured-meat burger. Thus, one of the major challenges faced by the scientists involved in this new ambitious and fascinating field, is how to efficiently scale-up cell manufacture. Considering the great potential presented by cultured meat, audiences from different backgrounds are very interested in this topic and eager to be informed of the challenges and possible solutions in this area. In light of this, we will provide an overview of the main existing bioprocessing technologies used to scale-up adherent cells at a small and large scale. Thus, giving a brief technical description of these bioprocesses, with the main associated advantages and disadvantages. Moreover, we will introduce an alternative solution we believe has the potential to revolutionize the way adherent cells are grown, helping cultured meat become a reality.

Keywords: adherent cell manufacture; bioprocessing; bioreactors; continuous bioprocessing; cultured meat; scale-up cells manufacture.

Figure 1

Cell harvesting: (A) Anchorage dependent (adherent) cells require adherence to a surface for sustained and healthy culture, this anchorage can be to either common tissue culture plastics or to microcarriers. Although microcarriers themselves are in suspension it is important to note that the cells are still anchored to the microcarrier and thus require the same cleavage of anchorage proteins as cells anchored to tissue culture plastics. Classically, anchorage proteins are cleaved either via enzymatic or mechanical means. Such cleavage releases the cells from the surface and into suspension for collection. Anchorage dependent cells typically cannot survive long in suspension conditions, hence the requirement for batch cleavage. (B) Anchorage independent (suspension) cells do not require surface adherence to be viable and proliferate, thus they are readily available for collection and easily adaptable to bioprocessing.

Figure 2

Differences between traditional and High intensity scale up: (Top) Traditional and (Bottom) High intensity methods.

Figure 3

Obtaining a high-density cell bank: High density cell banks are used to reduce the required steps in the traditional scale-up process to generate larger numbers of cells. Initially, cells are grown at a high cell density in a perfusion bioreactor, a cryopreservation is added to the culture and the volume is reduced. Cells are then banked as high-density aliquots in cryovials. When required, a test vial is revived. If the revival is successful, the high-density aliquots are seeded into a perfusion bioreactor which is subsequently seeded into a larger bioreactor.

Figure 4

Scale-up and Scale-out. Cultures are typically initiated from cryopreserved stocks or biopsies and cultivated in small-scale cultures such as flasks. These flasks can be scaled up with the use of microcarriers or scaled out with the use of hyper or multilayered flasks. The process then enters the bench scale stage. Here, the use of larger vessels such as roller bottles, perfusion bioreactors and spinner flasks can be deployed to both scale-up and scale-out the process. When required, the process then enters the industrial scale. This stage of bioprocessing enters two distinct streams: bioreactors scale-out (multiple bioreactors of the same size) or scale-up (further processing up to a single bioreactor).

Figure 5

Small scale technologies. Schematic representing the discussed compact technologies: (A) T-flask; (B) Multi-layered flask; (C) Roller bottle; and (D) Spinner flask.

Figure 6

Large scale technologies. Schematic representing the discussed industrial technologies: (A) Wave; (B) Stirred tank; (C) Packed bed; and (D) Hollow fiber bioreactors.

Figure 7

Continuous process. Schematic showing an example of how a continuous process for the manufacture of adherent cells could work. Such system will allow adherent cells to grow onto a surface and detach continuously as single-cells at a steady-state. The detaching cells will leave an empty space for neighboring cells to grow into maintaining a stable number of cells in culture whilst continuously harvesting single cells from the system.