Metabolic Reprogramming and the Recovery of Physiological Functionality in 3D Cultures in Micro-Bioreactors

Abstract

The recovery of physiological functionality, which is commonly seen in tissue mimetic three-dimensional (3D) cellular aggregates (organoids, spheroids, acini, etc.), has been observed in cells of many origins (primary tissues, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and immortal cell lines). This plurality and plasticity suggest that probably several basic principles promote this recovery process. The aim of this study was to identify these basic principles and describe how they are regulated so that they can be taken in consideration when micro-bioreactors are designed. Here, we provide evidence that one of these basic principles is hypoxia, which is a natural consequence of multicellular structures grown in microgravity cultures. Hypoxia drives a partial metabolic reprogramming to aerobic glycolysis and an increased anabolic synthesis. A second principle is the activation of cytoplasmic glutaminolysis for lipogenesis. Glutaminolysis is activated in the presence of hypo- or normo-glycaemic conditions and in turn is geared to the hexosamine pathway. The reducing power needed is produced in the pentose phosphate pathway, a prime function of glucose metabolism. Cytoskeletal reconstruction, histone modification, and the recovery of the physiological phenotype can all be traced to adaptive changes in the underlying cellular metabolism. These changes are coordinated by mTOR/Akt, p53 and non-canonical Wnt signaling pathways, while myc and NF-kB appear to be relatively inactive. Partial metabolic reprogramming to aerobic glycolysis, originally described by Warburg, is independent of the cell's rate of proliferation, but is interwoven with the cells abilities to execute advanced functionality needed for replicating the tissues physiological performance.

Keywords: 3D cell culture; Warburg; aerobic glycolysis; bioreactors; glutaminolysis; hypoxia; metabolic reprogramming; organoids; physiological performance; spheroids.

Figure 1

(A) an assembled bioreactor containing >300 21 day old spheroids. (B) The open bioreactor with (left) a 10 mL petri-dish like culture chamber and (right) the gas exchange membrane. Behind the membrane is a water reservoir and humidification labyrinth. White stoppers allow access for media change or filling the reservoir. This type of bioreactor has a gas membrane exchange area of 13.2 cm² and a fixed volume (nominally 10 mL) and is available from CelVivo (Denmark).

Figure 2

Ratios of the protein abundance of central metabolic enzymes and membrane transporter in 3D compared to two-dimensional (2D) cultures. Metabolites are marked in black, enzymes in green, transporters in blue and selected cofactors in red. Negative ratios indicate that the protein is present in higher amounts in 2D cultures. Arrows connecting metabolites are marked in bold if the enzyme expression is increased by a factor of 1.5 or greater. Arrows connecting metabolites are dotted if the enzyme expression is decreased. (Raw data taken from [57]).

Figure 3

The diffusion depleted zone and its consequences for the oxygen, nutrient and catabolite gradients in (A) passive and (B) irrigated 3D culture. The size shown indicates the approximate maximum size above which anoxia develops in the spheroid core. Volumes, number of cells and amounts of protein are indicated for each. The apparent increase in cell volume is attributed in part to increased ECM and the development of sinusoidal and bile cannalicular spaces between the cells.

Figure 4

Effects of hypoxia on HIF-1α. The thickness of the line indicates the ratio of protein amount in thre—dimensional (3D) cultures compared to 2D cultures. Dotted lines indicate reduced expression. Green lines ending in arrowheads indicate activators, while red lines ending in a bar indicate inhibitory activity. Dotted-boxes indicate links to other pathway figures.

Figure 5

Relationship of glucose consumption from the media with the level of glycogen present in the spheroids. The amount of glucose in the spheroids prior to glycogen hydrolysis was negligible.

Figure 6

mTOR signalling in 3D spheroids. See legend to Figure 4 for nomenclature.

Figure 7

Myc signalling in 3D spheroids. See legend to Figure 4 for nomenclature.

Figure 8

p53 signalling in 3D spheroids. See legend to Figure 4 for nomenclature.