Bioengineering the liver: scale-up and cool chain delivery of the liver cell biomass for clinical targeting in a bioartificial liver support system

Abstract

Acute liver failure has a high mortality unless patients receive a liver transplant; however, there are insufficient donor organs to meet the clinical need. The liver may rapidly recover from acute injury by hepatic cell regeneration given time. A bioartificial liver machine can provide temporary liver support to enable such regeneration to occur. We developed a bioartificial liver machine using human-derived liver cells encapsulated in alginate, cultured in a fluidized bed bioreactor to a level of function suitable for clinical use (performance competence). HepG2 cells were encapsulated in alginate using a JetCutter to produce ∼500 μm spherical beads containing cells at ∼1.75 million cells/mL beads. Within the beads, encapsulated cells proliferated to form compact cell spheroids (AELS) with good cell-to-cell contact and cell function, that were analyzed functionally and by gene expression at mRNA and protein levels. We established a methodology to enable a ∼34-fold increase in cell density within the AELS over 11-13 days, maintaining cell viability. Optimized nutrient and oxygen provision were numerically modeled and tested experimentally, achieving a cell density at harvest of >45 million cells/mL beads; >5×10(10) cells were produced in 1100 mL of beads. This process is scalable to human size ([0.7-1]×10(11)). A short-term storage protocol at ambient temperature was established, enabling transport from laboratory to bedside over 48 h, appropriate for clinical translation of a manufactured bioartificial liver machine.

Keywords: HepG2 cells; alginate encapsulation; fluidized bed bioreactor.

FIG. 1.

Cell density in beads during proliferation phase of the biomass (n=5, mean±SEM). Inset: Average proliferation, (46.57±6.3)×10⁶ cells/mL at harvest (SEM).

FIG. 2.

Viability and phase contrast microscopy of HepG2s encapsulated in alginate beads. (A, D) Fluorescein diacetate (FDA)-stained viable cells. (B, E) Propidium iodide (PI)-stained dead cells. (C, F) Phase contrast images. (A–C) Day of encapsulation, (D–F) after 10 days of proliferation in fluidized bed bioreactor.

FIG. 3.

Metabolite levels during culture of proliferating biomass. (A) Glucose concentration during proliferation phase (n=5, mean±SEM), peaks match media changes on days 4, 6, 8, and 10). (B) Lactate profile: Lactate concentration during proliferation (n=5, mean±SEM). Falls in lactate correspond to media changes (days 4, 6, 8, and 10). (C) Alpha-fetoprotein (AFP) concentration; vertical lines represent samples before and after media change.

FIG. 4.

Amino acid levels during biomass production. (A) Amino acid levels during 12 days of biomass cultivation. Graph shows consumption of five key amino acids being only partially replenished by media changes on days 5, 7, 9, and 11. (B) Successful maintenance of depleted amino acids during fermentation. Control was by use of a model relating cell proliferation and amino acid consumption to predict media supplementation; in all cases the minimum amino acid concentrations remained above 60% of initial value.

FIG. 5.

Oxygenation of a typical fluidized bed bioreactor experiment over 11 days. (A) Dissolved oxygen (DO)1 (dO₂ in fermentor), DO2 (dO₂ immediately after the biomass); additional oxygenation (dashed line) supplied directly to the biomass chamber. (B) Cumulative oxygen consumption of biomass. (C) Oxygen consumption in millimoles per hour.