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; >5×1010 cells were produced in 1100 mL of beads. This process is scalable to human size ([0.7–1]×1011). 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.

Cell encapsulation: technical and clinical advances

“…3D spherical culture systems allow enrichment of cancer stem-like cells for cancer research and therapy development [91], and allow the production of a readily scalable liver cell biomass for the development of a bioartificial livers [92].…”

GMP Cryopreservation of Large Volumes of Cells for Regenerative Medicine: Active Control of the Freezing Process

“…2,14 For small volume experiments, ELS were maintained in static culture for 7 days in six-well plates in media supplemented with glucose to a final concentration of 22.5 mM, with 10% FFP in place of FCS. 2 For large volume experiments, ELS were maintained in culture for 11 days in a fluidized bed bioreactor. Fifty percent, 70%, and 80% media changes were performed on days 6, 8, and 10 respectively.…”

“…Fifty percent, 70%, and 80% media changes were performed on days 6, 8, and 10 respectively. 2 For all experiments, cell density within ELS was *20 · 10 6 /mL alginate. The exact cell densities within each set of experiments are given in Tables 1, 2, and 3 under ''unfrozen control.''…”

“…These include retention of good functionality, sterility, and scale up: for example, estimates for a bioartificial liver indicate that cryopreservation of 2 L of alginate-encapsulated cell spheroids (ELS) are required. 2 Cryopreservation in bags of large volumes ( > 100 mL) of adult stem cells 3 and of mammalian tissue culture cells [4][5][6] using conventional liquid nitrogen-based controlled rate freezers (CRFs) is established. However, the use of liquid nitrogen is problematic in a GMP environment; following manufacture, liquid nitrogen contains very low levels of contaminants, but during transport and storage it can become contaminated by ice, inanimate debris, and viable microorganisms [7][8][9] that can transfer to the local environment.…”

Problems and prospects of creation of extracorporal systems for support of functional livers status

“…Эмбриональные тельца человека, культивируемые в микрочастицах альгината в присутствии экзогенных факторов роста, дифференцировались в гепатоциты с экспрессией альфа-фетопротеина, альбумина, CYP7A1 и цитокератина 18 [75], а культивирование клеточной линии С3А гепатоцитов человека внутри альгинатных матриксов приводило к образованию многоклеточных сфероидов с гораздо более выраженной активностью цитохромов P450 по сравнению с монослойными культурами [76]. Разработано устройство типа "ЭБИП" с использованием инкапсулированных в альгинате и культивируемых в жидкостно-пластинчатом биореакторе гепатоцитов человека линии HepG2 [77], которые инкапсулировались с помощью аппарата JetCutter с образованием сферических частиц размером около 500 мкм, содержащих 1,75 млн клеток/мл в каждой. Клетки внутри частиц пролиферировали до образования компактных клеточных сфероидов с хорошими межклеточными контактами и клеточными функциями.…”