Dimethyl sulfoxide for cryopreservation of alginate encapsulated liver cell spheroids in bioartificial liver support; assessments of cryoprotectant toxicity tolerance and dilution strategies.

The Bioartificial Liver (BAL) is an extra-corporeal liver support designed to support the function of the Liver in patients with impaired liver function. The BAL biomass consists of alginate encapsulated liver spheroids (AELS). To facilitate rapid delivery of a BAL to patients the AELS are cryopreserved using a DMSO-containing cryoprotectant solution. This study assesses toxicity of DMSO in AELS at concentrations and temperatures relevant to the cryopreservation and recovery process of a cellular biomass. Additionally, it develops a process to remove DMSO from AELS before delivery of cell product to patients. Exposure of AELS to DMSO, at a concentration of 12% (v/v) for 10 min did not have a negative effect on the viability of the AELS up to 24 h after exposure, irrespective of the exposure temperature between 37 C and 0 C. Evidence of toxicity was only seen with exposure to 40% (v/v) DMSO, which was more notable at warm temperatures. Post-Thaw removal of DMSO was measured by determining the DMSO concentration of the post-thaw washes using refractometry. Washing AELS 3 times in tapering concentrations of Glucose supplemented DMEM at an AELS:wash ratio of 1:2 was sufficient to reduce DMSO to undetectable levels (<1%). The study demonstrated that the thawing method minimised DMSO toxicity to the BAL biomass, and the post-thaw washing protocol successfully removed all the DMSO present in the cryopreserved BAL. Thereby enabling effective cryopreservation of the BAL for future clinical translation.

Decisional tool for cost of goods analysis of bioartificial liver devices for routine clinical use.

BACKGROUND AIMS: Bioartificial liver devices (BALs) are categorized as advanced therapy medicinal products (ATMPs) with the potential to provide temporary liver support for liver failure patients. However, to meet commercial demands, next-generation BAL manufacturing processes need to be designed that are scalable and financially feasible. The authors describe the development and application of a process economics decisional tool to determine the cost of goods (COG) of alternative BAL process flowsheets across a range of industrial scales. METHODS: The decisional tool comprised an information database linked to a process economics engine, with equipment sizing, resource consumption, capital investment and COG calculations for the whole bioprocess, from cell expansion and encapsulation to fluidized bed bioreactor (FBB) culture to cryopreservation and cryorecovery. Four different flowsheet configurations were evaluated across demands, with cell factories or microcarriers in suspension culture for the cell expansion step and single-use or stainless steel technology for the FBB culture step. RESULTS: The tool outputs demonstrated that the lowest COG was achieved with microcarriers and stainless steel technology independent of the annual demand (1500-30 000 BALs/year). The analysis identified the key cost drivers were parameters impacting the medium volume and cost. CONCLUSIONS: The tool outputs can be used to identify cost-effective and scalable bioprocesses early in the development process and minimize the risk of failing to meet commercial demands due to technology choices. The tool predictions serve as a useful benchmark for manufacturing ATMPs.

Small-Scale Fluidized Bed Bioreactor for Long-Term Dynamic Culture of 3D Cell Constructs and in vitro Testing.

With the increasing interest in three-dimensional (3D) cell constructs that better represent native tissues, comes the need to also invest in devices, i.e., bioreactors, that provide a controlled dynamic environment similar to the perfusion mechanism observed in vivo. Here a laboratory-scale fluidized bed bioreactor (sFBB) was designed for hydrogel (i.e., alginate) encapsulated cells to generate a dynamic culture system that produced a homogenous milieu and host substantial biomass for long-term evolution of tissue-like structures and "per cell" performance analysis. The bioreactor design, conceptualized through scale-down empirical similarity rules, was initially validated through computational fluid dynamics analysis for the distributor capacity of homogenously dispersing the flow with an average fluid velocity of 4.596 × 10-4 m/s. Experimental tests then demonstrated a consistent fluidization of hydrogel spheres, while maintaining shape and integrity (606.9 ± 99.3 µm diameter and 0.96 shape factor). It also induced mass transfer in and out of the hydrogel at a faster rate than static conditions. Finally, the sFBB sustained culture of alginate encapsulated hepatoblastoma cells for 12 days promoting proliferation into highly viable (>97%) cell spheroids at a high final density of 27.3 ± 0.78 million cells/mL beads. This was reproducible across multiple units set up in parallel and operating simultaneously. The sFBB prototype constitutes a simple and robust tool to generate 3D cell constructs, expandable into a multi-unit setup for simultaneous observations and for future development and biological evaluation of in vitro tissue models and their responses to different agents, increasing the complexity and speed of R&D processes.

Evaluation of encapsulated liver cell spheroids in a fluidised-bed bioartificial liver for treatment of ischaemic acute liver failure in pigs in a translational setting.

Liver failure is an increasing problem. Donor-organ shortage results in patients dying before receiving a transplant. Since the liver can regenerate, alternative therapies providing temporary liver-support are sought. A bioartificial-liver would temporarily substitute function in liver failure buying time for liver regeneration/organ-procurement. Our aim: to develop a prototype bioartificial-liver-machine (BAL) comprising a human liver-derived cell-line, cultured to phenotypic competence and deliverable in a clinical setting to sites distant from its preparation. The objective of this study was to determine whether its use would improve functional parameters of liver failure in pigs with acute liver failure, to provide proof-of-principle. HepG2 cells encapsulated in alginate-beads, proliferated in a fluidised-bed-bioreactor providing a biomass of 4-6 × 10(10)cells, were transported from preparation-laboratory to point-of-use operating theatre (6000 miles) under perfluorodecalin at ambient temperature. Irreversible ischaemic liver failure was induced in anaesthetised pigs, after portal-systemic-shunt, by hepatic-artery-ligation. Biochemical parameters, intracranial pressure, and functional-clotting were measured in animals connected in an extracorporeal bioartificial-liver circuit. Efficacy was demonstrated comparing outcomes between animals connected to a circuit containing alginate-encapsulated cells (Cell-bead BAL), and those connected to circuit containing alginate capsules without cells (Empty-bead BAL). Cells of the biomass met regulatory standards for sterility and provenance. All animals developed progressive liver-failure after ischaemia induction. Efficacy of BAL was demonstrated since animals connected to a functional biomass (+ cells) had significantly smaller rises in intracranial pressure, lower ammonia levels, more bilirubin conjugation, improved acidosis and clotting restoration compared to animals connected to the circuit without cells. In the +cell group, human proteins accumulated in pigs' plasma. Delivery of biomass using a short-term cold-chain enabled transport and use without loss of function over 3 days. Thus, a fluidised-bed bioreactor containing alginate-encapsulated HepG2 cell-spheroids improved important parameters of acute liver failure in pigs. The system can readily be up-scaled and transported to point-of-use justifying development at clinical scale.

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

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.

Altered mitochondrial function and cholesterol synthesis influences protein synthesis in extended HepG2 spheroid cultures.

Cultures of hepatocytes and HepG2 cells provide useful in vitro models of liver specific function. In this study, we investigated metabolic and biosynthetic function in 3-D HepG2 spheroid cultures, in particular to characterise changes on prolonged culture. We show that HepG2 cells cultured in spheroids demonstrate a reduction in mitochondrial membrane potential and respiration following 10 days of culture. This coincides with a modest reduction in glycolysis but an increase in glucose uptake where increased glycogen synthesis occurs at the expense of the intracellular ATP pool. Lowered biosynthesis coincides with and is linked to mitochondrial functional decline since low glucose-adapted spheroids, which exhibit extended mitochondrial function, have stable biosynthetic activity during extended culture although biosynthetic function is lower. This indicates that glucose is required for biosynthetic output but sustained mitochondrial function is required for the maintenance of biosynthetic function. Furthermore, we show that cholesterol synthesis is markedly increased in spheroids cf. monolayer culture and that inhibition of cholesterol synthesis by lovastatin extends mitochondrial and biosynthetic function. Therefore, increased cholesterol synthesis and/or its derivatives contributes to mitochondrial functional decline in extended HepG2 spheroid cultures.

Epithelial colonies cultured from human explanted liver in subacute hepatic failure exhibit hepatocyte, biliary epithelial, and stem cell phenotypic markers.

The liver in subacute hepatic failure may become enriched for hepatic progenitor cells. Liver tissue from such a patient was collagenase digested and, from the nonparenchymal cell fraction, epithelioid colonies were developed. Albumin and alpha-1-antitrypsin (AAT) were secreted for greater than 120 days from these colonies. Reverse transcription-polymerase chain reaction showed expression of markers of both hepatocyte and biliary epithelial phenotypes (cytokeratins 7, 18, and 19, albumin and AAT, hepatocyte growth factor receptor, transforming growth factor beta receptor type II, gamma-glutamyl transpeptidase, biliary glycoprotein). The cell cycle regulator p21 was also expressed. The POU domain transcription factor octamer-binding protein 4 was present in these cells, but not in RNA or cDNA prepared from adult human liver. These markers were maintained even after 165 days culture. Proliferating epithelial-like cells with combined hepatocyte- and biliary-epithelial-specific functional markers and a stem cell marker can be isolated from the nonparenchymal fraction of liver cells in subacute hepatic failure.