Isobel Massie

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.

Tissue Engineering the Cornea: The Evolution of RAFT.

Corneal blindness affects over 10 million people worldwide and current treatment strategies often involve replacement of the defective layer with healthy tissue. Due to a worldwide donor cornea shortage and the absence of suitable biological scaffolds, recent research has focused on the development of tissue engineering techniques to create alternative therapies. This review will detail how we have refined the simple engineering technique of plastic compression of collagen to a process we now call Real Architecture for 3D Tissues (RAFT). The RAFT production process has been standardised, and steps have been taken to consider Good Manufacturing Practice compliance. The evolution of this process has allowed us to create biomimetic epithelial and endothelial tissue equivalents suitable for transplantation and ideal for studying cell-cell interactions in vitro.

Protein engineered variants of hepatocyte growth factor/scatter factor promote proliferation of primary human hepatocytes and in rodent liver.

BACKGROUND & AIMS Hepatocyte growth factor/scatter factor (HGF/SF) stimulates hepatocyte DNA synthesis and protects against apoptosis; in vivo it promotes liver regeneration and reduces fibrosis. However, its therapeutic value is limited by its complex domain structure, high cost of production, instability, and poor tissue penetration due to sequestration by heparin sulfate proteoglycans (HSPGs). METHODS Using protein engineering techniques, we created a full-length form of HGF/SF (called HP21) and a form of the small, naturally occurring HGF/SF fragment, NK1 (called 1K1), which have reduced affinity for HSPG. We characterized the stability and proliferative and anti-apoptotic effects of these variants in primary human hepatocytes and in rodents. RESULTS Analytical ultracentrifugation showed that 1K1 and NK1 were more stable than the native, full-length protein. All 4 forms of HGF/SF induced similar levels of DNA synthesis in human hepatocytes; 1K1 and NK1 required heparin, an HSPG analogue, for full agonistic activity. All the proteins reduced levels of Fas ligand-mediated apoptosis, reducing the activity of caspase-3/7 and cleavage of poly(adenosine diphosphate-ribose) polymerase. 1K1 was more active than NK1 in rodents; in healthy mice, 1K1 significantly increased hepatocyte DNA synthesis, and in mice receiving carbon tetrachloride, it reduced fibrosis. In rats, after 70% partial hepatectomy, daily administration of 1K1 for 5 days significantly increased liver mass and the bromodeoxyuridine labeling index compared with mice given NK1. CONCLUSIONS 1K1, an engineered form of the small, naturally occurring HGF/SF fragment NK1, has reduced affinity for HSPG and exerts proliferative and antiapoptotic effects in cultured hepatocytes. In rodents, 1K1 has antifibrotic effects and promotes liver regeneration. The protein has better stability and is easier to produce than HGF/SF and might be developed as a therapeutic for acute and chronic liver disease.

Culture and Characterization of Oral Mucosal Epithelial Cells on a Fibrin Gel for Ocular Surface Reconstruction

Abstract Aim of the study: To develop a clinical grade fibrin gel for the culture of oral mucosal epithelial cells (OMEC) intended for ocular surface reconstruction in the treatment of limbal stem cell deficiency (LSCD). Materials and methods: Transparent fibrin gels composed of fibrinogen and thrombin were developed for the culture of epithelial cells. Oral mucosa was harvested from the buccal region of healthy volunteers and cultured as explants on fibrin gels. Tranexamic acid (TA), a clinically approved anti-fibrinolytic agent was added to prevent the fibrin gel from digesting due to cellular activity. The gels were stained for p63α (as a marker of poorly differentiated epithelial cells), CK19, CK13 and CK3 (expressed by OMEC). Epithelial cell stratification was observed using hematoxylin–eosin staining. Results: Addition of TA prevented gels from dissolving during the culture period. OMEC proliferated on the fibrin gel and attained confluence over a 2-week period (±2 d) and exhibited a typical epithelial, cobblestone morphology. Basal OMEC exhibited positive staining for p63α while the superficial cells exhibited positive staining for CK3. The cells expressed a strong immunoreactivity for CK19 and CK13 suggesting that they retained a normal oral epithelial phenotype. Conclusion: Fibrin gels, maintained in the presence of TA, to control the rate of substrate degradation, provide a more robust yet transparent substrate for the culture and transplantation of cultured OMEC. The fibrin gels are easily standardized, the components commercially available, and produced from clinically approved materials. The resulting stratified OMEC-derived epithelium displays characteristics similar to that of a human cornea, e.g. CK3 expression. The conventional dependence on a murine feeder layer for support of epithelial cells is unnecessary with this technique and hence, provides for an attractive alternative for treatment of LSCD.

Response of human limbal epithelial cells to wounding on 3D RAFT tissue equivalents: Effect of airlifting and human limbal fibroblasts

Limbal epithelial stem cell deficiency can cause blindness but may be treated by human limbal epithelial cell (hLE) transplantation, normally on human amniotic membrane. Clinical outcomes using amnion can be unreliable and so we have developed an alternative tissue equivalent (TE), RAFT (Real Architecture for 3D Tissue), which supports hLE expansion, and stratification when airlifted. Human limbal fibroblasts (hLF) may be incorporated into RAFT TEs, where they support overlying hLE and improve phenotype. However, the impact of neither airlifting nor hLF on hLE function has been investigated. hLE on RAFT TEs (±hLF and airlifting) were wounded using heptanol and re-epithelialisation (fluorescein diacetate staining), and percentage putative stem cell marker p63α and proliferative marker Ki67 expression (wholemount immunohistochemistry), measured. Airlifted, hLF- RAFT TEs were unable to close the wound and p63α expression was 7 ± 0.2% after wounding. Conversely, non-airlifted, hLF- RAFT TEs closed the wound within 9 days and p63α expression was higher at 22 ± 5% (p < 0.01). hLE on both hLF- and hLF+ RAFT TEs (non-airlifted) closed the wound and p63α expression was 26 ± 8% and 36 ± 3% respectively (ns). Ki67 expression by hLE increased from 1.3 ± 0.5% before wounding to 7.89 ± 2.53% post-wounding for hLF- RAFT TEs (p < 0.01), and 0.8 ± 0.08% to 17.68 ± 10.88% for hLF+ RAFT TEs (p < 0.05), suggesting that re-epithelialisation was a result of proliferation. These data suggest that neither airlifting nor hLF are necessarily required to maintain a functional epithelium on RAFT TEs, thus simplifying and shortening the production process. This is important when working towards clinical application of regenerative medicine products.

Advanced imaging and tissue engineering of the human limbal epithelial stem cell niche.

The limbal epithelial stem cell niche provides a unique, physically protective environment in which limbal epithelial stem cells reside in close proximity with accessory cell types and their secreted factors. The use of advanced imaging techniques is described to visualize the niche in three dimensions in native human corneal tissue. In addition, a protocol is provided for the isolation and culture of three different cell types, including human limbal epithelial stem cells from the limbal niche of human donor tissue. Finally, the process of incorporating these cells within plastic compressed collagen constructs to form a tissue-engineered corneal limbus is described and how immunohistochemical techniques may be applied to characterize cell phenotype therein.

Optimization of optical and mechanical properties of real architecture for 3-dimensional tissue equivalents: Towards treatment of limbal epithelial stem cell deficiency.

Graphical abstract

Evaluation of Decellularized Porcine Jejunum as a Matrix for Lacrimal Gland Reconstruction In Vitro for Treatment of Dry Eye Syndrome

Purpose Dry eye syndrome (DES) can cause blindness in severe cases, but mainly palliative treatments exist. A tissue-engineered lacrimal gland (LG) could provide a curative treatment. We aimed to evaluate decellularized porcine jejunum (SIS-Muc) as a scaffold for porcine LG epithelial cells. Methods To evaluate SIS-Muc as a potential scaffold, basement membrane proteins in SIS-Muc and native LG were compared (immunohistochemistry [IHC]). Porcine LG epithelial cells cultured on plastic were characterized (immunocytochemistry), and their culture supernatant was compared with porcine tears (proteomics). Epithelial cells were then seeded onto SIS-Muc in either a static (cell crown) or dynamic culture (within a perfusion chamber) and metabolic (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and secretory capacities (β-hexosaminidase assay), protein expression (IHC), and ultrastructure transmission electron microscopy (TEM) compared in each. Results Collagen IV and laminin were found in both native LG and SIS-Muc. When cultured on plastic, LG epithelial cells expressed pan-cytokeratin, Rab3D, HexA, and produced mucins, but lysozyme and lactoferrin expression was nearly absent. Some porcine tear proteins (lipocalin-2 and lactoferrin) were found in LG epithelial cell culture supernatants. When LG cells were cultured on SIS-Muc, metabolic and β-hexosaminidase activities were greater in dynamic cultures than static cultures (P < 0.05). In both static and dynamic cultures, cells expressed pan-cytokeratin, Rab3D, lysozyme, and lactoferrin and produced mucins, and TEM revealed cell polarization at the apical surface and cell-cell and cell-scaffold contacts. Conclusions SIS-Muc is a suitable scaffold for LG cell expansion and may be useful toward reconstruction of LG tissue to provide a curative treatment for DES. Dynamic culture enhances cell metabolic and functional activities.

1080 OXYGEN CARRIERS FOR BIOMASS PRESERVATION DURING THE TRANSPORT OF A BIOARTIFICIAL LIVER

Background and Aims: A variety of potential Bioartificial liver (BAL) support systems have been developed over the years, to provide adequate conditions to support the liver cells required to replace liver function. A significant challenge for clinical utility is the preservation of the biomass with adequate viability during transport and set-up prior to use. Whilst cryopreservation proffers an ideal tool for maintaining frozen cells for unlimited periods until they are required, unfortunately this technique currently fails to optimally preserve organised tissues in a satisfactory manner. Oxygen carriers such as perfluorocarbons (PFCs) are routinely used for organ preservation before transplantation, and we have developed these as potentially ideal candidates for BAL preservation. We describe here the technique applied to our BAL support system, based on alginate encapsulated human liver cells (HepG2 line), with preserved function using PFC emulsion and antioxidants in the media during transport. Methods: Alginate encapsulated HepG2 cell beads are grown in a fluidised bed bioreactor into performance competent. Cells proliferate within ~450–500um size hydrogel beads generating spheroid-shaped tissue, including liver specific cell structures. During the transport cells are dispensed into polystyrene flasks containing Hepes (25mM) buffered media and oxygenated PFC. Catalase (500U/ml), N-acetyl cysteine (3mM) and Trolox (0.85mM) antioxidants are added to the standard media to prevent cell damage. Iterative experimentation defined the optimal ratio of alginate beads, PFC and media as 1:1:10. 50% of the media was replenished after 27hours. Following storage, viability was analyzed by vital dye staining (FDA/PI). Cell functionality after storage was evaluated. Results: The BAL biomass of ~2.5×1010 cells remained >90% viable 48 hours after storage and transportation. Analyses of the media showed lactate accumulation and sustained glucose consumption over the storage time, indicating persistence of low level metabolism, using normal HepG2 pathway of anaerobic glucose metabolism. After storage, cells were cultured in media and plasma, showing restored synthetic function. Conclusions: We describe a method for optimal maintenance of alginate entrapped biomass capable of preserving 2.5×1010 viable cells in a volume of ~1200ml alginate beads. The performance of this biomass is being tested in an animal ischemic model of acute liver failure.

Development of lacrimal gland spheroids for lacrimal gland tissue regeneration

Severe dry eye syndrome resulting from lacrimal gland (LG) dysfunction can cause blindness, yet treatments remain palliative. In vitro reconstruction of LG tissue could provide a curative treatment. We aimed to combine epithelial cells with endothelial cells and mesenchymal stem cells (MSCs) to form a 3D functional unit.