A New High Throughput Screening Platform for Cell Encapsulation in Alginate Hydrogel Shows Improved Hepatocyte Functions by Mesenchymal Stromal Cells Co-encapsulation.

Hepatocyte transplantation has emerged as an alternative to liver transplant for liver disease. Hepatocytes encapsulated in alginate microbeads have been proposed for the treatment of acute liver failure, as they are able to provide hepatic functions while the liver regenerates. Furthermore, they do not require immunosuppression, as the alginate protects the hepatocytes from the recipient's immune cells. Mesenchymal stromal cells are very attractive candidates for regenerative medicine, being able to differentiate into cells of the mesenchymal lineages and having extensive proliferative ability. When co-cultured with hepatocytes in two-dimensional cultures, they exert a trophic role, drastically improving hepatocytes survival and functions. In this study we aimed to (i) devise a high throughput system (HTS) to allow testing of a variety of different parameters for cell encapsulation and (ii) using this HTS, investigate whether mesenchymal stromal cells could have beneficial effects on the hepatocytes when co-encapsulated in alginate microbeads. Using our HTS platform, we observed some improvement of hepatocyte behavior with MSCs, subsequently confirmed in the low throughput analysis of cell function in alginate microbeads. Therefore, our study shows that mesenchymal stromal cells may be a good option to improve the function of hepatocytes microbeads. Furthermore, the platform developed may be used for HTS studies on cell encapsulation, in which several conditions (e.g., number of cells, combinations of cells, alginate modifications) could be easily compared at the same time.

Hepatocyte transplantation and advancements in alternative cell sources for liver-based regenerative medicine.

Human hepatocyte transplantation has been actively perused as an alternative to liver replacement for acute liver failure and liver-based metabolic defects. Current challenges in this field include a limited cell source, reduced cell viability following cryopreservation and poor engraftment of cells into the recipient liver with consequent limited life span. As a result, alternative stem cell sources such as pluripotent stem cells, fibroblasts, hepatic progenitor cells, amniotic epithelial cells and mesenchymal stem/stromal cells (MSCs) can be used to generate induced hepatocyte like cells (HLC) with each technique exhibiting advantages and disadvantages. HLCs may have comparable function to primary human hepatocytes and could offer patient-specific treatment. However, long-term functionality of transplanted HLCs and the potential oncogenic risks of using stem cells have yet to be established. The immunomodulatory effects of MSCs are promising, and multiple clinical trials are investigating their effect in cirrhosis and acute liver failure. Here, we review the current status of hepatocyte transplantation, alternative cell sources to primary human hepatocytes and their potential in liver regeneration. We also describe recent clinical trials using hepatocytes derived from stem cells and their role in improving the phenotype of several liver diseases.

Cryopreserved neonatal hepatocytes may be a source for transplantation: Evaluation of functionality toward clinical use.

Neonatal livers are a potential source of good-quality hepatocytes for clinical transplantation. We compared viability and function of neonatal hepatocytes (NHs) and adult hepatocytes (AHs) and report their clinical use both intraportally and in alginate microbeads. Following isolation from donor livers, hepatocyte function was assessed using albumin, alpha-1-antitrypsin, and factor VII. Metabolic function was investigated by measuring resorufin conjugation, ammonia metabolism, uridine diphosphate glucuronosyltransferase enzyme activity, and cytochrome P450 (CYP) function following induction. Activation of the instant blood-mediated inflammatory reaction by NHs and AHs was investigated using an in vitro blood perfusion model, and tissue factor expression was analyzed using real-time polymerase chain reaction (RT-PCR). Clinical hepatocyte transplantation (HT) was undertaken using standard protocols. Hepatocytes were isolated from 14 neonatal livers, with an average viability of 89.4% ± 1.8% (mean ± standard error of the mean) and average yield of 9.3 × 106 ± 2.0 × 106 cells/g. Hepatocytes were isolated from 14 adult livers with an average viability of 78.6% ± 2.4% and yield 2.2 × 106 ± 0.5 × 105 cells/g. NHs had significantly higher viability after cryopreservation than AHs, with better attachment efficiency and less plasma membrane leakage. There were no differences in albumin, alpha-1-antitrypsin, and factor VII synthesis between NHs and AHs (P > 0.05). Neonatal cells had inducible phase 1 enzymes as assessed by CYP function and functional phase 2 enzymes, in which activity was comparable to AHs. In an in vitro blood perfusion model, AHs elicited increased thrombus formation with a greater consumption of platelets and white cells compared with NHs (28.3 × 109 versus 118.7 × 109 and 3.3 × 109 versus 6.6 × 109 ; P < 0.01). Intraportal transplantation and intraperitoneal transplantation of alginate encapsulated hepatocytes was safe, and preliminary data suggest the cells may activate the immune response to a lesser degree than adult cells. In conclusion, we have shown NHs have excellent cell viability, function, and drug metabolism making them a suitable alternative source for clinical HT. Liver Transplantation 24 394-406 2018 AASLD.

Instant Blood-Mediated Inflammatory Reaction in Hepatocyte Transplantation: Current Status and Future Perspectives.

Hepatocyte transplantation (HT) is emerging as a promising alternative to orthotopic liver transplantation (OLT) in patients with certain liver-based metabolic disease and acute liver failure. Hepatocytes are generally infused into the portal venous system, from which they migrate into the liver cell plates of the native organ. One of the major hurdles to the sustained success of this therapy is early cell loss, with up to 70% of hepatocytes lost immediately following infusion. This is largely thought to be due to the instant blood-mediated inflammatory reaction (IBMIR), resulting in the activation of complement and coagulation pathways. Transplanted hepatocytes produce and release tissue factor (TF), which activates the coagulation pathway, leading to the formation of thrombin and fibrin clots. Thrombin can further activate a number of complement proteins, leading to the activation of the membrane attack complex (MAC) and subsequent hepatocyte cell death. Inflammatory cells including granulocytes, monocytes, Kupffer cells, and natural killer (NK) cells have been shown to cluster around transplanted hepatocytes, leading to their rapid clearance shortly after transplantation. Current research aims to improve cell engraftment and prevent early cell loss. This has been proven successful in vitro using pharmacological interventions such as melagatran, low-molecular-weight dextran sulphate, and N-acetylcysteine (NAC). Effective inhibition of IBMIR would significantly improve hepatocyte engraftment, proliferation, and function, providing successful treatment for patients with liver-based metabolic diseases.

Atrial septal aneurysm.

As we begin to see more echocardiograms in the medical records that are available to us, we are faced with assessing some findings that leave us with questions about their significance. One such finding is the atrial septal aneurysm (ASA).

Chiari I malformation.

As MRI's are becoming more popular for evaluating the brain and cranial vault, we are beginning to see the diagnosis of Chiari I malformation being made rather frequently. This case study describes Chiari I malformation of the brainstem and its implications for mortality.

Osteopetrosis.

This case study describes the fairly rare entity of osteopetrosis and points out how a seemingly insignificant "anemia" can, on thorough investigation, be found to be a very significant disease with a poor prognosis.

Kawasaki disease.

Kawasaki disease is currently the leading cause of acquired heart disease in children in the United States and Japan. The case of an 18-year-old insurance applicant is used to review the pathogenesis, complications, treatment and prognostic implications of this disorder.