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Blastocyst complementation combined with gene editing is an emerging approach in the field of regenerative medicine that could potentially solve the worldwide problem of organ shortages for transplantation. In theory, blastocyst complementation can generate fully functional human organs or tissues, grown within genetically engineered livestock animals. Targeted deletion of a specific gene(s) using gene editing to cause deficiencies in organ development can open a niche for human stem cells to occupy, thus generating human tissues. Within this review, we will focus on the pancreas, liver, heart, kidney, lung, and skeletal muscle, as well as cells of the immune and nervous systems. Within each of these organ systems, we identify and discuss (i) the common causes of organ failure; (ii) the current state of regenerative therapies; and (iii) the candidate genes to knockout and enable specific exogenous organ development via the use of blastocyst complementation. We also highlight some of the current barriers limiting the success of blastocyst complementation.

Primary hepatocytes form spheroids under some culture conditions. These spheroids exhibit many tissuelike ultrastructures and retain many liver-specific functions over a long period of time. They are attractive for many applications employing liver cells. The ability to maintain their viability and functions at a reduced temperature to allow for transportation to the site of their application will facilitate their use. Furthermore, with their structural and functional similarity, they could possibly be used as a model system for studying various liver ischemias. The effect of hypothermic treatment was assessed by oxygen consumption rate, ATP, H 2 O 2 , and caspase 8 content, as well as albumin and urea synthesis, during and posttreatment. No single outcome variable gives a superlative quantification of hypothermic damage. Taken together, the hypothermic treatment can be seen as increasingly damaging as the temperature decreases from 21°C to 15°C and 4°C. The addition of the chemical protectants glutathione, N-acetyl-L-cystein (NAC), and tauroursodeoxycholic acid (TUDCA) decreased the damaging effect of hypothermic treatment. This protection effect was even more profound when spheroids were preincubated with the protectant for 24 h, and was most prominent at 4°C. The viability of the hypothermically treated hepatocyte spheroids was confirmed by laser scanning confocal microscopy. The method reported provides a means of maintaining spheroids' viability and may allow for their distribution to application sites at a distance.

Cytochrome P450 (CYP450) enzymes are essential for xenobiotic metabolism. Although CYP450s are found in many tissues, CYP2B1/2 are primarily expressed in the rat liver. The constitutive expression in vivo of CYP2B1/2 is low but it is induced in the presence of various drugs such as phenobarbital (PB). In this study, CYP2B1/2 activity in cultured hepatocytes was assessed in situ with the introduction of a fluorogenic sub-strate, pentoxyresorufin. The product of 7-pentoxyresorufin-O-dealkylation (PROD), which is catalyzed specifically by CYP2B1/2, was detected using confocal laser scanning microscopy (CLSM). Primary hepatocytes cultured as monolayers on collagen-coated surfaces exhibited background PROD activity and minimal PB inducibility after 4 days in culture. In contrast, rat hepatocytes organized in compacted aggregates, or spheroids, exhibited higher levels of PROD activity and retained their ability for PB induction. The results from the CLSM analysis were verified by RT-PCR and Western immunoblotting analysis. Furthermore, CLSM in conjunction with image processing techniques and three-dimensional reconstruction revealed the localization of enhanced PROD activity in the center of spheroids. The results support the use of CLSM as a powerful tool for investigating CYP2B1/2 activity in cultured rat hepatocytes.

Primary rat hepatocytes can self-assemble to form multicellular spheroids when plated onto Primaria petri dishes. Spheroids have been observed to exhibit enhanced liver-specific functions and differentiated ultrastructure compared to monolayer cultures on dry collagen. With confocal scanning laser microscopy, CYP1A1 activity was evaluated in situ by detecting resorufin. This highly fluorescent molecule is the P450-mediated product of 7-ethoxyresorufin O-dealkylation (EROD). Significantly higher P450 activity was observed in spheroids compared to monolayers on collagen upon induction with 50 μM β-naphthoflavone (BNF), a CYP1A inducer. This was confirmed by measuring microsomal EROD activity. The distribution of CYP1A1 activity within spheroids was heterogeneous, with higher activity localized to the hepatocytes in the interior. During the process of spheroid formation, cells were initially seen to attach and spread out as a monolayer. This stage was associated with relatively low CYP1A1 activity. As cells formed multicellular structures and aggregated into spheroids, the level of CYP1A1 activity increased over time. At least a fivefold higher fluorescence intensity was observed in spheroids compared to that of monolayers maintained on collagen. The higher P450 activity within spheroids may be associated with their ability to maintain a greater degree of differentiation compared to monolayers. These studies demonstrate the potential of hepatocyte spheroids as a model system for investigating drug metabolism, tissue engineering, and tissue self-assembly.

Xenogeneic hepatocytes have recently been used in a bioartificial liver device as a potential short-term extracorporeal support of acute liver failure. Scaling up the system requires large quantities of viable and highly active cells. Hepatocytes grown as spheroids manifest higher metabolic activities for longer time periods as compared to those in monolayer cultures. Use of hepatocyte spheroids for application in a bioartificial liver can possibly alleviate the need of scaling up. Porcine hepatocytes when cultured under stirred conditions, form multicellular spheroids in a defined culture medium. Spheroids were formed 24 h after cell inoculation with an efficiency of 80-90% and a mean diameter of about 135 μm. Scanning electron microscopy revealed numerous microvilli projecting from the entire surface of the spheroids. Transmission electron microscopy revealed differentiated hepatocytes which displayed well-developed cytoplasmic structures separated by bile canaliculus-like structures. The morphological studies show a resemblance between cells in the spheroids and in the liver in vivo. Ureagenesis by spheroids was twice as active and was sustained for a longer culture period than that by hepatocytes cultured as monolayers. Preparation of porcine hepatocyte spheroids in an agitated vessel is simple efficient and reproducible. It will allow for preparation of large quantities of spheroids to be employed in a bioartificial liver device as well as in liver metabolism studies.

Metabolic activity of a gel-entrapment, hollow fiber, bioartificial liver was evaluated in vitro and during extracorporeal hemoperfusion in an anhepatic rabbit model. The bioartificial liver contained either 100 million rat hepatocytes (n = 12), fibroblasts (n = 3), or no cells (n = 7) during hemoperfusion of anhepatic rabbits. Eight other anhepatic rabbits were studied without hemoperfusion as anhepatic controls, and three sham rabbits served as normal controls. Albumin production rates (mean ± SEM) were similar during in vitro (17.0 ± 2.8 μg/h) and extracorporeal (18.0 ± 4.0 μg/h) application of the hepatocyte bioartificial liver. Exogenous glucose requirements were reduced (p < 0.01) and euglycemia was prolonged (p < 0.001) in anhepatic rabbits treated with the hepatocyte bioartificial liver. The maximum rate of glucose production by the hepatocyte bioartificial liver ranged from 50-80 μg/h. Plasma concentrations of aromatic amino acids, proline, alanine, and ammonia were normalized in anhepatic rabbits during hepatocyte hemoperfusion. Gel-entrapped hepatocytes in the bioartificial liver performed sulfation and glucuronidation of 4-methylumbelliferone. P450 activity was demonstrated during both in vitro and extracorporeal application of the BAL device by the formation of 3-hydroxy-lidocaine, the major metabolite of lidocaine biotransformation by gel-entrapped rat hepatocytes. In summary, a gel-entrapment, bioartificial liver performed multiple hepatocyte-specific functions without adverse side effects during extracorporeal application in an anhepatic, small animal model. With its potential for short term support of acute liver failure, scale-up of the current bioartificial liver device is indicated for further investigations in large animal, preclinical trials.

In vitro culture of islets of Langerhans decreases their immunogenecity, presumably by eliminating passenger leukocytes and other Ia+ presenting cells within the islets. Islets cultivated in petri dishes either at 37°C or at 25°C gradually disintegrate during culture in a time-dependent manner which is related to the free-floating condition of the islets. Also, a fraction of the islets disperse as single cells and beta-cell aggregates or adhere to the bottom of the culture dishes. Thus, the retrieval rate of transplantable islets is dampened due to their disintegration and spontaneous dispersion in conventional petri dish cultures. Entrapment of freshly harvested islets of Langerhans in a three-dimensional collagen matrix was studied as an alternative method for islet cultivation. The contraction of collagen fibrils during in vitro culture counteracts the dispersion of islets and helps in maintaining their integrity while in culture. It was observed that the entrapped islets maintain satisfactory morphology, viability, and capability of glucose-dependent insulin secretion for over 2 wk. The oxygen consumption rate and glucose metabolism of these islets was not deranged when entrapped in collagen. Also, the retrieval of islets is easier and more efficient than that observed in conventional culture systems. Our results indicate that culture of islets in three-dimensional collagen gels can potentially develop into an ideal system applicable to clinical transplantation of cultured islets or beta-cell aggregates.