Bioengineering Liver Organoids: Comparison
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Please note this is a comparison between Version 2 by Jason Zhu and Version 1 by Junzhi Li.

Organoids as three-dimension (3D) cellular organizations partially mimic the physiological functions and micro-architecture of native tissues and organs, holding great potential for clinical applications. Advances in the identification of essential factors including physical cues and biochemical signals for controlling organoid development have contributed to the success of growing liver organoids from liver tissue and stem/progenitor cells. However, to recapitulate the physiological properties and the architecture of a native liver, one has to generate liver organoids that contain all the major liver cell types in correct proportions and relative 3D locations as found in a native liver.

  • bioengineering
  • liver organoids
  • liver disease models

1. Introduction

The significant events of bioengineering liver organoids are illustrated in the timeline. The 3D spheroidal aggregate derived from rat liver cells could be the beginning of the journey of liver organoid development [27][1]. Long-term cultures of rat hepatocytes were achieved by using liver biomatrix [28][2] and a coculture system consisting of fibroblasts and hepatic parenchymal cells could be applied in forming 3D liver tissue [29][3]. The 1980s witnessed the start of culturing 3D liver structures and laying the groundwork for the emergence of liver organoids in the 1990s. Biodegradable scaffolds [30][4], hydrogels [31][5], and polymeric microcarriers [32,33,34][6][7][8] had been employed to engineer liver organoids, which could be the starting of bioengineering liver organoids, meanwhile, coculture systems were developed to reconstruct liver organoids in the period of 1990s [31,35][5][9]. In the 2000s, more and more techniques such as bioreactor [36][10], light patterning [37,38][11][12], and bioprinting [39][13] were induced for engineering a liver organoid. Extracellular matrix combined with polymeric scaffolds were used to improve the survival and morphological integrity of hepatocytes [40][14]. When it came to the 2010s, advanced liver organoids with a vascular network [41][15] and gene-modified liver organoids [42][16] were presented in the laboratories. The long-term culture and expansion of liver organoids play a key role in constructing reliable and replicable liver development and disease models, which was achieved by Hans Clevers’ group [23,24][17][18]. Liver cancer organoid models that can be used to understand liver cancer biology and develop therapeutics have also been established in the 2010s [4][19]. Currently, in the 2020s liver organoids were extended to biliary atresia [43][20] and virus-infected models [44][21]. The functional hydrogels [45][22] and the advanced printing technique [46][23] have attracted more and more attention to their application in engineering liver organoids.

2. Liver Organoids Derived from Different Cell Sources

Liver primary cells which express a surface receptor Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) were identified to be self-renewable and a single LGR5-expressing cell was able to produce liver organoids of functional cholangiocytes and hepatocytes [47][24]. Transplantable liver organoids have been generated from long-term clonally expanded LGR5+ cells using an R-Spondin1 (Rspo1)-based culture medium [48][25]. Large-scale expansion of LGR5+ human liver cells was achievable using spinner flasks to improve oxygenation of the culture resulting in a 40-fold cell expansion after 2 weeks of culture [47][24]. Long-term culture of feline bile duct tissue-derived liver organoids retained the characteristics of adult liver stem cells [49][26]. Primary bile duct cells have been expanded in vitro to form 3D liver organoids and these cells also can differentiate into functional hepatocyte cells [23][17]. All these findings indicated that the liver LGR5+ primary cells can maintain their “stemless” properties after long-term culture and expansion and can be induced to form liver organoids.
Induced pluripotent stem cells (iPSC) are capable of differentiating into many different cell types, and iPSC-derived liver organoids can well replace the conventional liver tissue-derived organoid systems [50][27]. Protocols have been established to induce iPSC to differentiate into liver organoids of cholangiocytes and hepatocytes [51,52,53,54,55][28][29][30][31][32]. Typically, iPSCs were cultured in a medium containing bone morphogenetic protein 4 (BMP4), fibroblast growth factor-4 (FGF4), and B-27 supplements to induce differentiation of iPSC into hepatoblast-like cells, and subsequently cultured with epidermal growth factor (EGF) in matrigel to encourage cholangiocyte differentiation [56][33]. Careful selection of extracellular matrix (ECM) is a key step toward cholangiocyte differentiation from human iPSC: laminin 411 and laminin 511 have been shown to promote cholangiocyte differentiation [56][33]. The construction of iPSC-derived cholangiocytes for disease modelling (Alagille syndrome) and drug validation (cystic fibrosis drug VX809) have been reported [57][34]. There are two steps to generate a liver organoid from iPSCs. Initially, iPSCs are differentiated into hepatic endoderm and progenitor cells using specific protocols under a 2-dimension (2D) culture environment. Thereafter, the cells will be seeded in a 3D culture to construct a complex 3D organoid. However, to engineer a transplantable and functional in vitro organoid, the vasculature plays a significant role in producing suitable engraftment with biological functions. Takebe et al. reported a protocol for generating liver buds from human iPSCs and investigated the vascularization and maturation of implanted liver buds in immuno-deficient mice [58][35].
Multiple cell-type liver organoid systems allow more sophisticated structures to be engineered with the vasculature and biliary system to mimic a native liver. Vascularization is a crucial step toward organogenesis, which is essential for oxygen and nutrition transportation and distribution. A liver organoid platform involving iPSCs, mesenchymal stem cells (MSCs), and human umbilical vein endothelial cells (HUVECs) was employed to investigate the paracrine effects of stem cell therapies for regenerative medicine [25,59][36][37]. Liver organoid models containing hepatocytes and cholangiocytes derived from iPSCs were employed to study human genetic disorders [60][38].

3. Biological Factors for Directing Liver and Biliary Organoid Formation

Biological factors play a pivotal role in the regulation of cell expansion, differentiation and self-organization in in vitro organoid cultures. As liver is developed from embryonic endoderm, which also give rise to the endoderm tissues in pancreas and gut tube [61][39]. The biological factors implicated in some of the important molecular signaling pathways underlying the proliferation/migration/differentiation of endoderm such as Noggin, epidermal growth factor (EGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), R-spondin 1 (a Wnt pathway potentiator), TGF-β inhibitor (A83-01), a cAMP pathway agonist forskolin (FSK), and retinoic acid (RA) have been used in the generation of organoids of endoderm origin including liver organoid [62][40]. A medium containing these factors supported the long-term culturing of liver organoids for genetic manipulation [42][16].

4. Biomaterials for Engineering Liver Organoids

The ultimate goal of liver tissue engineering is to create a transplantable artificial liver for transplantation to patients with end-stage liver failure. The liver ECM can act as a scaffold supporting cell survival, proliferation, and differentiation [63][41]. A 3D environment influences cellular behaviors via the interactions between cells and the ECM, and an absence of cell-ECM interaction could lead to a specific type of cell apoptosis called anoikis [64][42]. Cell migration and differentiation are also influenced by the physical properties such as the rigidity, adhesion, confinement, topology, and biochemical signals of the ECM [65,66][43][44]. A variety of biomaterials have been investigated and employed to reconstruct an appropriate microarchitecture with suitable stiffness and liver-specific ECM protein signals, for supporting liver organoid in vitro expansion.
Traditional polymers such as polyhydroxyalkanoate and poly(lactic-co-glycolic acid) (PLGA) polylactic acid (PLA), polycaprolactone (PCL), and poly(ethylene glycol) diacrylate (PEGDA) have been employed for liver tissue scaffold fabrication [67,68][45][46]. These polymeric materials have good long-term biocompatibility, quality mechanical properties, and biodegradable feature for liver organoid expansion. However, most of these polymers have a high mechanical stiffness that is not applicable for 3D liver organoid culture. In contrast, hydrogels, with their soft mechanical properties, become the best candidate materials to mimic the mechanical environment of soft tissue like liver [69][47].
Alginate is a natural polysaccharide, which is widely used as biomedical hydrogel scaffolds due to its good biocompatibility and affordable price [70][48]. It can be crosslinked by divalent cations to provide a stable architecture supporting liver organoid growth and proliferation [71][49]. Alginate is a flexible material for a number of fabrication and modification techniques, which enables organoid encapsulation for biomedical applications. For example, microporous alginate scaffolds were fabricated to promote the development of infant hepatic cells into hepatic tissue for drug screening [72][50]. Gelatin methacryloyl (GelMA), a hydrogel that can be photo crosslinked, is typically prepared by a direct reaction of gelatin and methacrylic anhydride (MA). Many preparation methods were developed for its widespread applications in the biomedicine [73][51]. Organoid-laden complex scaffolds which are composed of GelMA, polyisocyanopeptides (PIC), and laminin-111 enable long-term expansion of human liver organoids up to 14 passages with a proliferation rate comparable to commercial Matrigels [46][23]. Hyaluronic acid (HA) and collagen are the main components of native organ ECM, which have been widely used in regenerative medicine and tissue engineering scaffolds. A host-liver colorectal-tumor organoid model was established using HA-based microcarriers loaded with ECM elements and liver-specific growth factors, which demonstrated a possible application of the HA-based hydrogel for liver tumor model [74,75][52][53]. Condensed collagen fibril scaffolds have been employed to encapsulate liver organoids into a transplantable hepatic tissue, which provides a microenvironment enhancing the cell-cell and cell-ECM interactions, and maintaining liver-specific functions of the organoids [76][54]. The native organ decellularization matrix could be an implantable scaffold for transplantable organoid tissue in vitro reconstruction. The whole organ decellularization matrix was used to produce vascularized liver organoids [41][15], and various decellularized ECM (dECM) liver organoid systems were reported [77,78,79][55][56][57].
Compared with the above-mentioned hydrogels, hybrid hydrogels can address some of the limitations of using a single component matrix for liver organoid culture. To date, the most prevalent commercial matrix is Matrigel, a basement membrane extracellular matrix, that mainly consists of collagen IV, nidogen, perlecan, and a high content of glycosylated molecules [80][58]. Matrigel has been widely used for liver organoid expansion (for review see [50,81][27][59]). The reasons for the widespread use of commercial Matrigel for liver organoid generation are due to its (i) good biocompatible properties; (ii) stable thermal-sensitive gelation properties; and (iii) optically transparent properties for real-time monitoring of organoid growth. The clinical applicability of Matrigel is, however, severely limited by the variability in its composition and the presence of xenogenic contaminants. Other hybrid-hydrogel scaffolds have also been used to fabricate organoid-laden scaffolds, which diversify the materials for the organoid generation [45,82][22][60].

5. Current Fabrication Techniques for Engineering Liver Organoid

With the development of material processing technology, large-scale fabrication of liver organoids for transplantation is not unachievable. An engineering technique for building implantable liver organoids should fulfill the criteria not only in the material processing aspect but also in the biological aspect. Three-D printing technology allows the creation of physical objects from a geometrical representation by successive addition of materials. Meanwhile, 3D bioprinting, a combination of 3D printing with biological content, has emerged as a powerful tool for the fabrication of biomimetic and implantable scaffolds [83[61][62],84], which is also used for the production of organoid-laden structures [85][63]. Manon et al. reported an extrusion-based bioprinting approach using GelMA hydrogel as a printing ink to construct liver tissue-derived epithelial organoids [86][64]. The viability of printed organoids in GelMA hydrogel could be maintained at not less than 88% up to 10 days, which enabled the hepatotoxin exposure experiment to be conducted. A nozzle-free volumetric bioprinting technique has been developed to fabricate gelatin-based scaffolds encapsulating hepatic organoids [46][23].
Suspension culture is a well-developed technique for 3D organoid expansion for the scalable production of organoids. Researchers have developed a two-step protocol for the large-scale production of liver organoids from human iPSCs, in that homogeneous and uniform-sized human embryoid bodies (hEBs) were first generated and the hEBs were then cultured in a suspension culture platform to form liver organoids [87][65]. Saskia and his colleagues have developed a protocol for the scalable production of hepatocytes and hepatic organoids; furthermore, their protocol also included a cryopreservation method to store intermediate hepatic organoids for the efficient production of a large number of organoids [88][66].
Organ on a chip is an effective in vitro platform to reflect human liver-specific functions, as well as the complex process of detoxication and bile production. Organ on a chip platform can provide a dynamic microenvironment by manipulating the compositions of the perfusion fluids. To mimic liver functions and the native liver microenvironment, a chip with a specific design can be fabricated for a variety of in vitro studies. In a microfluidic 3D human liver sinusoid, faster albumin and urea responses were observed under continuous perfusion, allowing drug screening on a liver-on-a-chip platform [89][67]. Microfluidic organ-on-a-chip devices were designed and produced to study the in vitro liver metabolism and assess cardiac safety of drugs (e.g., clomipramine) through a co-culture model of liver and cardiac organoids on the chip [90][68]. Using this multi-organoid platform, the urea synthesis was measured for evaluating the liver-specific function of liver organoids and the expression of liver-specific CYP450 enzyme genes.

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