Assessing bioartificial organ function: the 3P model framework and its validation.

The rapid advancement in the fabrication and culture of in vitro organs has marked a new era in biomedical research. While strides have been made in creating structurally diverse bioartificial organs, such as the liver, which serves as the focal organ in our study, the field lacks a uniform approach for the predictive assessment of liver function. Our research bridges this gap with the introduction of a novel, machine-learning-based "3P model" framework. This model draws on a decade of experimental data across diverse culture platform studies, aiming to identify critical fabrication parameters affecting liver function, particularly in terms of albumin and urea secretion. Through meticulous statistical analysis, we evaluated the functional sustainability of the in vitro liver models. Despite the diversity of research methodologies and the consequent scarcity of standardized data, our regression model effectively captures the patterns observed in experimental findings. The insights gleaned from our study shed light on optimizing culture conditions and advance the evaluation of the functional maintenance capacity of bioartificial livers. This sets a precedent for future functional evaluations of bioartificial organs using machine learning.

A Computational Template for Three-Dimensional Modeling of the Vascular Scaffold of the Human Thyroid Gland.

We recently designed an innovative scaffold-bioreactor unit for the bioengineering of a three-dimensional (3D) bioartificial human thyroid gland or its miniaturized replica as a part of a microfluidic chip test system. This device is based on the evidence that the 3D geometry of the intraglandular stromal/vascular scaffold (SVS; i.e., the fibrous and vascular matrix) of mammalian viscera plays a key role in guiding growth and differentiation of in vitro seeded cells. Therefore, we initiated a research program focused on computer-aided reconstruction of the 2nd to 4th order intralobar arterial network (IAN) of the human thyroid gland as a reliable surrogate for its 3D SVS, to be used as an input for rapid prototyping of a biomaterial replica. To this end, we developed a computational template that works within the Mathematica environment, giving rise to a quasi-fractal growth of the IAN distribution, constrained within an approximation of the thyroid lobe shape as a closed surface. Starting from edge detection of planar images of real human thyroid lobes acquired by in vivo real-time ultrasonography, we performed data approximation of the lobar profiles based on splines and Bezier curves, providing 3D lobar shapes as geometric boundaries for vessel growth by a diffusion-limited aggregation model. Our numerical procedures allowed for a robust connection between development of lobar arterial trees and thyroid lobe shape, led to a vascular self-similarity consistent with that of a cadaveric lobar arterial cast, and reproduced arterial vessels in a proportion not statistically different from that described for the real human thyroid gland. We conclude that our algorithmic template offers a reliable reproduction of the extremely complex IAN of the adult human thyroid lobe, potentially useful as a computational guidance for bioprinting of thyroid lobe matrix replicas. In addition, due to the simplicity and limited number of morphometrical parameters required by our system, we predict its application to the design of a number of patient-tailored human bioartificial organs and organs-on-chip, including parenchymal viscera and bones. Impact statement The study introduces the computer simulation of the three-dimensional (3D) intrinsic vascular matrix of the human thyroid gland, offering a general concept applicable to a number of other human viscera. Indeed, it provides a flexible software tool for reproduction of a 3D surrogate of the organ's 3D stromal matrix, suitable for eventual 3D bioprinting with biomaterials, and recellularization with organ-specific stem cells/progenitors. The final expectation is the design of patient-tailored 3D organ's matrices upon clinical request.

Tubular Bioartificial Organs: From Physiological Requirements to Fabrication Processes and Resulting Properties. A Critical Review.

In this featured review manuscript, the aim is to present a critical survey on the processes available for fabricating bioartificial organs (BAOs). The focus will be on hollow tubular organs for the transport of anabolites and catabolites, i.e., vessels, trachea, esophagus, ureter and urethra, and intestine. First, the anatomic hierarchical structures of tubular organs, as well as their principal physiological functions, will be presented, as this constitutes the mandatory requirements for effectively designing and developing physiologically relevant BAOs. Second, 3D bioprinting, solution electrospinning, and melt electrowriting will be introduced, together with their capacity to match the requirements imposed by designing scaffolds compatible with the anatomical and physiologically relevant environment. Finally, the intrinsic correlation between processes, materials, and cells will be critically discussed, and directives defining the strengths, weaknesses, and opportunities offered by each process will be proposed for assisting bioengineers in the selection of the appropriate process for the target BAO and its specific required functions.

Reconstruction of functional uterine tissues through recellularizing the decellularized rat uterine scaffolds by MSCs in vivo and in vitro.

Infertile people who suffered from loss of uterine structures and/or functions can be treated through gestational surrogacy or uterus transplantation, which remains challenging due to the ethical and social issues, the lack of donor organs as well as technical and safety risks. One promising solution is to regenerate and reconstruct a bioartificial uterus for transplantation through the engineering of uterine architecture and appropriate cellular constituents. Here, we developed a well-defined system to regenerate a functional rat uterine through recellularization of the decellularized uterine matrix (DUM) patches reseeded with human mesenchymal stem cells (hMSCs). Engraftment of the recellularized DUMs on the partially excised uteri yielded a functional rat uterus with a pregnancy rate and number of fetuses per uterine horn comparable to that of the control group with an intact uterus. Particularly, the recellularized DUMs enhanced the regeneration of traumatic uterine in vivo because of MSC regulation. The established system here will shed light on the treatment of uterine infertility with heterogeneous DUMs/cell resources through tissue engineering in the future.

Bioengineering lungs - current status and future prospects.

INTRODUCTION: Once pulmonary disease progresses to end-stage pulmonary disease, treatment options are very limited. An important advance in the field is the development of a bioartificial lung derived from a generic matrix scaffold populated with patients' own cells. Significant progress has already been made in the engineering of bioartificial lungs. AREAS COVERED: This review explains how previous and current research contributes to the goal of creating a successful bioartificial lung, and the barriers faced in doing so. We will also highlight some of the design considerations being explored to optimize bioartificial lungs and considerations for clinical translation. EXPERT OPINION: While current bioartificial lungs are able to provide short-term gas exchange in large-animal studies, much work is still required to combine the disciplines of cell biology, materials science, and tissue engineering to create such clinically useful and functioning artificial lungs.

Bioprinting of Complex Vascularized Tissues.

Functional vasculature is crucial for the maintenance of living tissues via the transport of oxygen, nutrients, and metabolic waste products. As a result, insufficient vascularization in thick engineered tissues will lead to cell death and necrosis due to mass transport and diffusional constraints. To circumvent these limitations, we describe the development of a microscale continuous optical bioprinting (µCOB) platform for 3D printing complex vascularized tissues with superior resolution and speed. By using the µCOB system, endothelial cells and other supportive cells can be printed directly into hydrogels with precisely controlled distribution and subsequent formation of lumen-like structures in vitro.

Dual Function of iPSC-Derived Pericyte-Like Cells in Vascularization and Fibrosis-Related Cardiac Tissue Remodeling In Vitro.

Myocardial interstitial fibrosis (MIF) is characterized by excessive extracellular matrix (ECM) deposition, increased myocardial stiffness, functional weakening, and compensatory cardiomyocyte (CM) hypertrophy. Fibroblasts (Fbs) are considered the principal source of ECM, but the contribution of perivascular cells, including pericytes (PCs), has gained attention, since MIF develops primarily around small vessels. The pathogenesis of MIF is difficult to study in humans because of the pleiotropy of mutually influencing pathomechanisms, unpredictable side effects, and the lack of available patient samples. Human pluripotent stem cells (hPSCs) offer the unique opportunity for the de novo formation of bioartificial cardiac tissue (BCT) using a variety of different cardiovascular cell types to model aspects of MIF pathogenesis in vitro. Here, we have optimized a protocol for the derivation of hPSC-derived PC-like cells (iPSC-PCs) and present a BCT in vitro model of MIF that shows their central influence on interstitial collagen deposition and myocardial tissue stiffening. This model was used to study the interplay of different cell types-i.e., hPSC-derived CMs, endothelial cells (ECs), and iPSC-PCs or primary Fbs, respectively. While iPSC-PCs improved the sarcomere structure and supported vascularization in a PC-like fashion, the functional and histological parameters of BCTs revealed EC- and PC-mediated effects on fibrosis-related cardiac tissue remodeling.

Preliminary evaluations of 3-dimensional human skin models for their ability to facilitate in vitro the long-term development of the debilitating obligatory human parasite Onchocerca volvulus.

Onchocerciasis also known as river blindness is a neglected tropical disease and the world's second-leading infectious cause of blindness in humans; it is caused by Onchocerca volvulus. Current treatment with ivermectin targets microfilariae and transmission and does not kill the adult parasites, which reside within subcutaneous nodules. To support the development of macrofilaricidal drugs that target the adult worm to further support the elimination of onchocerciasis, an in-depth understanding of O. volvulus biology especially the factors that support the longevity of these worms in the human host (>10 years) is required. However, research is hampered by a lack of access to adult worms. O. volvulus is an obligatory human parasite and no small animal models that can propagate this parasite were successfully developed. The current optimized 2-dimensional (2-D) in vitro culturing method starting with O. volvulus infective larvae does not yet support the development of mature adult worms. To overcome these limitations, we have developed and applied 3-dimensional (3-D) culture systems with O. volvulus larvae that simulate the human in vivo niche using in vitro engineered skin and adipose tissue. Our proof of concept studies have shown that an optimized indirect co-culture of in vitro skin tissue supported a significant increase in growth of the fourth-stage larvae to the pre-adult stage with a median length of 816-831 µm as compared to 767 µm of 2-D cultured larvae. Notably, when larvae were co-cultured directly with adipose tissue models, a significant improvement for larval motility and thus fitness was observed; 95% compared to 26% in the 2-D system. These promising co-culture concepts are a first step to further optimize the culturing conditions and improve the long-term development of adult worms in vitro. Ultimately, it could provide the filarial research community with a valuable source of O. volvulus worms at various developmental stages, which may accelerate innovative unsolved biomedical inquiries into the parasite's biology.

Mesenchymal Stem Cells as a Promising Cell Source for Integration in Novel In Vitro Models.

The human-relevance of an in vitro model is dependent on two main factors-(i) an appropriate human cell source and (ii) a modeling platform that recapitulates human in vivo conditions. Recent years have brought substantial advancements in both these aspects. In particular, mesenchymal stem cells (MSCs) have emerged as a promising cell source, as these cells can differentiate into multiple cell types, yet do not raise the ethical and practical concerns associated with other types of stem cells. In turn, advanced bioengineered in vitro models such as microfluidics, Organs-on-a-Chip, scaffolds, bioprinting and organoids are bringing researchers ever closer to mimicking complex in vivo environments, thereby overcoming some of the limitations of traditional 2D cell cultures. This review covers each of these advancements separately and discusses how the integration of MSCs into novel in vitro platforms may contribute enormously to clinical and fundamental research.

ß1 integrin, ILK and mTOR regulate collagen synthesis in mechanically loaded tendon cells.

Tendons are specialized tissues composed primarily of load-responsive fibroblasts (tenocytes) embedded in a collagen-rich extracellular matrix. Habitual mechanical loading or targeted exercise causes tendon cells to increase the stiffness of the extracellular matrix; this adaptation may occur in part through collagen synthesis or remodeling. Integrins are likely to play an important role in transmitting mechanical stimuli from the extracellular matrix to tendon cells, thereby triggering cell signaling pathways which lead to adaptive regulation of mRNA translation and protein synthesis. In this study, we discovered that mechanical stimulation of integrin ß1 leads to the phosphorylation of AKT, an event which required the presence of integrin-linked kinase (ILK). Repetitive stretching of tendon cells activates the AKT and mTOR pathways, which in turn regulates mRNA translation and collagen expression. These results support a model in which integrins are an upstream component of the mechanosensory cellular apparatus, regulating fundamental tendon cell functions relevant to exercise-induced adaptation and mechanotherapy.

Human-scale lung regeneration based on decellularized matrix scaffolds as a biologic platform.

Lung transplantation is currently the only curative treatment for patients with end-stage lung disease; however, donor organ shortage and the need for intense immunosuppression limit its broad clinical application. Bioartificial lungs created by combining native matrix scaffolds with patient-derived cells might overcome these problems. Decellularization involves stripping away cells while leaving behind the extracellular matrix scaffold. Cadaveric lungs are decellularized by detergent perfusion, and histologic examination confirms the absence of cellular components but the preservation of matrix proteins. The resulting lung scaffolds are recellularized in a bioreactor that provides biomimetic conditions, including vascular perfusion and liquid ventilation. Cell seeding, engraftment, and tissue maturation are achieved in whole-organ culture. Bioartificial lungs are transplantable, similarly to donor lungs, because the scaffolds preserve the vascular and airway architecture. In rat and porcine transplantation models, successful anastomoses of the vasculature and the airway were achieved, and gas exchange was evident after reperfusion. However, long-term function has not been achieved because of the immaturity of the vascular bed and distal lung epithelia. The goal of this strategy is to create patient-specific transplantable lungs using induced pluripotent stem cell (iPSC)-derived cells. The repopulation of decellularized scaffolds to create transplantable organs is one of possible future clinical applications of iPSCs.

Decellularized scaffolds for tissue engineering: Current status and future perspective.

Based on Organ Procurement and Transplantation Network data as of December 2019, more than 113 000 patients require an organ transplantation, yet, over the course of this past year, only 36 000 patients received a transplant. This discrepancy between those that need a donor organ and those that receive one remains one of medicine's biggest challenges. Multiple solutions, both biologic and artificial, have been proposed to mitigate this difference. One of the most promising approaches to generate bioartificial organs for transplantation involves re-seeding decellularized scaffolds with appropriate cells. Decellularization involves physical, chemical, or biological methods that typically require intimate contact of various decellularization solutions with each cell. Consequently, conventional submersion decellularization has been limited to simple tissues such as heart valves. The invention of perfusion decellularization was a breakthrough that allowed the generation of tissue-engineered scaffolds from tissues with higher structural organization and entire organs. Such scaffolds are composed of a myriad of extracellular matrix (ECM) components that include collagen, elastin, proteoglycans, and glycoproteins. Together, these components allow the scaffolds to fulfill specialized functions, such as structural functions as well as biological functions including the regulation of cellular processes and extracellular molecules. These specialized functions of decellularized scaffolds can increasingly be harnessed for applications in tissue engineering.

Assessment of biological functions for C3A cells interacting with adverse environments of liver failure plasma.

BACKGROUND: For its better differentiated hepatocyte phenotype, C3A cell line has been utilized in bioartificial liver system. However, up to now, there are only a few of studies working at the metabolic alternations of C3A cells under the culture conditions with liver failure plasma, which mainly focus on carbohydrate metabolism, total protein synthesis and ureagenesis. In this study, we investigated the effects of acute liver failure plasma on the growth and biological functions of C3A cells, especially on CYP450 enzymes. METHODS: C3A cells were treated with fresh DMEM medium containing 10% FBS, fresh DMEM medium containing 10% normal plasma and acute liver failure plasma, respectively. After incubation, the C3A cells were assessed for cell viabilities, lactate dehydrogenase leakage, gene transcription, protein levels, albumin secretion, ammonia metabolism and CYP450 enzyme activities. RESULTS: Cell viabilities decreased 15%, and lactate dehydrogenase leakage had 1.3-fold elevation in acute liver failure plasma group. Gene transcription exhibited up-regulation, down-regulation or stability for different hepatic genes. In contrast, protein expression levels for several CYP450 enzymes kept constant, while the CYP450 enzyme activities decreased or remained stable. Albumin secretion reduced about 48%, and ammonia accumulation increased approximately 41%. CONCLUSIONS: C3A cells cultured with acute liver failure plasma showed mild inhibition of cell viabilities, reduction of albumin secretion, and increase of ammonia accumulation. Furthermore, CYP450 enzymes demonstrated various alterations on gene transcription, protein expression and enzyme activities.

The ins and outs of engineering functional tissues and organs: evaluating the in-vitro and in-situ processes.

PURPOSE OF REVIEW: For many disorders that result in loss of organ function, the only curative treatment is organ transplantation. However, this approach is severely limited by the shortage of donor organs. Tissue engineering has emerged as an alternative solution to this issue. This review discusses the concept of tissue engineering from a technical viewpoint and summarizes the state of the art as well as the current shortcomings, with the aim of identifying the key lessons that we can learn to further advance the engineering of functional tissues and organs. RECENT FINDINGS: A plethora of tissue-engineering strategies have been recently developed. Notably, these strategies put different emphases on the in-vitro and in-situ processes (i.e. preimplantation and postimplantation) that take place during tissue formation. Biophysical and biomechanical interactions between the cells and the scaffold/biomaterial play a crucial role in all steps and have started to be exploited to steer tissue regeneration. SUMMARY: Recent works have demonstrated the need to better understand the in-vitro and in-situ processes during tissue formation, in order to regenerate complex, functional organs with desired cellular organization and tissue architecture. A concerted effort from both fundamental and tissue-specific research has the potential to accelerate progress in the field.

Immune responses towards bioengineered tissues and strategies to control them.

PURPOSE OF REVIEW: Research into development of artificial tissues and bioengineered organs to replace physiological functions of injured counterparts has highlighted a previously underestimated challenge for its clinical translatability: the immune response against biomaterials. Herein, we will provide an update and review current knowledge regarding this important barrier to regenerative medicine. RECENT FINDINGS: Although a clear understanding of the immune reactivity against biomaterials remains elusive, accumulating evidence indicates that innate immune cells, primarily neutrophils and macrophages, play a key role in the initial phases of the immune response. More recently, data have shown that in later phases, T and B cells are also involved. The use of physicochemical modifications of biomaterials and cell-based strategies to modulate the host inflammatory response is being actively investigated for effective biomaterial integration. SUMMARY: The immune response towards biomaterials and bioengineered organs plays a crucial role in determining their utility as transplantable grafts. Expanding our understanding of these responses is necessary for developing protolerogenic strategies and delivering on the ultimate promise of regenerative medicine.

Unravelling effects of relative humidity on lipid barrier formation in human skin equivalents.

Relative humidity (RH) levels vary continuously in vivo, although during in vitro generation of three-dimensional human skin equivalents (HSEs) these remain high (90-95%) to prevent evaporation of the cell-culture medium. However, skin functionality is directly influenced by environmental RH. As the barrier formation in HSEs is different, there is a need to better understand the role of cell-culture conditions during the generation of HSEs. In this study, we aim to investigate the effects of RH on epidermal morphogenesis and lipid barrier formation in HSEs. Therefore, two types of HSEs were developed at 90% or at 60% RH. Assessments were performed to determine epidermal morphogenesis by immunohistochemical analyses, ceramide composition by lipidomic analysis, and lipid organization by Fourier transform infrared spectroscopy and small-angle X-ray diffraction. We show that reduction of RH mainly affected the uppermost viable epidermal layers in the HSEs, including an enlargement of the granular cells and induction of epidermal cell activation. Neither the composition nor the organization of the lipids in the intercorneocyte space were substantially altered at reduced RH. In addition, lipid processing from glucosylceramides to ceramides was not affected by reduced RH in HSEs as shown by enzyme expression, enzyme activity, and substrate-to-product ratio. Our results demonstrate that RH directly influences epidermal morphogenesis, albeit the in vitro lipid barrier formation is comparable at 90% and 60% RH.

Decellularization of the mouse ovary: comparison of different scaffold generation protocols for future ovarian bioengineering.

BACKGROUND: In order to preserve fertility in young women with disseminated cancer, e.g. leukemia, an approach that has been suggested is to retransplant isolated small follicles within an ovarian matrix free from malignant cells and with no risk for contamination. The present study evaluates the first step to create a bioengineered ovarian construct that can act as growth-supporting tissue for isolated small follicles that are dependent on a stroma for normal follicular maturation. The present study used the intact mouse ovary to develop a mouse ovarian scaffold through various protocols of decellularization. MATERIAL AND METHODS: Potential Immunogenic DNA and intracellular components were removed from whole mouse ovaries by agitation in a 0.5% sodium dodecyl sulfate solution (Protocol 1; P1), or in a 2% sodium deoxycholate solution (P2) or by a combination of the two (P3). The remaining decelluralized ovarian extracellular matrix structure was then assessed based on the DNA- and protein content, and was further evaluated histologically by haematoxylin and eosin-, Verhoeff's van gieson- (for elastin), Masson's trichrome- (for collagens) and Alcian blue (for glycosaminoglycans) staining. We also evaluated the decellularization efficiency using the mild detergent Triton-X100 (1%). RESULTS: Sodium dodecyl sulfate efficiently removed DNA and intracellular components from the ovarian tissue but also significantly reduced the integrity of the remaining ovarian extracellular matrix. Sodium deoxycholate, a considerably milder detergent compared to sodium dodecyl sulfate, preserved the ovarian extracellular matrix better, evident by a more distinct staining for glycosaminoglycan, collagen and elastic fibres. Triton-X100 was found ineffective as a decellularization reagent for mouse ovaries in our settings. CONCLUSIONS: The sodium dodecyl sulfate generated ovarian scaffolds contained minute amounts of DNA that may be an advantage to evade a detrimental immune response following engraftment. The sodium deoxycholate generated ovarian scaffolds had higher donor DNA content, yet, retained the extracellular composition better and may therefore have improved recellularization and other downstream bioengineering applications. These two novel types of mouse ovarian scaffolds serve as promising scaffold-candidates for future ovarian bioengineering experiments.