Modelling tissues in 3D: the next future of pharmaco-toxicology and food research?

The development and validation of reliable in vitro methods alternative to conventional in vivo studies in experimental animals is a well-recognised priority in the fields of pharmaco-toxicology and food research. Conventional studies based on two-dimensional (2-D) cell monolayers have demonstrated their significant limitations: the chemically and spatially defined three-dimensional (3-D) network of extracellular matrix components, cell-to-cell and cell-to-matrix interactions that governs differentiation, proliferation and function of cells in vivo is, in fact, lost under the simplified 2-D condition. Being able to reproduce specific tissue-like structures and to mimic functions and responses of real tissues in a way that is more physiologically relevant than what can be achieved through traditional 2-D cell monolayers, 3-D cell culture represents a potential bridge to cover the gap between animal models and human studies. This article addresses the significance and the potential of 3-D in vitro systems to improve the predictive value of cell-based assays for safety and risk assessment studies and for new drugs development and testing. The crucial role of tissue engineering and of the new microscale technologies for improving and optimising these models, as well as the necessity of developing new protocols and analytical methods for their full exploitation, will be also discussed.     

A human corneal equivalent constructed from SV40-immortalised corneal cell lines

Within the last decade, extensive research in the field of tissue and organ engineering has focused on the development of in vitro models of the cornea. The use of organotypic, three-dimensional corneal equivalents has several advantages over simple monolayer cultures. The aim of this study was to develop a corneal equivalent model composed of the same cell types as in the natural human tissue, but by using immortalised cell lines to ensure reproducibility and to minimise product variation. We report our success in the establishment of an SV40-immortalised human corneal keratocyte cell line (designated HCK). A collagen matrix, built up with these cells, displayed the morphological characteristics of the human stromal tissue and served as a biomatrix for the immortalised human corneal epithelial and endothelial cells. Histological cross-sections of the whole-cornea equivalents resemble human corneas in tissue structure. This organotypic in vitro model may serve as a research tool for the ophthalmic science community, as well as a model system for testing for eye irritancy and drug efficacy.     

"Culture shock" from the bone cell's perspective: emulating physiological conditions for mechanobiological investigations

Bone physiology can be examined on multiple length scales. Results of cell-level studies, typically carried out in vitro, are often extrapolated to attempt to understand tissue and organ physiology. Results of organ- or organism-level studies are often analyzed to deduce the state(s) of the cells within the larger system(s). Although phenomena on all of these scales—cell, tissue, organ, system, organism—are interlinked and contribute to the overall health and function of bone tissue, it is difficult to relate research among these scales. For example, groups of cells in an exogenous, in vitro environment that is well defined by the researcher would not be expected to function similarly to those in a dynamic, endogenous environment, dictated by systemic as well as organismal physiology. This review of the literature on bone cell culture describes potential causes and components of cell "culture shock," i.e., behavioral variations associated with the transition from in vivo to in vitro environment, focusing on investigations of mechanotransduction and experimental approaches to mimic aspects of bone tissue on a macroscopic scale. The state of the art is reviewed, and new paradigms are suggested to begin bridging the gap between two-dimensional cell cultures in petri dishes and the three-dimensional environment of living bone tissue. osteoblast; osteocyte; tissue engineering; mechanobiology; mechanochemical transduction; fluid flow     

Canine embryonic stem cells: State of the art

Embryonic stem cells (ESCs) are permanent cell lines that can be maintained in a pluripotent, undifferentiated state. Appropriate environmental stimuli can cause them to differentiate into cell types of all three germ layers both in vitro and in vivo. Embryonic stem cells bear many opportunities for clinical applications in tissue engineering and regenerative medicine. Whereas most of our knowledge on the biology and technology of ESCs is derived from studies with mouse cells, large animal models mimicking important aspects of human anatomy, physiology, and pathology more closely than mouse models are urgently needed for studies evaluating the safety and efficacy of cell therapies. The dog is an excellent model for studying human diseases, and the availability of canine ESCs would open new possibilities for this model in biomedical research. In addition, canine ESCs could be useful for the development of cell-based approaches for the treatment of dogs. Here, we discuss the features of recently reported canine embryo-derived cells and their potential applications in basic and translational biomedical research.     

n-3 Polyunsaturated fatty acids—Physiological relevance of dose

n-3 Polyunsaturated fatty acids (PUFA) are widely used for chemotheraphy/chemoprevention of chronic diseases. However, the molecular mechanism(s) by which the bioactive n-3 PUFA (eicosapentaenoic acid and docosahexaenoic acid) modulate effector pathways are not fully elucidated. Multiple experimental approaches, including use of animal models, cell lines, and human clinical trials, have been utilized to dissect the complex effectors. It is imperative to link these different experimental approaches together in order to interpret outcomes in the context of human physiology and pathophysiology. Unfortunately, the adoption of a broad array of model systems and a wide range of fatty acid exposures (i.e. doses) has made it difficult to interpret biological outcomes. Therefore, in this mini-review we discuss the impact of (a) molecular structure of bioactive fatty acids, (b) dose relevance relative to human consumption, (c) enrichment of fatty acids in sera and tissues following dietary intake, and (d) limitations of cell/tissue culture studies.     

Alternatives to animal experimentation for hormonal compounds research

Alternatives to animal testing and the identification of reliable methods that may decrease the need for animals are currently the subject of intense investigation worldwide. Alternative testing procedures are particularly important for synthetic and natural chemicals that exert their biological actions through binding nuclear receptors, called nuclear receptors-interacting compounds (NR-ICs), for which research is increasingly emphasizing the limits of several models in the accurate estimation of the physiological consequences of exposure to these compounds. In particular, estrogen receptor interacting compounds (ER-ICs) have a great impact on human health from the therapeutic, nutritional, and toxicological point of view due to the highly permissive nature of the estrogen receptors towards a large number of natural and synthetic compounds. Similar to in vitro systems, recently generated animal models (e.g., animal models generated for the study of estrogen receptor ligands) may fulfill the 3R principles: refine, reduce, and replace. If used correctly, NR-regulated models, such as reporter mice, xenopus, or zebrafish, and models obtained by somatic gene transfer in reporter systems, combined with imaging technologies, may contribute to strongly decreasing the overall number of animals required for NR-IC testing and research. With these models, flexible and highly standardized parameters and reporter marker quantification can be obtained. Here, we highlight the need for the substitution of currently used testing models with more appropriate ones that can reproduce the features and reactivity of specific mammalian target tissue/organs. We consider the promotion of this advancement a research priority bearing scientific, economic, social, and ethical relevance.     

Engineered human cardiac tissues for modeling heart diseases

Heart disease is one of the major life-threatening diseases with high mortality and incidence worldwide. Several model systems, such as primary cells and animals, have been used to understand heart diseases and establish appropriate treatments. However, they have limitations in accuracy and reproducibility in recapitulating disease pathophysiology and evaluating drug responses. In recent years, three-dimensional (3D) cardiac tissue models produced using tissue engineering technology and human cells have outperformed conventional models. In particular, the integration of cell reprogramming techniques with bioengineering platforms (e.g., microfluidics, scaffolds, bioprinting, and biophysical stimuli) has facilitated the development of heart-on-a-chip, cardiac spheroid/organoid, and engineered heart tissue (EHT) to recapitulate the structural and functional features of the native human heart. These cardiac models have improved heart disease modeling and toxicological evaluation. In this review, we summarize the cell types for the fabrication of cardiac tissue models, introduce diverse 3D human cardiac tissue models, and discuss the strategies to enhance their complexity and maturity. Finally, recent studies in the modeling of various heart diseases are reviewed.     

Adipose tissue engineering with cells in engineered matrices

Tissue engineering has shown promise for the development of constructs to facilitate large volume soft tissue augmentation in reconstructive and cosmetic plastic surgery. This article reviews the key progress to date in the field of adipose tissue engineering. In order to effectively design a soft tissue substitute, it is critical to understand the native tissue environment and function. As such, the basic physiology of adipose tissue is described and the process of adipogenesis is discussed. In this article, we have focused on tissue engineering using a cell-seeded scaffold approach, where engineered extracellular matrix substitutes are seeded with exogenous cells that may contribute to the regenerative response. The strengths and limitations of each of the possible cell sources for adipose tissue engineering, including adipose-derived stem cells, are detailed. We briefly highlight some of the results from the major studies to date, involving a range of synthetic and naturally derived scaffolds. While these studies have shown that adipose tissue regeneration is possible, more research is required to develop optimized constructs that will facilitate safe, predictable, and long-term augmentation in clinical applications.     

Expression of mediators of purinergic signaling in human liver cell lines

Purinergic signaling regulates a diverse and biologically relevant group of processes in the liver. However, progress of research into functions regulated by purinergic signals in the liver has been hampered by the complexity of systems probed. Specifically, there are multiple liver cell subpopulations relevant to hepatic functions, and many of these have been effectively modeled in human cell lines. Furthermore, there are more than 20 genes relevant to purinergic signaling, each of which has distinct functions. Hence, we felt the need to categorize genes relevant to purinergic signaling in the best characterized human cell line models of liver cell subpopulations. Therefore, we investigated the expression of adenosine receptor, P2X receptor, P2Y receptor, and ecto-nucleotidase genes via RT-PCR in the following cell lines: LX-2, hTERT, FH11, HepG2, Huh7, H69, and MzChA-1. We believe that our findings will provide an excellent resource to investigators seeking to define functions of purinergic signals in liver physiology and liver disease pathogenesis.     

The use of non-human primates in biological and medical research: evidence submitted by FRAME to the Academy of Medical Sciences/Medical Research Council/Royal Society/Wellcome Trust Working Group

The Academy of Medical Sciences, the Medical Research Council, the Royal Society and the Wellcome Trust are undertaking a study into the use of non-human primates in biological and medical research. An independent working group of scientific experts, led by Sir David Weatherall, aims to produce a report summarising the findings of this study, early in 2006. The trends in primate research, and the nature and effects of recent and proposed changes in the global use of non-human primates in research, will be investigated. The associated ethical, welfare and regulatory issues, and the role and impact of the Three Rs principles of refinement, reduction and replacement will also be reviewed. As part of this study, a call for evidence was made. The evidence submitted by FRAME emphasised that the use of non-human primates for fundamental research or for regulatory testing still fails to take into account the fact that, although non-human primates are anatomically and physiologically similar to humans, they are not necessarily relevant models for studies on human disease or human physiology. FRAME continues to believe that we have a duty to ensure that these animals are not used without overwhelming evidence that they are the only suitable and relevant models for use in work of undeniable significance.     

体外模型的发展及其在鲸豚研究中的应用

体外补充替代模型“细胞系”为生命科学研究提供了新的平台,在一定程度上突破了科学研究中伦理、法律、动物福利和动物保护等的限制,从细胞和分子视角更深层次地揭示复杂生命体的生物效应和 调控机制。尤其对于濒危动物,细胞系的建立与超低温冷冻技术相结合,既可以保存濒危动物具有生物表达活性的遗传种质,又可以提供体外保育研究的新平台（如动物毒理学实验）,对动物保护意义 重大。目前细胞培养体系已作为多功能平台被应用于鲸豚类细胞遗传学、病毒学和毒理学的相关研究中,但从物种和组织来源以及细胞类型来看,能长期稳定传代的鲸豚类细胞系仍较单一。优化细胞培 养条件,运用鲸豚类体外细胞揭示更多的生命机制问题,仍是当前鲸豚类体外细胞模型研究所面临的挑战。本文对动物体外模型及其在鲸豚研究中的应用进行了概述,以期推动该技术在鲸豚保育研究中 的创新和发展。     

CHO microRNA engineering is growing up: Recent successes and future challenges

microRNAs with their ability to regulate complex pathways that control cellular behavior and phenotype have been proposed as potential targets for cell engineering in the context of optimization of biopharmaceutical production cell lines, specifically of Chinese Hamster Ovary cells. However, until recently, research was limited by a lack of genomic sequence information on this industrially important cell line. With the publication of the genomic sequence and other relevant data sets for CHO cells since 2011, the doors have been opened for an improved understanding of CHO cell physiology and for the development of the necessary tools for novel engineering strategies. In the present review we discuss both knowledge on the regulatory mechanisms of microRNAs obtained from other biological models and proof of concepts already performed on CHO cells, thus providing an outlook of potential applications of microRNA engineering in production cell lines.     

Adipose tissue engineering with cells in engineered matrices

Tissue engineering has shown promise for the development of constructs to facilitate large volume soft tissue augmentation in reconstructive and cosmetic plastic surgery. This article reviews the key progress to date in the field of adipose tissue engineering. In order to effectively design a soft tissue substitute, it is critical to understand the native tissue environment and function. As such, the basic physiology of adipose tissue is described and the process of adipogenesis is discussed. In this article, we have focused on tissue engineering using a cell-seeded scaffold approach, where engineered extracellular matrix substitutes are seeded with exogenous cells that may contribute to the regenerative response. The strengths and limitations of each of the possible cell sources for adipose tissue engineering, including adipose-derived stem cells, are detailed. We briefly highlight some of the results from the major studies to date, involving a range of synthetic and naturally derived scaffolds. While these studies have shown that adipose tissue regeneration is possible, more research is required to develop optimized constructs that will facilitate safe, predictable and long-term augmentation in clinical applications.Key words: tissue engineering, regenerative medicine, adipose tissue, adipose-derived stem cells, adipogenesis, cell culture, scaffolds, cell-biomaterial interactions     

Organ-on-Chip platforms to study tumor evolution and chemosensitivity

Despite tremendous advancements in oncology research and therapeutics, cancer remains a primary cause of death worldwide. One of the significant factors in this critical challenge is a precise diagnosis and limited knowledge on how the tumor microenvironment (TME) behaves to the treatment and its role in chemo-resistance. Therefore, it is critical to understand the contribution of a heterogeneous TME in cancer drug response in individual patients for effective therapy management. Micro-physiological systems along with tissue engineering have facilitated the development of more physiologically relevant platforms, known as Organ-on-Chips (OoC). OoC platforms recapitulate the critical hallmarks of the TME in vitro and subsequently abet in sensitivity and efficacy testing of anti-cancer drugs before clinical trials. The OoC platforms incorporating conventional in vitro models enable researchers to control the cellular, molecular, chemical, and biophysical parameters of the TME in precise combinations while analyzing how they contribute to tumor progression and therapy response. This review discusses the application of OoC platforms integrated with conventional 2D cell lines, 3D organoids and spheroid models, and the organotypic tissue slices, including patient-derived and xenograft tumor slice cultures in cancer treatment responses. We summarize the relevance and drawbacks of conventional in vitro models in assessing cancer treatment response, challenges and limitations associated with OoC models, and future opportunities enabled by the OoC technologies towards developing personalized cancer diagnostics and therapeutics.     

Genetic Modification of Cancer Cells Using Non-Viral,Episomal S/MAR Vectors for In Vivo Tumour Modelling

The development of genetically marked animal tumour xenografts is an area of ongoing research to enable easier and more reliable testing of cancer therapies. Genetically marked tumour models have a number of advantages over conventional tumour models, including the easy longitudinal monitoring of therapies and the reduced number of animals needed for trials. Several different methods have been used in previous studies to mark tumours genetically, however all have limitations, such as genotoxicity and other artifacts related to the usage of integrating viral vectors. Recently, we have generated an episomally maintained plasmid DNA (pDNA) expression system based on Scaffold/Matrix Attachment Region (S/MAR), which permits long-term luciferase transgene expression in the mouse liver. Here we describe a further usage of this pDNA vector with the human Ubiquitin C promoter to create stably transfected human hepatoma (Huh7) and human Pancreatic Carcinoma (MIA-PaCa2) cell lines, which were delivered into “immune deficient” mice and monitored longitudinally over time using a bioluminometer. Both cell lines revealed sustained episomal long-term luciferase expression and formation of a tumour showing the pathological characteristics of hepatocellular carcinoma (HCC) and pancreatic carcinoma (PaCa), respectively. This is the first demonstration that a pDNA vector can confer sustained episomal luciferase transgene expression in various mouse tumour models and can thus be readily utilised to follow tumour formation without interfering with the cellular genome.     

From 3D cell culture to organs-on-chips

3D cell-culture models have recently garnered great attention because they often promote levels of cell differentiation and tissue organization not possible in conventional 2D culture systems. We review new advances in 3D culture that leverage microfabrication technologies from the microchip industry and microfluidics approaches to create cell-culture microenvironments that both support tissue differentiation and recapitulate the tissue-tissue interfaces, spatiotemporal chemical gradients, and mechanical microenvironments of living organs. These 'organs-on-chips' permit the study of human physiology in an organ-specific context, enable development of novel in vitro disease models, and could potentially serve as replacements for animals used in drug development and toxin testing.     

The blastema and epimorphic regeneration in mammals

Studying regeneration in animals where and when it occurs is inherently interesting and a challenging research topic within developmental biology. Historically, vertebrate regeneration has been investigated in animals that display enhanced regenerative abilities and we have learned much from studying organ regeneration in amphibians and fish. From an applied perspective, while regeneration biologists will undoubtedly continue to study poikilothermic animals (i.e., amphibians and fish), studies focused on homeotherms (i.e., mammals and birds) are also necessary to advance regeneration biology. Emerging mammalian models of epimorphic regeneration are poised to help link regenerative biology and regenerative medicine. The regenerating rodent digit tip, which parallels human fingertip regeneration, and the regeneration of large circular defects through the ear pinna in spiny mice and rabbits, provide tractable, experimental systems where complex tissue structures are regrown through blastema formation and morphogenesis. Using these models as examples, we detail similarities and differences between the mammalian blastema and its classical counterpart to arrive at a broad working definition of a vertebrate regeneration blastema. This comparison leads us to conclude that regenerative failure is not related to the availability of regeneration-competent progenitor cells, but is most likely a function of the cellular response to the microenvironment that forms following traumatic injury. Recent studies demonstrating that targeted modification of this microenvironment can restrict or enhance regenerative capabilities in mammals helps provide a roadmap for eventually pushing the limits of human regeneration.     

Induced pluripotent stem cells from swine (Sus scrofa): Why they may prove to be important

Three recent papers, published almost simultaneously by different groups, have described the generation of induced pluripotent stem (iPS) cells from the pig, a species whose size, anatomy, and physiology render them attractive as clinical models for the human. The approach used in each case was to infect somatic cells with integrating retroviral vectors designed to express four reprogramming genes (POU5F1, SOX2, cMYC and KLF4). The cell lines generated met the standard criteria for pluripotency, including the ability to differentiate along multiple tissue lineages. In most respects, the porcine iPS cells more resembled human embryonic stem cells and human iPS cells than their murine equivalents. Provided such porcine iPS cells can be “personalized” to specific pigs and then coaxed to differentiate along specific lineages, it should be possible to use such animals to test transplantation therapies with iPS cells for safety and efficacy before the procedures are applied to human patients.