3D cell culture models and organ-on-a-chip: Meet separation science and mass spectrometry

In vitro derived simplified 3D representations of human organs or organ functionalities are predicted to play a major role in disease modeling, drug development, and personalized medicine, as they complement traditional cell line approaches and animal models. The cells for 3D organ representations may be derived from primary tissues, embryonic stem cells or induced pluripotent stem cells and come in a variety of formats from aggregates of individual or mixed cell types, self-organizing in vitro developed “organoids” and tissue mimicking chips. Microfluidic devices that allow long-term maintenance and combination with other tissues, cells or organoids are commonly referred to as “microphysiological” or “organ-on-a-chip” systems. Organ-on-a-chip technology allows a broad range of “on-chip” and “off-chip” analytical techniques, whereby “on-chip” techniques offer the possibility of real time tracking and analysis. In the rapidly expanding tool kit for real time analytical assays, mass spectrometry, combined with “on-chip” electrophoresis, and other separation approaches offer attractive emerging tools. In this review, we provide an overview of current 3D cell culture models, a compendium of current analytical strategies, and we make a case for new approaches for integrating separation science and mass spectrometry in this rapidly expanding research field.     

3D Bioprinting of Artificial Tissues: Construction of Biomimetic Microstructures

It is promising that artificial tissues/organs for clinical application can be produced via 3D bioprinting of living cells and biomaterials. The construction of microstructures biomimicking native tissues is crucially important to create artificial tissues with biological functions. For instance, the fabrication of vessel‐like networks to supply cells with initial nutrient and oxygen, and the arrangement of multiple types of cells for creating lamellar/complex tissues through 3D bioprinting are widely reported. The current advances in 3D bioprinting of artificial tissues from the view of construction of biomimetic microstructures, especially the fabrication of lamellar, vascular, and complex structures are summarized. In the end, the conclusion and perspective of 3D bioprinting for clinical applications are elaborated.     

3D打印生物医用材料研究进展

3D打印(亦称增材制造)技术因其独特的材料成型优势,在组织工程、航空航天、汽车制造、以及电子工业等众多领域显示出巨大的应用潜力。然而,在实际生物医学应用中,3D打印生物器件和组织器官除了要求具有复杂的结构和优异的生物学性能外,其打印结构的表面性质也需满足某些特定的要求,如3D打印组织骨架和器官必须具有生物相容性、抗菌性及细胞粘附性等。因此,将3D打印与传统表面修饰技术相结合,在不改变材料三维结构的基础上调控其表面生物化学性质,从而赋予3D打印生物骨架器官多功能化,可实现更为广泛的应用。本文以3D打印生物骨架及器官的表面修饰为主要内容对就近年来3D打印生物医用材料的最新研究进展进行了综述。     

Dynamic surface tensiometry of tissues using Langmuir films

Langmuir monolayers are useful models of biomembranes as they allow simulation of biological conditions and rigorous thermodynamic analysis. This technique was used to characterize tissues at body temperature for the first time in our study. The organs studied include liver, kidney, stomach, testis, heart and brain from goat and certain human cancerous as well as their corresponding normal biopsies to reveal the potential of the tissue monolayer technique. Monolayers were formed on the surface of deionized water by spreading monolayer amounts of the tissue homogenates. The parameters calculated were minimum surface tension, relative lift off area, relative limiting area, compressibility and hysteresis area. Our results reveal that the parameters can differentiate between tissues obtained from different organs and were statistically significant using one-way ANOVA and Newman Keul's test (P<0.05). For example goat's stomach tissue had the lowest hysteresis area (DeltaG) value (27.6 microJ) whereas brain DeltaG value was nine folds higher than stomach value. Brain had the lowest minimum surface tension of 30.3+/-1.0 mN/m whereas stomach had a value of 40.5+/-0. 2 mN/m. Interestingly, the DeltaG values of human normal neck and esophageal tissues were 3.4 and 3.2 folds greater than that of their respective cancer tissues whereas the DeltaG values of vulval and breast cancer tissues were 4.6 and 4 folds greater than that of their respective normal tissues. While the gammamin values of neck cancer tissue showed 95% increase from normal tissue values, those of vulval and breast cancer tissues were 46 and 50% less compared to their respective normal tissue values. Though all the surface tensiometric parameters showed significant changes, minimum surface tension and hysteresis area were the most sensitive indicators of tissue types and diseased states. Further, the effects of therapeutics could also be monitored by this technique. This is evidenced by the post-radiotherapy tissue isotherms of neck and vulval cancers, where clinical radio-sensitivity was associated with a shift in the tensiometry towards their respective normal isotherms. The small sample amounts required, precision of the technique, very low within group variability, organ specificity and sensitivity to detect changes in diseased states make it a promising tool for prognostic evaluation of diseased states and monitoring effects of therapeutics. Further research is warranted in this promising and hitherto unexplored field of tissue tensiometry.     

Use of Three‐Dimensional Arterial Models To Predict the In Vivo Behavior of Nanoparticles for Drug Delivery

Nanomaterials have been widely used for applications in biomedical fields and could become indispensable in the near future. However, since it is difficult to optimize in vivo biological behavior in a 3D environment by using a single cell in vitro, there have been many failures in animal models. In vitro prediction systems using 3D human‐tissue models reflecting the 3D location of cell types may be useful to better understand the biological characteristics of nanomaterials for optimization of their function. Herein we demonstrate the potential ability of 3D engineered human‐arterial models for in vitro prediction of the in vivo behavior of nanoparticles for drug delivery. These models enabled optimization of the composition and size of the nanoparticles for targeting and treatment efficacy for atherosclerosis. In vivo experiments with atherosclerotic mice suggested excellent biological characteristics and potential treatment effects of the nanoparticles optimized in vitro.     

Synthetic chemoselective rewiring of cell surfaces: generation of three-dimensional tissue structures

Proper cell-cell communication through physical contact is crucial for a range of fundamental biological processes including, cell proliferation, migration, differentiation, and apoptosis and for the correct function of organs and other multicellular tissues. The spatial and temporal arrangements of these cellular interactions in vivo are dynamic and lead to higher-order function that is extremely difficult to recapitulate in vitro. The development of three-dimensional (3D), in vitro model systems to investigate these complex, in vivo interconnectivities would generate novel methods to study the biochemical signaling of these processes, as well as provide platforms for tissue engineering technologies. Herein, we develop and employ a strategy to induce specific and stable cell-cell contacts in 3D through chemoselective cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. This strategy uses liposome fusion for the delivery of ketone or oxyamine groups to different populations of cells for subsequent cell assembly via oxime ligation. We demonstrate how this method can be used for several applications including, the delivery of reagents to cells for fluorescent labeling and cell-surface engineering, the formation of small, 3D spheroid cell assemblies, and the generation of large and dense, 3D multilayered tissue-like structures for tissue engineering applications.     

Recent Progress in Fabrication of Electrospun Nanofiber Membranes for Developing Physiological In Vitro Organ/Tissue Models

Nanofiber membranes (NFMs), which have an extracellular matrix-mimicking structure and unique physical properties, have garnered great attention as biomimetic materials for developing physiologically relevant in vitro organ/tissue models. Recent progress in NFM fabrication techniques immensely contributes to the development of NFM-based cell culture platforms for constructing physiological organ/tissue models. However, despite the significance of the NFM fabrication technique, an in-depth discussion of the fabrication technique and its future aspect is insufficient. This review provides an overview of the current state-of-the-art of NFM fabrication techniques from electrospinning techniques to postprocessing techniques for the fabrication of various types of NFM-based cell culture platforms. Moreover, the advantages of the NFM-based culture platforms in the construction of organ/tissue models are discussed especially for tissue barrier models, spheroids/organoids, and biomimetic organ/tissue constructs. Finally, the review concludes with perspectives on challenges and future directions for fabrication and utilization of NFMs.     

A 3D bio-printed spheroids based perfusion in vitro liver on chip for drug toxicity assays

Liver is the foremost organ of human being for drug metabolism, and it played a significant role in toxicity evaluation of drugs. Establishing a liver model in vitro can accelerate the process of the drug screening and new drug research and development. We provide a 3D printing based hepatic sinusoid-on-a-chip microdevice that reconstitutes organ-level liver functions to create a drug screening model of toxicity evaluation on chip. The microfluidic device, which recapitulates the hepatic sinusoi...     

Three‐Dimensional Tissue Models Constructed by Cells with Nanometer‐ or Micrometer‐Sized Films on the Surfaces

Living tissues or organ modules consist of different types of highly organized cells and extracellular matrices (ECMs) in a hierarchical manner, such as the multilayered structure of blood vessels and the radial structures of hepatic lobules. Due to animal examinations being banned in the EU since 2013 and a shortage in the demand for tissue repair or organ transplantation, the creation of artificial 3D tissues possessing specific structures and functions similar to natural tissues are key challenges in tissue engineering. To date, we have developed a simple but unique bottom‐up approach, a hierarchical cell manipulation technique, with a nanometer‐sized ECM matrix consisting of fibronectin (FN) and gelatin (G) on cell surfaces. About 10 nm thick FN/G ECM films on cell surfaces were coated successfully by using layer‐by‐layer coating methodology. Various 3D constructs with higher cell density with different types of cells were successfully constructed. In addition to the construction of tissues with higher cell densities, other tissues, such as cartilage or skin tissues, with different cell densities are also important tissue models for tissue engineering and pharmaceutical industries. Thus, we recently developed other methodologies, the collagen coating method and multiple coating method, to fabricate micrometer‐sized level ECM layers on cell surfaces. Various micro‐ or millimeter‐sized 3D constructs with lower cell densities were constructed successfully. By using these two methods, cell distances in 2D or 3D views can be controlled by different thicknesses of ECM layers on cell surfaces at the single‐cell level. Both FN/G and the collagen coating method resulted in homogenous 3D tissues with a controlled layer numbers, cell type, cell location, and properties; these will be promising to achieve different goals in tissue engineering.

     

Designer self-assembling peptide scaffolds for 3-d tissue cell cultures and regenerative medicine

Biomaterial science has made enormous progress in the last few decades. Nonetheless, innovative biomaterials are still urgently needed to provide in vitro cell-culture models that more closely resemble three-dimensional (3-D) cell interactions and cyto-architectures in bodies and tissues. In this review, the recent advances toward this goal through molecular engineering of various designer self-assembling peptide scaffolds are discussed. These peptide scaffolds can be commercially and custom-tailor synthesized materials with high purity and may be not only useful for specific 3-D tissue cell cultures but also for tissue repair and regenerative therapies. Furthermore, these designer self-assembling peptide scaffolds have recently become powerful tools for regenerative medicine to repair nervous tissue, to stop bleeding in seconds, to repair infarctuated myocardia, as well as being useful medical devices for slow drug release.     

Recent Advances in 3D Printing of Photocurable Polymers: Types,Mechanism, and Tissue Engineering Application

The conversion of liquid resin into solid structures upon exposure to light of a specific wavelength is known as photopolymerization. In recent years, photopolymerization-based 3D printing has gained enormous attention for constructing complex tissue-specific constructs. Due to the economic and environmental benefits of the biopolymers employed, photo-curable 3D printing is considered an alternative method for replacing damaged tissues. However, the lack of suitable bio-based photopolymers, their characterization, effective crosslinking strategies, and optimal printing conditions are hindering the extensive application of 3D printed materials in the global market. This review highlights the present status of various photopolymers, their synthesis, and their optimization parameters for biomedical applications. Moreover, a glimpse of various photopolymerization techniques currently employed for 3D printing is also discussed. Furthermore, various naturally derived nanomaterials reinforced polymerization and their influence on printability and shape fidelity are also reviewed. Finally, the ultimate use of those photopolymerized hydrogel scaffolds in tissue engineering is also discussed. Taken together, it is believed that photopolymerized 3D printing has a great future, whereas conventional 3D printing requires considerable sophistication, and this review can provide readers with a comprehensive approach to developing light-mediated 3D printing for tissue-engineering applications.     

Blood appearance of rat alkaline phosphatase originating from the duodenum in vitro

The major source of rat serum alkaline phosphatase (ALP) is well known to be from the intestinal enzyme, but it is still unclear whether it is from the duodenal or the ileal enzyme. The organic origin was investigated by means of two-dimensional electrophoresis. Major isoelectric points and molecular masses for activities of duodenal enzyme treated with both phosphatidylinositol-specific phospholipase C and neuraminidase were identified apparently with those of the major serum enzyme. In organ culture, the normal duodenal enzyme was released in the highest amounts to the culture medium. These results indicate that the major source of serum ALP in adult rats is basically from the duodenal enzyme. On the other hand, lectin affinity chromatography for ALPs showed that the ALP in the medium from culture duodenum and liver had the same complex-type sugar chain as with the ALP in the duodenal tissue. Although the duodenal ALP induced by glucosamine in vitro had the hybrid-type chain, sugar chains of the induced ALP in the culture medium were of the complex type, indicating that medial ALPs possessing the same sugar chain as the native duodenal enzyme, complex type, are mainly released from their tissues in normal conditions.     

Application of surface-enhanced laser desorption/ionization technology to the detection and identification of urinary parvalbumin-alpha: a biomarker of compound-induced skeletal muscle toxicity in the rat

In toxicity studies, compound-induced changes are typically evaluated using a combination of endpoints and there are often a number of potential markers in biological fluids which can indicate toxic change in tissues and organs. However, some biomarkers are not specific to the organ of injury and therefore there is a continuing search for more sensitive and specific indicators of target organ toxicity. In experiments to assess the potential diagnostic usefulness of surface-enhanced laser desorption/ionization (SELDI) ProteinChip technology, skeletal muscle toxicity was induced in Wistar Han rats by administering 2,3,5,6-tetramethyl-p-phenylenediamine (TMPD). The skeletal muscle toxicity was monitored using established endpoints such as increase in serum aldolase (Aldol), aspartate aminotransferase (AST) and histopathology, and also using SELDI retentate chromatography mass spectrometry of urine samples. Clear differences in urinary protein patterns between control and TMPD-treated animals were observed on the ProteinChip surfaces. Additionally a specific urine marker protein of 11.8 kDa was identified in TMPD-dosed rats, and the detection of the marker was related to the degree of skeletal muscle toxicity assessed by recognized clinical pathology endpoints. The 11.8 kDa protein was identified as parvalbumin-alpha. These experiments demonstrated the potential of urinary parvalbumin-alpha as a specific, noninvasive, and easily detectable biomarker for skeletal muscle toxicity in the rat and the potential of SELDI technology for biomarker detection and identification in toxicology studies.     

3D打印技术制备生物医用高分子材料的研究进展

3D打印技术能够根据不同患者需要,快速精确制备适合不同患者的个性化生物医用高分子材料,并能同时对材料的微观结构进行精确控制.因此,这种新兴的医用高分子材料制备技术在未来生物医学应用(尤其是组织工程应用)中具有独特的优势.近年来,对于3D打印技术制备生物医用高分子材料的研究开发受到了越来越多的关注.不同的生物相容高分子原料被应用于3D打印技术,而这些3D成型高分子材料被用于体外细胞培养,或动物模型的软组织或硬组织修复中.本文主要介绍了近年来3D打印技术在生物医用高分子材料制备中的研究进展,并对该领域的未来应用和挑战进行了展望.     

用于肿瘤成像的半乳糖/酞菁近红外荧光探针

本文首次报道半乳糖酞菁近红外荧光探针在肿瘤成像方面的应用。以酞菁为荧光发射基团,其在近红外区域有较高的量子产量和光学稳定性,可以解决探针的近红外光学特性;通过半乳糖对酞菁的修饰能够改善探针的溶解性和生物相容性,而且利用半乳糖的肿瘤靶向功能可以提高探针肿瘤主动靶向成像效果。结果表明它对恶性肿瘤具有高特异性结合和高灵敏度的分子探针体系,提高近红外光学分子成像效果。     

原位冷冻电子断层扫描的发展及在生物研究方面的应用

对生物大分子复合物的研究和结构分析对于全面了解其功能和生物学意义至关重要.冷冻电子显微镜在提供生物大分子结构及大分子分布等方面起到重要的作用.近年来,冷冻电子显微镜的硬件和软件的发展进一步提高了冷冻电子显微镜的有效性,使其对各种生物结构、蛋白质结构的解析更加准确快捷.但是,对于生物系统来说,蛋白质和大分子复合物等均处于复杂的生理环境中,因此原位检测生物分子的三维结构对于生物体系和结构生物学具有重要意义.冷冻电子断层扫描作为一种功能强大的技术,可以无需标记直接通过冷冻样品的固有衬度识别生物大分子的结构,并且可在原位生理环境中对生物分子进行纳米级分辨率的三维成像.本文综述了与冷冻电子断层扫描相关的样品制备和数据处理技术,并总结了冷冻电子断层扫描技术在分离的大分子复合物和整个细胞或组织中的生物学应用.     

Organ weaving: woven threads and sheets as a step towards a new strategy for artificial organ development

The concept of "organ weaving" is presented, a fabrication technique that can be an attractive option for the development of artificial tissues and organs. "Living threads" are created by immersing threads that are soaked in a CaCl(2) solution into a sodium-alginate-loaded cell suspension bath, encapsulating the cells and creating a bio-friendly, easily manageable starting material for building up larger scaffold structures. Such living threads have the advantage of being a particularly mild culturing medium for mammalian cells, protecting the cells during subsequent processing steps from dehydration and other rapid changes in the chemistry of the surrounding environment. Connecting different types of threads into 3D objects gives unique opportunities to address tissue engineering challenges.     

Therapeutic Potential of Dental Pulp Stem Cells According to Different Transplant Types

Stem cells are unspecialised cells capable of perpetual self-renewal, proliferation and differentiation into more specialised daughter cells. They are present in many tissues and organs, including the stomatognathic system. Recently, the great interest of scientists in obtaining stem cells from human teeth is due to their easy availability and a non-invasive procedure of collecting the material. Three key components are required for tissue regeneration: stem cells, appropriate scaffold material and growth factors. Depending on the source of the new tissue or organ, there are several types of transplants. In this review, the following division into four transplant types is applied due to genetic differences between the donor and the recipient: xenotransplantation, allotransplantation, autotransplantation and isotransplantation (however, due to the lack of research, type was not included). In vivo studies have shown that Dental Pulp Stem Cells (DPSCs)can form a dentin-pulp complex, nerves, adipose, bone, cartilage, skin, blood vessels and myocardium, which gives hope for their use in various biomedical areas, such as immunotherapy and regenerative therapy. This review presents the current in vivo research and advances to provide new biological insights and therapeutic possibilities of using DPSCs.     

Microstructured cocultures of cardiac myocytes and fibroblasts: a two-dimensional in vitro model of cardiac tissue.

Cardiac myocytes and fibroblasts are essential elements of myocardial tissue structure and function. In vivo, myocytes constitute the majority of cardiac tissue volume, whereas fibroblasts dominate in numbers. In vitro, cardiac cell cultures are usually designed to exclude fibroblasts, which, because of their maintained proliferative potential, tend to overgrow the myocytes. Recent advances in microstructuring of cultures and cell growth on elastic membranes have greatly enhanced in vitro preservation of tissue properties and offer a novel platform technology for producing more in vivo-like models of myocardium. We used microfluidic techniques to grow two-dimensional structured cardiac tissue models, containing both myocytes and fibroblasts, and characterized cell morphology, distribution, and coupling using immunohistochemical techniques. In vitro findings were compared with in vivo ventricular cyto-architecture. Cardiac myocytes and fibroblasts, cultured on intersecting 30-microm-wide collagen tracks, acquire an in vivo-like phenotype. Their spatial arrangement closely resembles that observed in native tissue: Strands of highly aligned myocytes are surrounded by parallel threads of fibroblasts. In this in vitro system, fibroblasts form contacts with other fibroblasts and myocytes, which can support homogeneous and heterogeneous gap junctional coupling, as observed in vivo. We conclude that structured cocultures of cardiomyocytes and fibroblasts mimic in vivo ventricular tissue organization and provide a novel tool for in vitro research into cardiac electromechanical function.