3D Tissue Models Research Articles

Piezoelectric Metamaterial Blood Pressure Sensor

Continuous blood pressure monitoring allows for detecting the early onset of cardiovascular disease and assessing personal health status. Conventional piezoelectric blood pressure monitoring techniques have the ability to sense biosignals due to their good dynamic responses but have significant drawbacks in terms of power consumption, which limits the operation of blood pressure sensors. Although piezoelectric materials can be used to enhance the self-powered blood pressure sensor responses, the structure of the piezoelectric element can be modified to achieve a higher output voltage. Here, a structural study on piezoelectric metamaterials in blood pressure sensors is demonstrated, and output voltages are computed and compared to other architectures. Next, a Bayesian optimization framework is defined to get the optimal design according to the metamaterial design space. Machine learning algorithms were used for applying regression models to a simulated dataset, and a 2D map was visualized for key parameters. Finally, a time-dependent blood pressure was applied to the inner surface of an artery vessel inside a 3D tissue skin model to compare the output voltage for different metamaterials. Results revealed that all types of metamaterials can generate a higher electric potential in comparison to normal square-shaped piezoelectric elements. Bayesian optimization showed that honeycomb metamaterials had the optimal performance in generating output voltage, which was validated according to regression model analysis resulting from machine learning algorithms. The simulation of time-dependent blood pressure in a 3D skin tissue model revealed that the design suggested by the Bayesian optimization process can generate an electric potential more than two times greater than that of a conventional square-shaped piezoelectric element.

A high-throughput 3D cantilever array to model airway smooth muscle hypercontractility in asthma.

Asthma is often characterized by tissue-level mechanical phenotypes that include remodeling of the airway and an increase in airway tightening, driven by the underlying smooth muscle. Existing therapies only provide symptom relief and do not improve the baseline narrowing of the airway or halt progression of the disease. To investigate such targeted therapeutics, there is a need for models that can recapitulate the 3D environment present in this tissue, provide phenotypic readouts of contractility, and be easily integrated into existing assay plate designs and laboratory automation used in drug discovery campaigns. To address this, we have developed DEFLCT, a high-throughput plate insert that can be paired with standard labware to easily generate high quantities of microscale tissues in vitro for screening applications. Using this platform, we exposed primary human airway smooth muscle cell-derived microtissues to a panel of six inflammatory cytokines present in the asthmatic niche, identifying TGF-β1 and IL-13 as inducers of a hypercontractile phenotype. RNAseq analysis further demonstrated enrichment of contractile and remodeling-relevant pathways in TGF-β1 and IL-13 treated tissues as well as pathways generally associated with asthma. Screening of 78 kinase inhibitors on TGF-β1 treated tissues suggests that inhibition of protein kinase C and mTOR/Akt signaling can prevent this hypercontractile phenotype from emerging, while direct inhibition of myosin light chain kinase does not. Taken together, these data establish a disease-relevant 3D tissue model for the asthmatic airway, which combines niche specific inflammatory cues and complex mechanical readouts that can be utilized in drug discovery efforts.

OC-001 THE ISOLATION, CULTURE AND CHARACTERISATION OF HUMAN RECTUS SHEATH FIBROBLASTS: THE BIRTH OF TISSUE ENGINEERING THE ABDOMINAL WALL AND A LIVING MESH?

Abstract Aim To isolate human rectus sheath fibroblasts (hRSFs) from tissue samples, and characterise those cells - establishing their capacity to contract collagen matrix. Methods Samples of posterior rectus sheath were taken from 3 patients undergoing stoma formation. Samples underwent collagenase digestion, and isolated cells grown in culture media. Cultures were grown to confluence at 2nd passage, then cryopreserved. Cells were successfully thawed and re-animated at 3rd passage. 3 Cell lines of hRSFs and one of human dermal fibroblasts (HDFs) were all studied. Cell lines were assessed for their morphology and underwent immunofluorescent staining for Vimentin and α-smooth muscle actin. Cell lines were also seeded into collagen matrices to assess stromal contraction. Results Our protocol produced confluent clusters of fibroblasts within 2 weeks, and healthy cultures of cells into their 3rd passage within 6 weeks. Cells were successfully cryopreserved in liquid nitrogen after 3rd passage and were thawed and reanimated thereafter. Notable differences in morphology were observed between different cell lines – all of which stained positively for vimentin and α-smooth muscle actin. All cell lines induced a different speed of contraction within a collagen matrix from the HDFs achieving 82% contraction after 5 days, to 54% in the slowest RSF group. Conclusion Our work is the first to describe the isolation and characterisation of human rectus sheath fibroblasts and prove the concept of their introduction into 3D tissue models. This work opens exciting future prospects of both forming 3D models of rectus sheath tissue and in vitro mesh testing.

Cyanobacterial toxins and components induce expression of pro-inflammatory cytokines/chemokines and secretion of soluble cell receptors in a 3D skin cell model

Abstract Exposure to cyanobacterial toxins has been linked to several diseases and disorders including skin rashes and damage to the liver and kidneys. Previous research has shown that Microcystin-LR, the most common and toxic microcystin in fresh water, induces the NLRP3 inflammasome in response to administration to mice by oral gavage. In this study, we used a Reconstructed Human Epidermis to investigate immune system responses to cyanobacterial exposure. This 3D tissue model consisting of normal, human-derived epidermal keratinocytes was exposed to Spirulina, anatoxin-contaminated lake water, and phycocyanin. The cell supernatant was probed with a multiplex of 38 cytokines/chemokines and 14 soluble receptors to elucidate the immunological response. Our results revealed that: while most proteins were down-regulated, a number were up-regulated; the expression of many proteins were highly correlated and clustered together; and across the case study datasets, multiple cytokines/chemokines (G-CSF, GRO, IL-6, IL-8, and MCP-1) and soluble cell receptors (sCD30, sEGFR, sTNFRII and sVEGFR1, sVEGFR2 and sVEGFR3) were highly expressed when exposed to the toxins. Our findings correspond with preliminarily histological results showing tissue damage when the skin cells were exposed to the cyanobacterial components. This work shows great promise in the ability to examine the immune response to toxic exposures and aids in the development of techniques useful for studying the impact of harmful algal blooms on public health. Future studies will focus on mapping the immune pathway in response to harmful algal bloom exposure and studying the roles of each of the expressed cytokines and soluble cell receptors in the inflammatory response. This work is supported by the United States Environmental Protection Agency.

Microwave hyperthermia represses human papillomavirus oncoprotein activity and induces cell death due to cell stress in 3D tissue models of anogenital precancers and cancers

Hyperthermia is a well-accepted cancer therapy. Microwaves provide a very precise, targeted means of hyperthermia and are currently used to treat plantar warts caused by cutaneous-infective human papillomaviruses (HPVs). Other HPV genotypes infecting the anogenital mucosa cause genital warts or preneoplastic lesions or cervical cancer. Effective, non-ablative therapies for these morbid HPV-associated lesions are lacking. The molecular consequences of microwave treatment were investigated in invitro cultured three-dimensional HPV-positive cervical tumour tissues, and tissues formed from HPV-infected normal immortalised keratinocytes. Microwave energy delivery to tissues was quantified. Quantitative reverse transcriptase PCR was used to quantify mRNA expression. Immunohistochemistry and fluorescence immunostaining was used to assess protein expression. Microwave energy deposition induced sustained, localised cell death at the treatment site. There was a downregulation in levels of HPV oncoproteins E6 and E7 alongside a reduction in cellular growth/proliferation and induction of apoptosis/autophagy. HSP70 expression confirmed hyperthermia, concomitant with induction of translational stress. The data suggest that microwave treatment inhibits tumour cell proliferation and allows the natural apoptosis of HPV-infected cells to resume. Precision microwave delivery presents a potential new treatment for treating HPV-positive anogenital precancerous lesions and cancers. Funding was through an Innovate UK Biomedical Catalyst grant (ID# 92138-556187), a Chief Scientist Office grant (TCS/19/11) and core support from Medical Research Council (MC_ UU_12014) core funding for the MRC-University of Glasgow Centre for Virus Research.

Evaluation of the cellular effects of silica particles used for dermal application

ABSTRACT The cellular effects of 5 types of spherical amorphous silica particles whose particle size were 4.2–12.8 μm for cosmetic use and two types of crystalline silica whose particle size were 2.4 and 7.1 μm particles for industrial use were examined. These silica particles were applied to HaCaT human keratinocytes for 24 hr. Crystalline silica enhanced IL-8 and IL-6 expression and caused cell membrane damage. Crystalline silica also enhanced HO-1 gene expression; however, the level of intracellular ROS did not change. Compared with crystalline silica, the cellular effects of the spherical silica employed in this study were minor. Cellular uptake of particles was observed for all of silica particle types. Cellular uptake of crystalline silica was observed 1 hr after exposure, and internalized silica particles were present in the cytoplasm. When HaCaT cells were exposed to crystalline silica for 1 hr and incubated for 23 hr in culture medium without silica particles, IL-8 expression was still detected. In addition, silica particles exerted negligible effects using a 3D skin tissue model. Thus, the following conclusions may be drawn. (1) cellular effects exerted by spherical silica are less compared to crystalline silica. (2) phagocytosis of particles is an important first step in the cellular effects of silica particles. (3) spherical silica particles might exert little, if any, effect on healthy skin attributed to no apparent cellular uptake.

Acoustic Printing of Patient‐Derived Organoids That Preserve Tumor Microenvironment for Personalized Drug Screening

AbstractPatient personalized treatment models can predict treatment response and assist in determining the optimal treatment option for individual patients. The tumor microenvironment mediates tumor development and prognosis. However, current tumor models such as patient‐derived tumor xenograft (PDX) and 2D culture models fail to preserve the patient‐derived tumor microenvironment. Herein, a technique for acoustic‐droplet‐printing‐of‐patient‐derived‐organoids (ADPDOs) is reported, which allows for high‐throughput, high‐viability, rapid, and uniform printing preparation of patient‐derived organoids. In particular, the ADPDOs are uniform in size and retain the parental tumor/fibroblast cell composition, mimicking a more realistic 3D environment of parent tissue in vitro, with histological features and protein expression closer to that of the tissue. Therefore, ADPDOs hold promise for application in personalized precision therapy for different patients. The authors test the clinical drug treatment response of different patients in a fast and convenient way to predict the therapeutic effect of various patients. Thus, this technique has great potential for predicting treatment response to personalized cancer therapy and constructing complex 3D tissue models.

3D Computed Tomography vs. 2D radiography: comparison of 3D direct anthropometry with 2D norm calculations and analysis of differences in soft tissue measurements.

The aim of this study was twofold: (1) to compare soft tissue measurements of the same distances obtained from 3D computed tomography reconstructions with 2D cephalometric radiograms, (2) to compare data from 3D measurements from direct anthropometry and 2D "norms" for the facial measurements. A total of 40 Caucasian patients that had their CBCT scans for various dental and dentoskeletal reasons were enrolled in this study. All the patients had large field of view (from the forehead to the chin). The data were stored in DICOM format and imported into a software for 3D reconstructions. After 3D facial soft tissue model generation, the distances between 18 soft tissue points were measured. The 3D soft tissue analysis was performed, and the facial indices were calculated. The mean 3D values were compared with 2D measurements performed on lateral cephalograms and Arnett's and Farkas' norms. The measurements were statistically compared using Student's t-test. Assessments from 2D and 3D measurements showed no statistical difference except for the distance Pogonion (for both male and female) and Labial superius prominence (females) to the True Vertical Line in 2D /Plane in case of 3D measurements. There was a significant difference between all 3D measurements and Arnett's and anthropometric Farkas' "norms". The mean difference between Farkas' "norms" and 3D measurements was within 3 mm for 70% of measurements. According to the results, 3D soft tissue analysis allows for complete diagnostic determination. The 3D "norms" are to be verified on a greater sample.

Synthesis of easily-processable collagen bio-inks using ionic liquid for 3D bioprinted liver tissue models with branched vascular networks

Synthesis of easily-processable collagen bio-inks using ionic liquid for 3D bioprinted liver tissue models with branched vascular networks

Alginate-Gelatin Self-Healing Hydrogel Produced via Static-Dynamic Crosslinking.

Alginate-gelatin hydrogels mimicking extracellular matrix (ECM) of soft tissues have been generated by static-dynamic double crosslinking, allowing fine control over the physical and chemical properties. Dynamic crosslinking provides self-healing and injectability attributes to the hydrogel and promotes cell migration and proliferation, while the static network improves stability. The static crosslinking was performed by enzymatic coupling of the tyrosine residues of gelatin with tyramine residues inserted in the alginate backbone, catalyzed by horseradish peroxidase (HRP). The dynamic crosslinking was obtained by functionalizing alginate with 3-aminophenylboronic acid which generates a reversible bond with the vicinal hydroxyl groups of the alginate chains. Varying the ratio of alginate and gelatin, hydrogels with different properties were obtained, and the most suitable for 3D soft tissue model development with a 2.5:1 alginate:gelatin molar ratio was selected. The selected hydrogel was characterized with a swelling test, rheology test, self-healing test and by cytotoxicity, and the formulation resulted in transparent, reproducible, varying biomaterial batch, with a fast gelation time and cell biocompatibility. It is able to modulate the loss of the inner structure stability for a longer time with respect to the formulation made with only covalent enzymatic crosslinking, and shows self-healing properties.

Novel Approach to the Treatment of Neuropathic Pain Using a Combination with Palmitoylethanolamide and Equisetum arvense L. in an In Vitro Study

Neuropathic pain is a typical patient disorder resulting from damage and dysfunction of the peripheral neuraxis. Injury to peripheral nerves in the upper extremities can result in a lifelong reduction in quality of life and a devastating loss of sensory and motor function. Since some standard pharmaceutical therapies can cause dependence or intolerance, nonpharmacological treatments have gained great interest in recent years. In this context, the beneficial effects of a new combination of palmitoylethanolamide and Equisetum arvense L. are evaluated in the present study. The bioavailability of the combination was initially analyzed in a 3D intestinal barrier simulating oral intake to analyze its absorption/biodistribution and exclude cytotoxicity. In a further step, a 3D nerve tissue model was performed to study the biological effects of the combination during the key mechanisms leading to peripheral neuropathy. Our results demonstrate that the combination successfully crossed the intestinal barrier and reached the target site, modulating the nerve recovery mechanism after Schwann cell injury and offering the initial response of relieving pain. This work supported the efficacy of palmitoylethanolamide and Equisetum arvense L. in reducing neuropathy and modifying the major pain mechanisms, outlining a possible alternative nutraceutical approach.

Three-dimensional analysis of the relationship between mandibular retromolar space and positional traits of third molars in non-hyperdivergent adults

BackgroundThe anatomical position of the mandibular third molars (M3s) is located in the distal-most portions of the molar area. In some previous literature, researchers evaluated the relationship between retromolar space (RS) and different classifications of M3 in three‑dimensional (3D) cone—beam computed tomography (CBCT).MethodsTwo hundred six M3s from 103 patients were included. M3s were grouped according to four classification criteria: PG-A/B/C, PG-I/II/III, mesiodistal angle and buccolingual angle. 3D hard tissue models were reconstructed by CBCT digital imaging. RS was measured respectively by utilizing the fitting WALA ridge plane (WP) which was fitted by the least square method and the occlusal plane (OP) as reference planes. SPSS (version 26) was used to analyze the data.ResultsIn all criteria evaluated, RS decreased steadily from the crown to the root (P < 0.05), the minimum was at the root tip. From PG-A classification, PG-B classification to PG-C classification and from PG-I classification, PG-II classification to PG-III classification, RS both appeared a diminishing tendency (P < 0.05). As the degree of mesial tilt decreased, RS appeared an increasing trend (P < 0.05). RS in classification criteria of buccolingual angle had no statistical difference (P > 0.05).ConclusionsRS was associated with positional classifications of the M3. In the clinic, RS can be evaluated by watching the Pell&Gregory classification and mesial angle of M3.

Gelatin-based cell culture device for construction and X-ray irradiation of a three-dimensional oral cancer model.

Bioassays using three-dimensional (3D) tissue models offer several advantages over 2D culture assays because they can reproduce the structure and function of native tissues. In this study, we used our newly designed gelatin device to generate a miniature 3D model of human oral squamous cell carcinoma with stroma and blood vessels. To enable air-liquid interface culture, we conceived a new device structure in which three wells were lined up and separated by a dividing thread; the wells could be connected by removing the dividing thread. Cells were seeded in the center well with the dividing thread to form a multilayer, followed by the supply of media from the side wells after thread removal. Human oral squamous cell carcinoma (HSC-4) cells, human umbilical vein endothelial cells (HUVECs), and normal human dermal fibroblasts (NHDFs) were successfully cocultured, resulting in structures that mimicked 3D-cancer tissues. This 3D-cancer model was subjected to an X-ray sensitivity assay, followed by the evaluation of DNA damage using confocal microscopy and section-scanning electron microscopy.

Cardiovascular Organoids/3D Models Review Series: an Introduction.

Cardiovascular Organoids/3D Models Review Series: an Introduction.

Myrtus communis and Celastrol enriched plant cell culture extracts control together the pivotal role of Cutibacterium acnes and inflammatory pathways in acne.

Acne is a multifactorial inflammatory disease of the pilosebaceous unit in which Cutibacterium acnes is one of the main triggers. A strong predominance of C.acnes phylotype IA1 is present in acne skin with higher biofilm organization and virulence, promoting local immuno-inflammation, especially the Th17 pathway. We evaluated the single and combined pharmacological properties of the plant extracts, Myrtus communis (Myrtacine®) and Celastrol enriched plant cell culture (CEE) extracts on the C.acnes/Th17 pathway. The effect of Myrtacine® on the virulence of C.acnes phylotype IA1 was quantified according to the expression of several related genes. The activity of Myrtacine® and CEE on the inflammatory cascade was assessed using monocytes-derived dendritic cells (Mo-DC) stimulated with membranes or biofilms of the C.acnes phylotype IA1. Finally, the effect of CEE on the Th17 pathway was studied using C.acnes stimulated sebocyte 2D cultures and 3D skin tissue models containing preactivated Th17 cells. Myrtacine® had an anti-virulence effect, evident as a significant and strong inhibition of the expression of several virulence factor genes by 60%-95% compared to untreated controls. Myrtacine® and CEE significantly inhibited proinflammatory cytokine (IL-6, IL-8, IL-12p40 and TNF-α) production by Mo-DC in response to C.acnes phylotype IA1. Interestingly, these two ingredients resulted in synergistic inhibition of most cytokines when used in combination. Finally, we demonstrated an inhibitory effect of CEE, in solution or formulated at 0.3%, specifically on IL-17 release by Th17 lymphocytes in a C.acnes-stimulated sebocyte 2D cultures and by Th17-lymphocytes integrated in a 3D skin models. 2D and 3D models were developed to represent relevant and specific pathways involved in acne. Myrtacine® and CEE were shown to alter one or more of these pathways, indicating their potential beneficial effects on this disease.

Localization of Multiple Hydrogels with MultiCUBE Platform Spatially Guides 3D Tissue Morphogenesis In Vitro

AbstractLocalization of multiple hydrogels is expected to develop the structure of 3D tissue models in a location‐specific manner. Here, 3D tissue morphogenesis is spatially guided by localizing different hydrogel conditions at different parts of a tissue. To achieve the localization, a unit‐based scaffold is developed with a unique frame design to trap hydrogel solutions inside their designated units. An optimal range of unit dimensions and surface wettabilities enables a solution trapping up to several cubic millimeters without any need for chemical additives. This capability allows spatial organization of biomolecular compositions and physical conditions of hydrogels, as well as the relative position of biological samples (cells, spheroids, and reconstituted tissues) within the scaffold. Successful localization of branching development on reconstituted human epithelial tissues is achieved by localizing growth factors or cross‐linked matrix proteins within hydrogels, demonstrating a direct dependence on local hydrogel conditions. Unlike 3D‐bioprinting or microfluidic techniques, this scaffold‐based localization of hydrogels requires only a manual pipetting and no specialized tools, making it ready‐to‐use for researchers from any field. This localization technique provides a new promising route to spatially control morphogenesis, differentiation, and other developmental processes within 3D organoids or tissue models for practical biomedical applications in the future.

Fabrication and Characterization Techniques of In Vitro 3D Tissue Models.

The culturing of cells in the laboratory under controlled conditions has always been crucial for the advancement of scientific research. Cell-based assays have played an important role in providing simple, fast, accurate, and cost-effective methods in drug discovery, disease modeling, and tissue engineering while mitigating reliance on cost-intensive and ethically challenging animal studies. The techniques involved in culturing cells are critical as results are based on cellular response to drugs, cellular cues, external stimuli, and human physiology. In order to establish in vitro cultures, cells are either isolated from normal or diseased tissue and allowed to grow in two or three dimensions. Two-dimensional (2D) cell culture methods involve the proliferation of cells on flat rigid surfaces resulting in a monolayer culture, while in three-dimensional (3D) cell cultures, the additional dimension provides a more accurate representation of the tissue milieu. In this review, we discuss the various methods involved in the development of 3D cell culture systems emphasizing the differences between 2D and 3D systems and methods involved in the recapitulation of the organ-specific 3D microenvironment. In addition, we discuss the latest developments in 3D tissue model fabrication techniques, microfluidics-based organ-on-a-chip, and imaging as a characterization technique for 3D tissue models.

A 3D multi-cellular tissue model of the human omentum to study the formation of ovarian cancer metastasis

Reliable and predictive experimental models are urgently needed to study metastatic mechanisms of ovarian cancer cells in the omentum. Although models for ovarian cancer cell adhesion and invasion were previously investigated, the lack of certain omental cell types, which influence the metastatic behavior of cancer cells, limits the application of these tissue models. Here, we describe a 3D multi-cellular human omentum tissue model, which considers the spatial arrangement of five omental cell types. Reproducible tissue models were fabricated combining permeable cell culture inserts and bioprinting technology to mimic metastatic processes of immortalized and patient-derived ovarian cancer cells. The implementation of an endothelial barrier further allowed studying the interaction between cancer and endothelial cells during hematogenous dissemination and the impact of chemotherapeutic drugs. This proof-of-concept study may serve as a platform for patient-specific investigations in personalized oncology in the future.

A New Model of Esophageal Cancers by Using a Detergent-Free Decellularized Matrix in a Perfusion Bioreactor.

The lack of physiologically relevant human esophageal cancer models has as a result that many esophageal cancer studies are encountering major bottleneck challenges in achieving breakthrough progress. To address the issue, here we engineered a 3D esophageal tumor tissue model using a biomimetic decellularized esophageal matrix in a customized bioreactor. To obtain a biomimetic esophageal matrix, we developed a detergent-free, rapid decellularization method to decellularize porcine esophagus. We characterized the decellularized esophageal matrix (DEM) and utilized the DEM for the growth of esophageal cancer cell KYSE30 in well plates and the bioreactor. We then analyzed the expression of cancer-related markers of KYSE30 cells and compared them with formalin-fixed, paraffin-embedded (FFPE) esophageal squamous cell carcinoma (ESCC) tissue biospecimens. Our results show that the detergent-free decellularization method preserved the esophageal matrix components and effectively removed cell nucleus. KYSE30 cancer cells proliferated well on and inside the DEM. KYSE30 cells cultured on the DEM in the dynamic bioreactor show different cancer marker expressions than those in the static well plate, and also share some similarities to the FFPE-ESCC biospecimens. These findings built a foundation with potential for further study of esophageal cancer behavior in a biomimetic microenvironment using this new esophageal cancer model.

Concentric-mineralized hybrid silk-based scaffolds for bone tissue engineering in vitro models.

There are many challenges in the development of 3D-tissue models for studying bone physiology and disease. Silk fibroin (SF), a natural fibrous protein used in biomedical applications has been studied for bone tissue engineering (TE) due to its mechanical properties, biocompatibility and biodegradability. However, low osteogenic capacity as well as the necessity to reinforce the protein mechanically for some orthopedic applications prompts the need for further designs for SF-based materials for TE bone. Concentric mineralized porous SF-based scaffolds were developed to improve mechanics and mineralization towards osteoregeneration. Hybrid SF silica microparticles (MP) or calcium carbonate nano-structured microparticles (NMP) were seeded with hMSCs co-cultured under osteogenic and osteoclastic conditions with THP-1 human monocytes up to 10 weeks to simulate and recapitulate bone regeneration. Scaffolds with appropriate pore size for cell infiltration, resulted in improved compressive strength, increased cell attachment and higher levels of expression of osteogenic markers and mineralization after adding the NMPs, compared to controls systems without these particles. These hybrid SF-based 3D-structures can provide improved scaffold designs for in vitro bone TE.