In vitro models最新文献

Titanium and its alloys are used to make different medical devices such as stents, artificial heart valves, and catheters for cardiovascular diseases due to their superior biocompatibility. Thrombus formation begins on the surface of these devices as soon as they encounter blood. This leads to the formation of blood clots, which obstructs the flow of blood that leads to severe complications. Recent advancements in nanoscale fabrication and superhydrophobic surface modification techniques have demonstrated that these surfaces have antiadhesive properties and the ability to reduce thrombosis. In this study, the interaction of erythrocytes and whole blood clotting kinetics on superhydrophobic titanium nanostructured surfaces was investigated. These surfaces were characterized for their wettability (contact angle), surface morphology and topography (scanning electron microscopy (SEM)), and crystallinity (glancing angled X-ray diffraction (GAXRD)). Erythrocyte morphology on different surfaces was characterized using SEM, and overall cell viability was demonstrated through fluorescence microscopy. The hemocompatibility of these surfaces was characterized using commercially available assays: thrombin generation assay thrombin generation, hemolytic assay hemolysis, and complement convertase assay complement activity. The results indicate that superhydrophobic titanium nanostructured surfaces had lower erythrocyte adhesion, less morphological changes in adhered cells, lower thrombin generation, lower complement activation, and were less cytotoxic compared to control surfaces. Thus, superhydrophobic titanium nanostructured surfaces may be a promising approach to prevent thrombosis for several medical devices.

ECIS is an impedance-based method to study the cellular responses to a stimulus. Manipulating the alternating current frequencies in ECIS helped reveal the adherent monolayer properties, including morphology, spreading, proliferation, changes in junctional proteins and barrier integrity. Our objective in the current study was to understand the progression of Pseudomonas infection in the airway epithelial cells using ECIS. The study also aimed at understanding the feasibility of using ECIS to study drug interactions on monolayer barrier functions. A significant reduction in impedance was noted in response to Pseudomonas infection, indicating loss of morphology and cell viability. At frequencies lower than 2000 Hz, a gradual decrease in impedance was observed during the early phase of infection, indicating a loss of junctional integrity. On the other hand, at frequencies above 16,000 Hz, a reduction in impedance was observed only during the later phases of infection. This suggested that Pseudomonas reduced the barrier integrity of cells during the early phase to gain access into the cells. Changes in cell morphology and subsequent loss of cell viability occur during the later phases of infection. Azithromycin is known to increase the barrier integrity of the monolayer (by increasing the expression of junctional proteins). We observed that pretreatment of A549 monolayers with azithromycin inhibited the progression of infection by Pseudomonas ATCC 27853 and delayed the infection of the epithelium by S373 clinical isolate. Our study is the report on the mechanism of bacterial infection progression using ECIS. It can be observed that an improvement in the barrier integrity reduces the susceptibility to bacterial infections. ECIS was demonstrated to be an effective tool for studying microbial-cell interactions and the role of drug molecules.

Objectives: This study aimed to fabricate porous PCL/GO scaffolds by adding graphene oxide (GO) which is a hydrophilic material to improve cell affinity of PCL. Calcium phosphate (CaP) coating was performed to enhance the bioactivity of the composite scaffold. The phase separation methods and the salt leaching process were used to impart high porosity and pores of various sizes in the scaffolds.

Methods: The scaffolds were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray spectrometry (EDS), water contact angle test, swelling test, and mechanical tests. For in vitro evaluation, cell morphology and viability test, alkaline phosphatase (ALP) activity, and double-stranded DNA (dsDNA) quantification were performed using mouse bone marrow stem cells (mBMSCs).

Results: All scaffolds had interconnected pore networks for transporting nutrients, oxygen, and waste products. GO addition and CaP coating improved hydrophilicity, swelling behavior, mechanical properties, and cell proliferation properties of the scaffolds by creating a biomimetic 3D microenvironment. The PCL/GO/CaP scaffold laden with mBMSCs had no clear cytotoxicity and further promoted osteogenic differentiation compared to the groups without GO or CaP.

Conclusions: Our results suggest that the porous PCL/GO/CaP scaffold showed enhanced hydrophilicity and swelling behavior and exerted beneficial effects on cell proliferation and differentiation. This composite scaffold shows potential for clinical application in bone tissue engineering.

Human organs are structurally and functionally complex systems. Their function is driven by the interactions between many specialised cell types, which is difficult to unravel on a standard Petri dish format. Conventional "Petri dish" approaches to culturing cells are static and self-limiting. However, current organ-on-a-chip technologies are difficult to use, have a limited throughput and lack compatibility with standard workflow conditions. We developed CELLBLOKS^® as a novel "plug-and-play" organ-on-a-chip platform that enables straightforward creation of multiple cell-type organ-specific microenvironments. Herein, we demonstrate its advantages by building a liver model representative of live tissue function. CELLBLOKS^® allows one to systematically test and identify various cell combinations that replicate optimal hepatic relevance. The combined interactions of fibroblasts, endothelial cells and hepatocytes were analysed using hepatic biochemistry (CYP3A4 and urea), cellular proliferation indices and transporter activities (albumin). The results demonstrate that optimal liver function can be achieved by exploiting crosstalk in co-culture combinations compared to conventional mono-culture. The optimised CELLBLOKS^® liver model was tested to analyse drug-induced liver toxicity using tamoxifen. The data suggests that our CELLBLOKS^® liver model is highly sensitive to toxic insult compared to mono-culture liver models. In summary, CELLBLOKS^® provides a novel cell culture technology for creating human-relevant organotypic models that are easy and straightforward to establish in laboratory settings.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-022-00027-8.

Whilst cancer is the single most researched disease by number of clinical trials, it remains a leading cause of death in middle- to high-income countries, with metastasis as the prime factor in cancer-associated mortality. The complex, multistep process of metastasis, in which cancer cells disseminate from the primary tumour and home to distant sites, is modulated by an extensive set of factors such as matrix properties, cancer cell plasticity, intercellular communication and oxygen tension. However, traditional treatment approaches have been focussed on the removal of the primary and secondary lesions, rather than interference with the metastatic cascade. Microfluidic platforms enable the deconvolution of the processes involved in metastasis by recapitulating key aspects of the tumour microenvironment in a controlled and reproducible fashion. Herein, we review recent developments in microfluidics for metastasis research and explain how these devices offer exceptional potential towards gaining a deeper understanding of this key aspect of malignancy.

The development and improvement of advanced intestinal in vitro models has received increasing attention in recent years. While the availability of relevant in vitro models is pivotal to advance the replacement and reduction of animal use in research, their robustness is a crucial determinant for intra- and interlaboratory reproducibility. We have developed a standard protocol to build a triple culture model combining two types of human intestinal epithelial cells (Caco-2, HT29-MTX-E12) and macrophages (THP-1), which was tested for transferability and reproducibility between three laboratories. The epithelial tissue barrier development and triple culture stability were investigated as well as the models' responses to the non-steroidal anti-inflammatory drug diclofenac in terms of barrier integrity, cytotoxicity, and cytokine release. The results of two partner laboratories were compared to previously established benchmark results and quality criteria. For the epithelial co-cultures, the results were overall highly comparable between the laboratories. The addition of THP-1 cells resulted in increased variability and reduced reproducibility. While good correlation was achieved in several endpoints, others showed substantial response differences between the laboratories. Some variations may be addressed with training or demonstrations, whereas others might be related to fundamental differences in the cell lines introduced during routine cell culture and maintenance. Our results underline the importance of interlaboratory transfer studies using standardised experimental procedures, including defined quality criteria and benchmarks, as well as of training when newly establishing complex in vitro models in laboratories.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-022-00025-w.

A 3D in vitro model of innervated skin would be a useful tool in dermatological research to study the effect of different chemicals and compounds on the sensory properties of skin. Current innervated skin models are limited in composition and often composed of ex vivo skin explants and/or animal-derived material. In this study, our aim was to develop a human innervated skin model with a better biomimicry composition for in vitro research. Fibrin hydrogel and aligned electrospun fibers of poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) were used as a scaffold to generate the 3D in vitro model. The skin component was made of primary human keratinocytes and primary human fibroblasts, while the neuronal component was composed of iPSC-derived sensory neurons. Our results showed that the dermal component consisted of fibroblasts and synthesized collagen. The epidermal component was characterized by the expression of keratins 10 and 14, and involucrin. Finally, sensory neurons extended axons throughout the scaffold and reached the epidermis. Treating the model with a capsaicin solution for 30 min, which was performed as a proof of concept test for sensitization studies, resulted into partial depletion of substance P and tubulin β3. This model could be used for studying skin-neuron interactions and cutaneous toxicity.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-022-00021-0.

Purpose: Blood compatibility of cardiovascular implants is still a major concern. Rapid endothelialization of these implant surfaces has emerged as a promising strategy to enhance hemocompatibility and prevent complications such as thrombus formation and restenosis. The successful endothelialization of implant surfaces mostly depends on the migration of endothelial cells (ECs), the differentiation of stem cells, and the inhibition of smooth muscle cell (SMC) proliferation. Our previous study demonstrated that nanostructured titania surfaces modified with polyelectrolyte multilayers based on tanfloc (a cationic tannin derivative) and glycosaminoglycans (heparin and hyaluronic acid) have improved antithrombogenic properties.

Methods: In this work, we used in vitro cell culture of ECs and SMCs to investigate the outcomes of these surface modifications on endothelialization. The cells were seeded on the surfaces, and their viability, adhesion, and proliferation were evaluated after 1, 3, and 5 days. Indirect immunofluorescent staining was used to determine the cellular expression of ECs through the presence of specific marker proteins after 7 and 10 days, and EC migration on the NT surfaces was also investigated.

Results: The surfaces modified with tanfloc and heparin showed enhanced EC adhesion, proliferation, and migration. However, SMC proliferation is not promoted by the surfaces. Therefore, these surfaces may promote endothelialization without stimulating SMC proliferation, which could improve the hemocompatibility without enhancing the risk of SMC proliferation leading to restenosis.

Conclusions: The surface modification here proposed is a promising candidate to be used in cardiovascular applications due to enhanced antithrombogenic and endothelialization properties.

An important hallmark in cancer research is the discovery of suitable features capable to reliably predict tumor invasiveness, and consequently, their metastatic potential at an early stage. Current methods are based on molecular biomarker screening and imaging that may not reveal the altered properties of tumor cells, being also labor-intensive and costly. Biophysical-based methodologies provide a new framework assessing-and even predicting-the invasion potential of tumors with improved accuracy. In particular, the stochastic fluctuations of cancer invasive protrusions can be used as a tumor-specific biophysical indicator of its aggressiveness. In this methodology, tumor micro-spheroids with different metastatic capabilities were employed as in vitro models to analyze protrusion activity. It is described the procedure for extracting the descriptive biophysical parameters characteristic of protrusion activity, which magnitude depends on the invasion capability of tumors. Next, a simple mathematical approach is employed to define a predictive index that correlates with tumor invasiveness. Overall, this innovative approach may provide a simple method for unveiling cancer invasiveness and complement existing diagnosis methodologies.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-022-00020-1.

WE43 magnesium alloy was modified using surface mechanical attrition treatment (SMAT) and characterized to evaluate the influence of sub-micron surface modification on degradation rate and in vitro behavior. Modified surface was characterized for wettability, hardness, roughness, degradation rate, in vitro biocompatibility, and antibacterial activity as per the ASTM standards. The treated substrates proved to have a significant decrease in the degradation profile by creating micro pockets of oxidation channels and reducing the total delamination in comparison to the conventional heterogeneous oxide layer formed on the untreated substrate surface. Biocompatibility studies showed that this modification did not induce any toxicity to human fetal osteoblast (hFOB) cells as demonstrated by cell proliferation and enhanced calcium deposition. In fact, results showed that between the 7^th day and 14^th day of culture, there was an eight time increase in calcium deposition for the surface-treated magnesium alloy. Bacterial adhesion and toxicity studies were carried out using Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. Bacterial toxicity studies showed that both treated and control samples were toxic to the bacteria with more dead cells. Hence, this treatment has developed a highly potential orthopedic surface with decreased biodegradability rate of WE43 and simultaneously enhanced antibacterial properties with good osteoblast cell growth and calcium deposition for faster in vitro bone growth.