In vitro models最新文献

Purpose: Alzheimer's disease (AD) early pathology needs better understanding and models. Here, we describe a human induced pluripotent stem cells (iPSCs)-derived 3D neural culture model to study certain aspects of AD biochemistry and pathology.

Method: iPSCs derived from controls and AD patients with Presenilin1 mutations were cultured in a 3D platform with a similar microenvironment to the brain, to differentiate into neurons and astrocytes and self-organise into 3D structures by 3 weeks of differentiation in vitro.

Results: Cells express astrocytic (GFAP), neuronal (β3-Tubulin, MAP2), glutamatergic (VGLUT1), GABAergic (GAD65/67), pre-synaptic (Synapsin1) markers and a low level of neural progenitor cell (Nestin) marker after 6 and 12 weeks of differentiation in 3D. The foetal 3R Tau isoforms and adult 4R Tau isoforms were detected at 6 weeks post differentiation, showing advanced neuronal maturity. In the 3D AD cells, total and insoluble Tau levels were higher than in 3D control cells.

Conclusion: Our data indicates that this model may recapitulate the early biochemical and pathological disease features and can be a relevant platform for studying early cellular and biochemical changes and the identification of drug targets.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-022-00038-5.

Background: Advanced cell culture techniques such as 3D bioprinting and hydrogel-based cell embedding techniques harbor many new and exciting opportunities to study cells in environments that closely recapitulate in vivo conditions. Researchers often study these environments using fluorescence microscopy to visualize the protein association with objects such as cells within the 3D environment, yet quantification of concentration profiles in the microenvironment has remained elusive.

Objective: Demonstrate an assay that enables near real-time in situ biomarker detection and spatiotemporal quantification of biomarker concentration in 3D cell culture.

Methods: A distributed bead-based immuno-assay was used in 3D cell culture to continuously measure the time-dependent concentration gradient of various biomarkers by sequestering soluble target molecules and concentrating the fluorescence intensity of these tagged proteins. Timelapse confocal microscopy was used to measure the in situ fluorescence intensity profile and a calibration curve was separately generated. Application of a calibration transfer function to in situ data is used to quantify spatiotemporal concentration.

Results: Example assays utilize an osteosarcoma spheroid as a case study for a quantitative single-plexed gel encapsulated assay, and a qualitative multi-plexed 3D-bioprinted assay. In both cases, a time-varying cytokine concentration gradient is measured. An estimation for the production rate of the IL-8 cytokine per second per osteosarcoma cell results from fitting an analytical function for continuous point source diffusion to the measured concentration gradient and reveals that spheroid production approaches nearly 0.18 fg/s of IL-8 after 18 h in culture.

Conclusions: Theoretical and experimental demonstration of bead-based immunoassays in diffusion-limited environments such as 3D cell culture is shown, and includes example measurements of various cytokines produced by an osteosarcoma spheroid. Proper calibration and use of this assay is exhaustively explored for the case of diffusion-limited Langmuir kinetics of a spherical adsorber.

Cancer cell spheroids are the simplest 3D in vitro cancer models and have been extensively used for cancer research. More recently, models have been becoming complex, with the introduction of a matrix and non-cancer cell types to mimic specific tumour aspects. However, applying drugs or agents in matrix-embedded cancer spheroids can be problematic. Most matrices can impede and also bind drugs or visualizing agents non-specifically, in the vicinity of the embedded spheroids. This may interfere with imaging or further analysis without breaking apart the 3D model into its constituents. Here, we developed a combined gelatin-carboxymethyl cellulose (G-CMC) hydrogel for initiating cancer spheroids that enabled intact harvesting pre/post treatment for further investigation, such as targeting and imaging. We combined CMC (1.25%) and gelatin (2.5%) at 25 °C and initiated polymerisation after autoclaving (121 °C) to obtain a mechanical strength (sheer stress) of 38 Pas versus 1.28 Pas for CMC alone. These matrix conditions facilitated separation of the spheroids from the G-CMC, using low centrifugation (100 g). We described growth of colorectal and breast cancer spheroids within the G-CMC matrix (with average diameters of 220 mm and 180 μm for representative cell lines HT29 and MCF7 at 10 days, respectively). As the cancer cells express the surface biomarker calreticulin (CRT), we manufactured anti-calreticulin IgG (anti-CRT) conjugated to fluorescent gold nanoclusters (anti-CRT-AuNC) as a probe. We harvested cancer spheroids and incubated live with the nanoclusters. Imaging demonstrated strong binding of CRT-targeted AuNCs compared to control AuNCs. This novel model preserves cancer spheroid integrity upon isolation and is well suited for targeted imaging and drug delivery of cancer in 3D.

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.