In vitro models最新文献

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.

Objective: Polycaprolactone (PCL) is a widely applied biomaterial in bone tissue engineering (BTE) due to its superior mechanical properties and biodegradability. However, the high hydrophobicity and low cell adhesion properties of PCL show limited cell interactions. Herein, we prepared the porous PCL/DBP composites with improved cell adhesion through the addition of demineralized bone powder (DBP). Three-dimensional scaffolds were fabricated by mixing various concentrations of DBP with PCL and applying non-solvent-induced phase separation (NIPS) and thermal-induced phase separation (TIPS) (N-TIPS) and solvent casting and particulate leaching (SCPL) to impart porosity.

Methods: A characteristic evaluation was performed through X-ray diffraction (XRD), morphological analysis, physicochemical analysis, bioactivity test, and mechanical test. Upon culture with mouse bone marrow stem cells (mBMSCs), proliferation and osteogenic differentiation of mBMSC were evaluated using quantitative dsDNA analysis and alkaline phosphatase (ALP) activity, respectively.

Results: The addition of DBP improved the physicochemical and mechanical properties of the scaffold and formed a large amount of hydroxyapatite (HAp). Also, cell proliferation and differentiation were increased by enhancing cell adhesion.

Conclusion: The porous PCL/DBP scaffolds could provide a favorable microenvironment for cell adhesion and be a promising biomaterial model for bone tissue engineering.

Graphical abstract:

The mechanical stimulation applied on engineered vascular constructs in perfusion bioreactors has been shown to be beneficial for their maturation. The level of mechanical stimulation applied on these constructs depends on the flow parameters of the circuit (e.g., fluid viscosity, flow rate, frequency, and pressure). As a group, these parameters are often overlooked in the literature, and they rarely meet the physiological values of the blood flow. For this reason, the level of circumferential stretching and shear stress that blood vessels experience in the human body are rarely reproduced. In this work, we reported the development of a physiologically relevant platform for (1) the in situ fabrication of vascular wall models based on collagen gel, and (2) their maturation under physiological levels of mechanical stimulation in a perfusion bioreactor (pulsatile flow rate of 100 mL/min, frequency of 1 Hz, pressure of 80-120 mmHg, and viscosity of 4 cP). One week of dynamic maturation oriented the seeded cells into the circumferential direction, increased the deposition of collagen and key elastin fiber-related proteins, and improved the mechanical properties in terms of tensile equilibrium elastic modulus (by 110%) and strength at break (by 63%) when compared to the static condition. In addition to the maturation study under selected physiologically relevant mechanical stimulation (such as adult, fetal, child, and hypertension conditions), the platform might also be used as a relevant in vitro testing system for new drugs or pro-active coating to medical devices (such as stents, endografts, and vascular prostheses) expected to trigger specific mechanisms or activities in vascular cells composing the arterial wall.