Glauco R. Souza

Three-dimensional tissue culture based on magnetic cell levitation

Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies.

Three-Dimensional In Vitro Co-Culture Model of Breast Tumor using Magnetic Levitation

In this study, we investigate a novel in vitro model to mimic heterogeneous breast tumors without the use of a scaffold while allowing for cell-cell and tumor-fibroblast interactions. Previous studies have shown that magnetic levitation system under conventional culturing conditions results in the formation of three-dimensional (3D) structures, closely resembling in vivo tissues (fat tissue, vasculature, etc.). Three-dimensional heterogeneous tumor models for breast cancer were designed to effectively model the influences of the tumor microenvironment on drug efficiency. Various breast cancer cells were co-cultured with fibroblasts and then magnetically levitated. Size and cell density of the resulting tumors were measured. The model was phenotypically compared to in vivo tumors and examined for the presence of ECM proteins. Lastly, the effects of tumor stroma in the 3D in vitro model on drug transport and efficiency were assessed. Our data suggest that the proposed 3D in vitro breast tumor is advantageous due to the ability to: (1) form large-sized (millimeter in diameter) breast tumor models within 24 h; (2) control tumor cell composition and density; (3) accurately mimic the in vivo tumor microenvironment; and (4) test drug efficiency in an in vitro model that is comparable to in vivo tumors.

Using space-based investigations to inform cancer research on Earth

Experiments conducted in the microgravity environment of space are not typically at the forefront of the mind of a cancer biologist. However, space provides physical conditions that are not achievable on Earth, as well as conditions that can be exploited to study mechanisms and pathways that control cell growth and function. Over the past four decades, studies have shown how exposure to microgravity alters biological processes that may be relevant to cancer. In this Review, we explore the influence of microgravity on cell biology, focusing on tumour cells grown in space together with work carried out using models in ground-based investigations.

Three-dimensional cell culturing by magnetic levitation

Recently, biomedical research has moved toward cell culture in three dimensions to better recapitulate native cellular environments. This protocol describes one method for 3D culture, the magnetic levitation method (MLM), in which cells bind with a magnetic nanoparticle assembly overnight to render them magnetic. When resuspended in medium, an external magnetic field levitates and concentrates cells at the air-liquid interface, where they aggregate to form larger 3D cultures. The resulting cultures are dense, can synthesize extracellular matrix (ECM) and can be analyzed similarly to the other culture systems using techniques such as immunohistochemical analysis (IHC), western blotting and other biochemical assays. This protocol details the MLM and other associated techniques (cell culture, imaging and IHC) adapted for the MLM. The MLM requires 45 min of working time over 2 d to create 3D cultures that can be cultured in the long term (>7 d).

A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis

There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures.

Bottom-up assembly of hydrogels from bacteriophage and Au nanoparticles: the effect of cis- and trans-acting factors.

Hydrogels have become a promising research focus because of their potential for biomedical application. Here we explore the long-range, electrostatic interactions by following the effect of trans-acting (pH) and cis-acting factors (peptide mutation) on the formation of Au-phage hydrogels. These bioinorganic hydrogels can be generated from the bottom-up assembly of Au nanoparticles (Au NP) with either native or mutant bacteriophage (phage) through electrostatic interaction of the phage pVIII major capsid proteins (pVIII). The cis-acting factor consists of a peptide extension displayed on the pVIII that mutates the phage. Our results show that pH can dictate the direct-assembly and stability of Au-phage hydrogels in spite of the differences between the native and the mutant pVIII. The first step in characterizing the interactions of Au NP with phage was to generate a molecular model that identified the charge distribution and structure of the native and mutant pVIII. This model indicated that the mutant peptide extension carried a higher positive charge relative to the native pVIII at all pHs. Next, by monitoring the Au-phage interaction by means of optical microscopy, elastic light scattering, fractal dimension analysis as well as Uv-vis and surface plasmon resonance spectroscopy, we show that the positive charge of the mutant peptide extension favors the opposite charge affinity between the phage and Au NP as the pH is decreased. These results show the versatility of this assembly method, where the stability of these hydrogels can be achieved by either adjusting the pH or by changing the composition of the phage pVIII without the need of phage display libraries.

A three-dimensional co-culture model of the aortic valve using magnetic levitation

The aortic valve consists of valvular interstitial cells (VICs) and endothelial cells (VECs). While these cells are understood to work synergistically to maintain leaflet structure and valvular function, few co-culture models of these cell types exist. In this study, aortic valve co-cultures (AVCCs) were assembled using magnetic levitation and cultured for 3 days. Immunohistochemistry and quantitative reverse-transcriptase polymerase chain reaction were used to assess the maintenance of cellular phenotype and function, and the formation of extracellular matrix. AVCCs stained positive for CD31 and α-smooth muscle actin (αSMA), demonstrating that the phenotype was maintained. Functional markers endothelial nitric oxide synthase (eNOS), von Willebrand factor (VWF) and prolyl-4-hydroxylase were present. Extracellular matrix components collagen type I, laminin and fibronectin also stained positive, with reduced gene expression of these proteins in three dimensions compared to two dimensions. Genes for collagen type I, lysyl oxidase and αSMA were expressed less in AVCCs than in 2-D cultures, indicating that VICs are quiescent. Co-localization of CD31 and αSMA in the AVCCs suggests that endothelial-mesenchymal transdifferentiation might be occurring. Differences in VWF and eNOS in VECs cultured in two and three dimensions also suggests that the AVCCs possibly have anti-thrombotic potential. Overall, a co-culture model of the aortic valve was designed, and serves as a basis for future experiments to understand heart valve biology.

A spheroid toxicity assay using magnetic 3D bioprinting and real-time mobile device-based imaging

An ongoing challenge in biomedical research is the search for simple, yet robust assays using 3D cell cultures for toxicity screening. This study addresses that challenge with a novel spheroid assay, wherein spheroids, formed by magnetic 3D bioprinting, contract immediately as cells rearrange and compact the spheroid in relation to viability and cytoskeletal organization. Thus, spheroid size can be used as a simple metric for toxicity. The goal of this study was to validate spheroid contraction as a cytotoxic endpoint using 3T3 fibroblasts in response to 5 toxic compounds (all-trans retinoic acid, dexamethasone, doxorubicin, 5′-fluorouracil, forskolin), sodium dodecyl sulfate (+control), and penicillin-G (−control). Real-time imaging was performed with a mobile device to increase throughput and efficiency. All compounds but penicillin-G significantly slowed contraction in a dose-dependent manner (Z’ = 0.88). Cells in 3D were more resistant to toxicity than cells in 2D, whose toxicity was measured by the MTT assay. Fluorescent staining and gene expression profiling of spheroids confirmed these findings. The results of this study validate spheroid contraction within this assay as an easy, biologically relevant endpoint for high-throughput compound screening in representative 3D environments.

An unrecognized extracellular function for an intracellular adapter protein released from the cytoplasm into the tumor microenvironment

Mammalian cell membranes provide an interface between the intracellular and extracellular compartments. It is currently thought that cytoplasmic signaling adapter proteins play no functional role within the extracellular tumor environment. Here, by selecting combinatorial random peptide libraries in tumor-bearing mice, we uncovered a direct, specific, and functional interaction between CRKL, an adapter protein [with Src homology 2 (SH2)- and SH3-containing domains], and the plexin-semaphorin-integrin domain of β1 integrin in the extracellular milieu. Through assays in vitro, in cellulo, and in vivo, we show that this unconventional and as yet unrecognized protein–protein interaction between a regulatory integrin domain (rather than a ligand-binding one) and an intracellular adapter (acting outside of the cells) triggers an alternative integrin-mediated cascade for cell growth and survival. Based on these data, here we propose that a secreted form of the SH3/SH2 adaptor protein CRKL may act as a growth-promoting factor driving tumorigenesis and may lead to the development of cancer therapeutics targeting secreted CRKL.

Nanoparticle Improved Stem Cell Therapy for Erectile Dysfunction in a Rat Model of Cavernous Nerve Injury

PURPOSE Recently intracavernous injection of stem cells has garnered great interest as a potential treatment of erectile dysfunction. However, most stem cells are washed out immediately after intracavernous injection. The goal of this study was to investigate using NanoShuttle™ magnetic nanoparticles to maintain stem cells in the corpus cavernosum after intracavernous injection, thereby improving stem cell therapy of erectile dysfunction in an animal model. MATERIALS AND METHODS Adipose derived stem cells were magnetized with NanoShuttle magnetic nanoparticles to create Nano-adipose derived stem cells. A total of 24 rats underwent bilateral cavernous nerve crush and were randomly assigned to 3 groups, including adipose derived stem cells, Nano-adipose derived stem cells and Nano-adipose derived stem cells plus magnet. Cells were tracked at days 1, 3, 5 and 9 after intracavernous injection. Another 40 rats with bilateral cavernous nerve crush were randomly assigned to 4 groups, including bilateral cavernous nerve crush, bilateral cavernous nerve crush plus adipose derived stem cell intracavernous injection, bilateral cavernous nerve crush plus Nano-adipose derived stem cell intracavernous injection and bilateral cavernous nerve crush plus Nano-adipose derived stem cell intracavernous injection plus magnet. Functional testing and histological analysis were performed 4 weeks after intracavernous injection. RESULTS In the in vitro study 1) NanoShuttle magnetic nanoparticles were successfully bound to adipose derived stem cells and 2) Nano-adipose derived stem cells migrated toward the magnet. In the in vivo study 1) cell tracking showed that Nano-adipose derived stem cells were successfully retained in the corpus cavernosum using the magnet for up to 3 days while most adipose derived stem cells were washed out in other groups by day 1 after intracavernous injection, and 2) intracavernous pressure/mean arterial pressure, and αSMA (α-smooth muscle actin) and PECAM-1 (platelet endothelial cell adhesion molecule 1) expression in the Nano-adipose derived stem cell group was significantly higher than in the other groups. CONCLUSIONS Magnetization of adipose derived stem cells with NanoShuttle magnetic nanoparticles kept adipose derived stem cells in the corpus cavernosum and improved adipose derived stem cell therapy of erectile dysfunction in an animal model.