Morphological dissimilarity and its evolution over time are one of the most unexpected variations found when comparing cell cultures in 2D and 3D. Monolayer cells appear to flatten in the lower part of the plate, adhering to and spreading in the horizontal plane while not extending vertically. Consequently, cells developed in two dimensions have a forced apex-basal polarity. Co-cultivation and crosstalking between multiple cell types, which control development and formation in the in vivo counterpart, are possible in 3D cultures. With or without a scaffold matrix, 3D model culture may exhibit more in vivo-like morphology and physiology. 3D cultures mimic relevant physiological cellular processes, transforming them into one-of-a-kind drug screening platforms. The structures and dynamics of regulatory networks, which are increasingly studied with live-imaging microscopy, must be considered to help and guarantee the functional maintenance of a 3D structure. However, commercially available technologies that can be used for current laboratory needs are minimal, despite the need to make it easier to acquire cellular kinetics with high spatial and temporal resolution, in order to improve visual efficiency and, as a result, experimentation performance. The CELLviewer is a newly developed multi-technology instrument that integrates and synchronizes the work of various scientific disciplines. The aim of this study is to test the device using two different models: a single Jurkat cell and an MCF-7 spheroid. The two models are loaded into the microfluidic cartridge for each experiment after they have been grown and captured in time-lapse for a total of 4 hours. The samples used are tracked under the operation of the optics after adaptive autofocus, while slipping inside the cartridge chamber, and the 3D rotation was successfully obtained experimentally. The MitoGreen dye, a fluorescence marker selectively permeable to live cells, was then used to determine cell viability. To measure the model diameter, construct fluorescence intensity graphs along a straight line passing through the cell, and visualize the spatial fluorescence intensity distribution in 3D, ImageJ software was used.

Conventional radiation therapy uses white X-rays that consist of a mixture of X-ray waves with various energy levels. In contrast, a monochromatic X-ray (monoenergetic X-ray) has a single energy level. Irradiation of high Z elements with a synchrotron generated monochromatic X-ray with the energy at or higher than the K-edge energy of the element results in the production of the Auger electrons that cause DNA damage leading to cell killing. Delivery of high Z elements into cancer cells and tumor mass can be facilitated by the use of nanoparticles. Mesoporous silica nanoparticles (MSNs) have been shown to be effective in delivering high Z elements to cancer cells. A proof of principle experiment was reported that demonstrated the feasibility of this approach. This opens up a possibility to pursue the Auger cancer therapy by the combined use of MSNs loaded with high Z elements and monochromatic X-rays. Similar cancer therapies using other types of quantum beams such as neutron, proton and carbon ion beams can be envisioned.

Cytoglobin has been implicated in a range of pathological conditions including fibrosis and cancer, where cytoglobin expression is linked to hypoxia- and drug-resistance. One disease where there is evidence of a role for cytoglobin is head and neck squamous cell carcinoma, a disease with a poor prognosis and where clinical resistance to platinum-based drugs is common. In the current study, we demonstrate that 3D-culture of head and neck cancer cells (PE/CA-PJ41, Liv-22K) and normal oral keratinocyte cells (NOK-hTERT) results in changes in sensitivity to cisplatin cytotoxicity compared to 2D-cultures. Relative to 2D-cultures, 3D-cultures of PE/CA-PJ41 and NOK-hTERT cells demonstrated increased cisplatin resistance. In contrast, 3D-cultures of Liv-22K cells were more sensitive to cisplatin. Evidence for cisplatin genotoxicity was observed in the form of GADD45A activation in PE/CA-PJ41 cells but not the other two cell types investigated. Furthermore, DNA-strand breaks were also detected by the alkaline comet assay in PE/CA-PJ41 cells although levels were not significantly affected by 3D-culture. Cytoglobin expression levels were elevated by 3D-culture in all three cell lines investigated and there were also changes in expression of genes related to cell division (MKI57, GJB6), cell adhesion (CDH1), stress response (NFkB, NQO1) and apoptosis (Casp3) but these changes were cell line specific. We also observed consistent transcriptional activation of HIF1a in 3D-cultures of all three cell lines, suggestive of hypoxic conditions in spheroids. In support of a direct role of hypoxia in cytoglobin induction, the HIF1a stabiliser cobalt chloride also induced cytoglobin expression in spheroids. Transcriptomic profiling of PE/CA-PJ41 cells over-expressing cytoglobin identified 121 differentially regulated genes, when cells were cultured under hypoxic conditions. Major changes identified included, upregulation of G1/S cell cycle regulation as well as wnt- and RhoGTPase-signalling pathways. In conclusion, 3D-cultures are a useful model to further study the biological function of cytoglobin in head and neck cancer and we provide evidence that further supports a role for cytoglobin in hypoxia-dependent cellular proliferation and phenotypic changes that could contribute to cisplatin resistance in vivo.

Three-dimensional (3D) cell culture is considered more clinically relevant in mimicking the structural and physiological conditions of tumors in vivo compared to two-dimensional cell cultures. In recent years, high-throughput screening (HTS) in 3D cell arrays has been extensively used for drug discovery because of its usability and applicability. Herein, we developed a microfluidic spheroid culture device (μFSCD) with a concentration gradient generator (CGG) that enabled cells to form spheroids and grow in the presence of cancer drug gradients. The device is composed of concave microwells with several serpentine micro-channels which generate a concentration gradient. Once the colon cancer cells (HCT116) formed a single spheroid (approximately 120 μm in diameter) in each microwell, spheroids were perfused in the presence of the cancer drug gradient irinotecan for 3 days. The number of spheroids, roundness, and cell viability, were inversely proportional to the drug concentration. These results suggest that the μFSCD with a CGG has the potential to become an HTS platform for screening the efficacy of cancer drugs.

This study aimed to assess the response of 3D printed PLA scaffolds biomimetically coated with apatite on human primary osteoblast spheroids and evaluate the biological response to its association with Bone Morphogenetic Protein 2 (rhBMP-2) in rat calvaria. PLA scaffolds were produced via 3D printing, soaked in simulated body fluid (SBF) solution, and characterized by physical-chemical, morphological, and mechanical properties. The in vitro biological response was assessed with human primary osteoblast (HOb) spheroids. The in vivo analysis was conducted through the implantation of 3D printed PLA scaffolds either alone, covered by apatite (PLA-CaP) or PLA-CaP loaded with rhBMP-2 (PLA-CaP+rhBMP-2) on critical-sized defects (8 mm) of rat calvaria. Increased cell adhesion and in vitro release of growth factors (PDGF, bFGF, VEGF) was observed for PLA-CaP scaffolds when pre-treated with FBS. PLA-CaP+BMP2 presented higher values of newly formed bone (NFB) than other groups at all experimental periods (p<0.05), attaining 44.85% of NFB after 6 months. These findings indicate that functionalization of PLA scaffolds with biomimetic apatite can improve its biological properties in the presence of complex biological media. Its association with BMP2 may enhance bone repair, suggesting this strategy as a promising candidate for bone tissue engineering.

Culture systems for 3-dimensional tissues, such as multicellular spheroids, are indispensable for high-throughput screening of primary or patient-derived xenograft (PDX)-expanded cancer tissues. Oxygen supply to the center of such spheroids is particularly critical for maintaining cellular functions as well as avoiding the development of a necrotic core. In this study, we evaluated 2 methods to enhance oxygen supply: (1) using culture plate with gas-permeable polydimethylsiloxane (PDMS) membrane at its bottom and (2) embedding hydrogel beads in the spheroids. Culturing spheroids on PDMS increased cell growth and affected glucose/lactate metabolism and CYP3A4 mRNA expression and subsequent enzyme activity. The spheroids comprised 5000 Hep G2 cells and 5000 20 µm-diameter hydrogel beads did not develop a necrotic core for 9 days when cultured on a gas-permeable sheet. In contrast, central necrosis in spheroids lacking hydrogel beads was observed after day 3 of culture, even when using PDMS. These results indicate that the combination of gas-permeable culture equipment and embedded hydrogel beads improves culture 3D spheroids produced from primary or PDX-expanded tumor cells.

Tri-dimensional (3D) cell aggregates or spheroids are considered to be closer to physiological conditions than traditional 2D cell culture. Mesenchymal stem cells (MSCs) assembling in spheroids have increased the survival of transplanted cells. The regulation of the biological processes that maintain crucial physiological reactions of MSCs is closely related to the functioning of ion channels. The pattern of expression, role and regulatory mechanisms of ion channels could be significantly different in 3D compared to 2D culture, and, thus, needed to be properly analyzed on the level of ionic currents. We developed a specific approach that allowed us to register, for the first time, endogenous ion channels in endometrial MSCs (eMSCs) assembled in spheroids. Particularly, using the single-channel patch-clamp technique, we have recorded the activity of ion channels and observed their functional interplay in mechanosensitive clusters. Our experimental protocol could be applied for identification and studying of ion channels in 3D cell cultures.

Interferon gamma (IFNg) is a pleiotropic cytokine that can potentially reprogramme the tumour microenvironment. However, the antitumour immunomodulatory properties of IFNg still need to be validated due to variable therapeutic outcomes in preclinical and clinical studies. We developed a replication-deficient Semliki Forest virus vector expressing IFNg (SFV/IFNg) and evaluated its immunomodulatory antitumour potential in vitro in a model of 3D spheroids and in vivo in immunocompetent 4T1 mouse breast cancer model. We demonstrated that SFV-derived IFN-g stimulated bone marrow macrophages to acquire the tumoricidal M1 phenotype in 3D nonattached conditions. Coculturing SFV/IFNg-infected 4T1 spheroids with BMDMs inhibited spheroid growth. In the orthotopic 4T1 mouse model, intratumoural administration of SFV/IFNg virus particles alone or in combination with the Pam3CSK4 TLR2/1 ligand led to significant inhibition of tumour growth compared to the administration of the control SFV/Luc virus particles. Analysis of the composition of intra-tumoural lymphoid cells isolated from tumours after SFV/IFNg treatment revealed an increase in CD4+ and CD8+ and a decrease in T-reg (CD4+/CD25+/FoxP3+) cell populations. Furthermore, a significant decrease in the populations of cells bearing myeloid cell markers CD11b, CD38 and CD206 was observed. In conclusion, the SFV/IFNg vector induces a therapeutic antitumour T-cell response and inhibits myeloid cell infiltration in treated tumours.

Three-dimensional (3D) cell culture systems can be regarded as suitable platforms to bridge the huge gap between animal studies and two-dimensional (2D) monolayer cell culture to study chronic diseases such as cancer. In particular, the preclinical platforms for multicellular spheroid formation and culture can be regarded as ideal in vitro tumor models. The complex tumor microenvironment such as hypoxic region and necrotic core can be recapitulated in 3D spheroid configuration. Cells aggregated in spheroid structures can better illustrate the performance of anti-cancer drugs as well. Various methods have been proposed so far to create such 3D spheroid aggregations. Both conventional techniques and microfluidic methods can be used for generation of multicellular spheroids. In this review paper, we first discuss various spheroid formation phases. Then, the conventional spheroid formation techniques such as bioreactor flasks, liquid overlay and hanging droplet technique are explained. Next, a particular topic of the hydrogel in spheroid formation and culture is explored. This topic has received less attention in the literature. Hydrogels entail some advantages to the spheroid formation and culture such as size uniformity, the formation of porous spheroids or hetero-spheroids as well as chemosensitivity and invasion assays and protecting from shear stress. Finally, microfluidic methods for spheroid formation and culture are briefly reviewed.

Gastroenteropancreatic neuroendocrine neoplasms (GEP NENs) are rare cancers consisting of neuroendocrine carcinomas (NECs) and neuroendocrine tumors (NETs), which have been increasing in incidence in recent years. Few cell lines and pre-clinical models exist for the study of GEP NECs and NETs, limiting the ability to discover novel imaging and treatment modalities. To address this gap, we isolated tumor cells from cryopreserved patient GEP NECs and NETs and injected them into the flanks of immunocompromised mice to establish patient-derived xenograft (PDX) models. Two of 6 mice developed tumors (NEC913 and NEC1452). Over 90% of NEC913 and NEC1452 tumor cells stained positive for Ki67. NEC913 PDX tumors expressed neuroendocrine markers such as chromogranin A (CgA), synaptophysin (SYP), and somatostatin receptor-2 (SSTR2) whereas NEC1452 PDX tumors do not express SSTR2. Exome sequencing revealed loss of p53 and RB1 in both tumors. To demonstrate an application of these novel NEC PDX models for SSTR2-targeted peptide imaging, the NEC913 and NEC1452 cells were bilaterally injected into mice. Near infrared-labelled octreotide was administered and fluorescent signal was specifically observed for the NEC913 SSTR2 positive tumors. These 2 GEP NEC PDX models serve as valuable resource for GEP NEN therapy testing.

We propose a non-invasive approach to control the quality of spheroids and their fusion into a complex bioconstruct. The proposed method is based on the union of the nutrient concentration change calculated using Fick's law and the "reaction-diffusion" equations, taking into account absorption coefficient with specifying the exact fusion geometry using implicit Function Repre-sentation (FRep) functions. The proposed approach allows us to analyze the viability of cells within the spheroid, predict the fusion of spheroids, and accurately model complex heteroge-neous biostructures as a future task for our research. These results will significantly accelerate the development of such a promising field of additive biotechnologies as 3D bioprinting.

Does cell clustering influence intrinsic and acquired multi-drug resistance (MDR) differently? To address this question, we studied cultured monolayers (representing individual cells) and cultured spheroids (representing clusters) formed by drug-naïve (intrinsic MDR) and drug-exposed (acquired MDR) lines of ovarian cancer A2780 cells by cytometry of reaction rate constant (CRRC). MDR efflux was characterized by accurate and robust “cell number vs. MDR efflux rate constant (kMDR)” histograms. Both drug-naïve and drug-exposed monolayer cells presented unimodal histograms; the histogram of drug-exposed cells was shifted towards higher kMDR value suggesting greater MDR activity. Spheroids of drug-naïve cells presented a bimodal histogram indicating the presence of two subpopulations with different MDR activity. In contrast, spheroids of drug-exposed cells presented a unimodal histogram qualitatively similar to that of the monolayers of drug-exposed cells but with a moderate shift towards greater MDR activity. The observed greater effect of cell clustering on intrinsic than on acquired MDR can help guide the development of new therapeutic strategies targeting clusters of circulating tumor cells.