Organotypic lung tissue culture as a preclinical model to study host- influenza A viral infection: A case for repurposing of nafamostat mesylate.

Reliable and effective models for recapitulation of host-pathogen interactions are imperative for the discovery of potential therapeutics. Ex vivo models can fulfill these requirements as the multicellular native environment in the tissue is preserved and be utilized for toxicology, vaccine, infection and drug efficacy studies due to the presence of immune cells. Drug repurposing involves the identification of new applications for already approved drugs that are not related to the prime medical indication and emerged as a strategy to cope with slow pace of drug discovery due to high costs and necessary phases to reach the patients. Within the scope of the study, broad-spectrum serine protease inhibitor nafamostat mesylate was repurposed to inhibit influenza A infection and evaluated by a translational ex vivo organotypic model, in which human organ-level responses can be achieved in preclinical safety studies of potential antiviral agents, along with in in vitro lung airway culture. The safe doses were determined as 10 µM for in vitro, whereas 22 µM for ex vivo to be applied for evaluation of host-pathogen interactions, which reduced virus infectivity, increased cell/tissue viability, and protected total protein content by reducing cell death with the inflammatory response. When the gene expression levels of specific pro-inflammatory, anti-inflammatory and cell surface markers involved in antiviral responses were examined, the significant inflammatory response represented by highly elevated mRNA gene expression levels of cytokines and chemokines combined with CDH5 downregulated by 5.1-fold supported the antiviral efficacy of NM and usability of ex vivo model as a preclinical infection model.

Receptor mediated targeting of EGF-conjugated alginate-PAMAM nanoparticles to lung adenocarcinoma: 2D/3D in vitro and in vivo evaluation.

Carboplatin (cis-diamine (1,1-cyclobutandicarboxylaso)­platinum (II)) is a second-generation antineoplastic drug, which is widely used for chemotherapy of lung, colon, breast, cervix, testicular and digestive system cancers. Although preferred over cisplatin due to the lower incidence of nephrotoxicity and ototoxicity, efficient carboplatin delivery remains as a major challenge. In this study, carboplatin loaded alginate- poly(amidoamine) (PAMAM) hybrid nanoparticles (CAPs) with mean sizes of 192.13 ± 4.15 nm were synthesized using a microfluidic platform, then EGF was conjugated to the surface of CAPs (EGF-CAPs) for the receptor-targeted delivery. Hence, increased FITC+ cell counts were observed in A549 spheroids after EGF-CAP treatment compared to CAP in the 3D cellular uptake study. As such, the cytotoxicity of EGF-CAP was approximately 2-fold higher with an IC50 value of 35.89 ± 10.37 µg/mL compared to the CAPs in A549 spheroids. Based on in vivo experimental animal model, anti-tumor activities of the group treated with CAP decreased by 61 %, whereas the group treated with EGF-CAP completely recovered. Additionally, EGF-CAP application was shown to induce apoptotic cell death. Our study provided a new strategy for designing a hybrid nanoparticle for EGFR targeted carboplatin delivery with improved efficacy both in vitro and in vivo applications.

Humanized brain organoids-on-chip integrated with sensors for screening neuronal activity and neurotoxicity.

The complex structure and function of the human central nervous system that develops from the neural tube made in vitro modeling quite challenging until the discovery of brain organoids. Human-induced pluripotent stem cells-derived brain organoids offer recapitulation of the features of early human neurodevelopment in vitro, including the generation, proliferation, and differentiation into mature neurons and micro-macroglial cells, as well as the complex interactions among these diverse cell types of the developing brain. Recent advancements in brain organoids, microfluidic systems, real-time sensing technologies, and their cutting-edge integrated use provide excellent models and tools for emulation of fundamental neurodevelopmental processes, the pathology of neurological disorders, personalized transplantation therapy, and high-throughput neurotoxicity testing by bridging the gap between two-dimensional models and the complex three-dimensional environment in vivo. In this review, we summarize how bioengineering approaches are applied to mitigate the limitations of brain organoids for biomedical and clinical research. We further provide an extensive overview and future perspectives of the humanized brain organoids-on-chip platforms with integrated sensors toward brain organoid intelligence and biocomputing studies. Such approaches might pave the way for increasing approvable clinical applications by solving their current limitations.

Differentiation of Neurons, Astrocytes, Oligodendrocytes and Microglia From Human Induced Pluripotent Stem Cells to Form Neural Tissue-On-Chip: A Neuroinflammation Model to Evaluate the Therapeutic Potential of Extracellular Vesicles Derived from Mesenchymal Stem Cells.

Advances in stem cell (SC) technology allow the generation of cellular models that recapitulate the histological, molecular and physiological properties of humanized in vitro three dimensional (3D) models, as well as production of cell-derived therapeutics such as extracellular vesicles (EVs). Improvements in organ-on-chip platforms and human induced pluripotent stem cells (hiPSCs) derived neural/glial cells provide unprecedented systems for studying 3D personalized neural tissue modeling with easy setup and fast output. Here, we highlight the key points in differentiation procedures for neurons, astrocytes, oligodendrocytes and microglia from single origin hiPSCs. Additionally, we present a well-defined humanized neural tissue-on-chip model composed of differentiated cells with the same genetic backgrounds, as well as the therapeutic potential of bone marrow mesenchymal stem cells (BMSCs)-derived extracellular vesicles to propose a novel treatment for neuroinflammation derived diseases. Around 100 nm CD9 + EVs promote a more anti-inflammatory and pro-remodeling of cell-cell interaction cytokine responses on tumor necrosis factor-α (TNF-α) induced neuroinflammation in neural tissue-on-chip model which is ideal for modeling authentic neural-glial patho-physiology.

Comprehensive Transcriptomic Investigation of Rett Syndrome Reveals Increasing Complexity Trends from Induced Pluripotent Stem Cells to Neurons with Implications for Enriched Pathways.

Rett syndrome (RTT) is a rare genetic neurodevelopmental disorder that has no cure apart from symptomatic treatments. While intense research efforts are required to fulfill this unmet need, the fundamental challenge is to obtain sufficient patient data. In this study, we used human transcriptomic data of four different sample types from RTT patients including induced pluripotent stem cells, differentiated neural progenitor cells, differentiated neurons, and postmortem brain tissues with an increasing in vivo-like complexity to unveil specific trends in gene expressions across the samples. Based on DEG analysis, we identified F8A3, CNTN6, RPE65, and COL19A1 to have differential expression levels in three sample types and also observed previously reported genes such as MECP2, FOXG1, CACNA1G, SATB2, GABBR2, MEF2C, KCNJ10, and CUX2 in our study. Considering the significantly enriched pathways for each sample type, we observed a consistent increase in numbers from iPSCs to NEUs where MECP2 displayed profound effects. We also validated our GSEA results by using single-cell RNA-seq data. In WGCNA, we elicited a connection among MECP2, TNRC6A, and HOXA5. Our findings highlight the utility of transcriptomic analyses to determine genes that might lead to therapeutic strategies.

Spatio-temporal dynamics enhance cellular diversity, neuronal function and further maturation of human cerebral organoids.

The bioengineerined and whole matured human brain organoids stand as highly valuable three-dimensional in vitro brain-mimetic models to recapitulate in vivo brain development, neurodevelopmental and neurodegenerative diseases. Various instructive signals affecting multiple biological processes including morphogenesis, developmental stages, cell fate transitions, cell migration, stem cell function and immune responses have been employed for generation of physiologically functional cerebral organoids. However, the current approaches for maturation require improvement for highly harvestable and functional cerebral organoids with reduced batch-to-batch variabilities. Here, we demonstrate two different engineering approaches, the rotating cell culture system (RCCS) microgravity bioreactor and a newly designed microfluidic platform (µ-platform) to improve harvestability, reproducibility and the survival of high-quality cerebral organoids and compare with those of traditional spinner and shaker systems. RCCS and µ-platform organoids have reached ideal sizes, approximately 95% harvestability, prolonged culture time with Ki-67 + /CD31 + /ß-catenin+ proliferative, adhesive and endothelial-like cells and exhibited enriched cellular diversity (abundant neural/glial/ endothelial cell population), structural brain morphogenesis, further functional neuronal identities (glutamate secreting glutamatergic, GABAergic and hippocampal neurons) and synaptogenesis (presynaptic-postsynaptic interaction) during whole human brain development. Both organoids expressed CD11b + /IBA1 + microglia and MBP + /OLIG2 + oligodendrocytes at high levels as of day 60. RCCS and µ-platform organoids showing high levels of physiological fidelity a high level of physiological fidelity can serve as functional preclinical models to test new therapeutic regimens for neurological diseases and benefit from multiplexing.

Aligned with sustainable development goals: microwave extraction of astaxanthin from wet algae and selective cytotoxic effect of the extract on lung cancer cells.

Astaxanthin is one of the most attractive carotenoid in the cosmetic, food, pharmaceutical, and aquaculture industries due to its strong bioactive properties. Among the various sources, several algae species are considered as rich sources of astaxanthin. Downstream processing of algae involves the majority of the total processing costs. Thus, elimination of high energy involved steps is imperative to achieve cost-effective scale in industry. This study aimed to determine operation conditions for astaxanthin extraction from wet Haematococcus pluvialis using microwave-assisted extraction. The isolated astaxanthin extract was evaluated for cytotoxicity on human lung cancer cells. The microwave-assisted extraction process at 75 °C under the power of 700 Watt for 7 min gave the highest astaxanthin yield (12.24 ± 0.54 mg astaxanthin/g wet cell weight). Based on MTT cell viability and Hoechst 33342 nuclear staining assays on A549 lung cancer cells, astaxanthin inhibited cell growth in dose- and time-dependent manners, where IC50 value was determined as 111.8 ± 14.8 µg/mL and apoptotic bodies were observed along with positive control group at 72 hr. These results showed that the treatment with astaxanthin extracted from wet H. pluvialis by microwave-assisted extraction exhibited anti-cancer activity on lung cancer cells indicating a newly potential to be utilized in industry.

Essential Oil and Supercritical Carbon Dioxide Extract of Grapefruit Peels Formulated for Candida albicans Infections: Evaluation by an in Vitro Model to Study Fungal-Host Interactions.

Resistance to currently available antifungal agents raises the need to develop alternative remedies. Candida albicans is the most common opportunistic pathogenic fungus of humans, colonizing in the genital and intestinal mucosa, skin, and oral-nasal cavity and reducing quality of life. Herein, essential oil from grapefruit (Citrus paradise) peels was obtained by hydrodistillation, and the remaining plant material was sequentially subjected to supercritical carbon dioxide (SC-CO2) extraction to determine the conditions for maximizing phenolic compounds. A statistical design was used to evaluate the effect of temperature (30, 50, 70 °C), pressure (80, 150, 220 bar), and ethanol as a cosolvent (0%, 10%, and 20% v/v). Essential oil and SC-CO2 extracts were mixed at various ratios to develop an effective antifungal formulation. Subsequently, fungal infection was modeled by coculturing C. albicans with human skin keratinocytes (HaCaT) to mimic dermal mycoses, endothelial cells (HUVEC) to evaluate vascular fate, and cervical adenocarcinoma (HeLa) cells to represent additional genital mycoses. Treatment with essential oil and extract (25:75%) formulation for 8 h exhibited slight cytotoxicity toward HeLa cells, no toxicity toward HaCaT and HUVECs, whereas inhibition of C. albicans. Considering the clinical significance, such in vitro models are essential to screen potential compounds for the treatment of opportunistic fungal infections.

Bilayered laponite/alginate-poly(acrylamide) composite hydrogel for osteochondral injuries enhances macrophage polarization: An in vivo study.

Addressing osteochondral defects, the objective of current study was to synthesize bilayered hydrogel, where the cartilage layer was formed by alginate (Alg)-polyacrylamide (PAAm) with and without the addition of TGF-ß3 and bone layer by laponite XLS/Alg-PAAm and characterize by in vitro and in vivo experiments. Exceeding the mechanical strength of Alg-PAAm (32.95 ± 1.23 kPa) and XLS based (317.5 ± 21.72 kPa) hydrogels, XLS/Alg-PAAm hydrogel (469.7 ± 6.1 kPa) activated macrophages towards M2 phenotype and stimulated the expression of anti-inflammatory factors. The addition of TGF-ß3 accelerated transition of macrophage polarization, especially between day 4 and 7. The expression levels of M1-related genes such as CD80, iNOS and TNF-α decreased gradually after day 4, reaching lowest values at day 13, whereas the expression levels of M2-related genes, CD206, Arg1 and STAT6 significantly increased promoting M2 macrophage polarization, which might be associated with accelerated bone repair. Moreover, bilayer structure exhibited a better cell viability as well as repairment thorough the XLS contents. In vivo histological examinations verified the significant surface regularity and hyaline like tissue formation employment, along with synchronized degradation profile of the hydrogel with tissue healing at the end of 12 weeks. A mechanically durable, biocompatible and immunocompatible hydrogel was formulated to be utilized in bone-cartilage engineering applications.

Immunogenicity of a xenogeneic multi-epitope HER2+ breast cancer DNA vaccine targeting the dendritic cell restricted antigen-uptake receptor DEC205.

Breast cancer was ranked first in global cancer incidence in 2020, and HER2 overexpression in breast cancer accounts for 20-30% of breast cancer patients. Current therapeutic strategies increase the survival rate, but resistance to them occurs frequently, and there is an urgent need to develop novel treatments such as DNA vaccines which can induce a specific and long-lasting immune response against HER2 antigens. To enhance the immunogenicity of DNA vaccines, dendritic cells (DCs) can be targeted using multi-epitope proteins that provide accurate immune focusing. For this purpose, we generated a DNA vaccine encoding a fusion protein composed of 1) in silico discovered antigenic epitopes of human and rat HER2 proteins (MeHer2) and 2) a single-chain antibody fragment (ScFv) specific for the DC-restricted antigen-uptake receptor DEC205 (ScFvDEC). The xenogeneic multi-epitope DNA vaccine (pMeHer2) encodes three only T-cell epitopes, two only B-cell epitopes, and two T and B cell epitopes, and pScFvDEC-MeHer2 vaccine additionally encodes ScFvDEC introduced at the N terminus of the MeHer2. Then, mouse groups were immunized with pScFvDEC-MeHer2, pMeHer2, pScFvDEC, pEmpty, and PBS to determine the elicited immune response. pScFvDEC-MeHer2 vaccinated mice showed a strong IgG response (P < 0.0001) and pScFvDEC-MeHer2 induced a significant IgG2a increase (P < 0.01). The percentages of both IFN-γ secreting CD4 and CD8 T cells were higher in mice immunized with pScFvDEC-MeHer2 compared with the pMeHer2. pScFvDEC-MeHer2 and pMeHer2 secreted significantly higher levels of extracellular IFN-γ compared with to control groups (P < 0.0001). In addition, the IFN-γ level of the pScFvDEC-MeHer2 vaccine group was approximately two times higher than the pMeHer2 group (P < 0.0001). Overall, this study identified the pScFvDECMeHer2 construct as a potential DNA vaccine candidate, supporting further studies to be conducted on HER2+ animal models.

Decellularised extracellular matrix-based biomaterials for repair and regeneration of central nervous system.

The central nervous system (CNS), consisting of the brain and spinal cord, regulates the mind and functions of the organs. CNS diseases, leading to changes in neurological functions in corresponding sites and causing long-term disability, represent one of the major public health issues with significant clinical and economic burdens worldwide. In particular, the abnormal changes in the extracellular matrix under various disease conditions have been demonstrated as one of the main factors that can alter normal cell function and reduce the neuroregeneration potential in damaged tissue. Decellularised extracellular matrix (dECM)-based biomaterials have been recently utilised for CNS applications, closely mimicking the native tissue. dECM retains tissue-specific components, including proteoglycan as well as structural and functional proteins. Due to their unique composition, these biomaterials can stimulate sensitive repair mechanisms associated with CNS damages. Herein, we discuss the decellularisation of the brain and spinal cord as well as recellularisation of acellular matrix and the recent progress in the utilisation of brain and spinal cord dECM.

Supercritical carbon dioxide dried double layer laponite XLS and alginate/polyacrylamide construct and immune response.

Fabrication of immunocompatible tissue constructs for bone-cartilage defect regeneration is of prime importance. In this study, a double layer hydrogel was successfully synthesized, where alginate/polyacrylamide were formulated to represent cartilage layer (5-10 % (w/w) total polymer ratio) and laponite XLS (2-5-8% (w/w))/alginate/polyacrylamide formed bone layer. Hydrogels were dried by supercritical CO2 at 100 and 200 bar, 45 °C, 5 g/min CO2 flow rate for 2 h. Constructs were treated with collagen, then cellularized and embedded in cell-laden GelMA to mimic the cellular microenvironment. The optimum weight ratio of alginate/polyacrylamide:laponite XLS was 10:5 based on mechanical strength test results. The constructs yielded high porosity (91.50 m2/g) and mesoporous structure, owing to the diffusivity of CO2 at 200 bar (0.49 × 10-7 m2/s). Constructs were then treated with collagen to increase cell adhesion and ATDC5 cells were seeded in the cartilage layer, whereas hFOB cells to the bone layer. About 10-15 % higher cell viability was attained. The porous structure of the construct allowed infiltration of macrophages, promoted polarization and positively affected the behavior of macrophages, yielding a decrease in M1 markers, whereas an increase in M2 on day 4. The formulated tissue constructs would be of value in tissue engineering applications.

Antimicrobial and wound healing properties of cotton fabrics functionalized with oil-in-water emulsions containing Pinus brutia bark extract and Pycnogenol® for biomedical applications.

Topical formulations containing 1-2% of Pinus brutia bark extract and Pycnogenol® have been prepared to investigate the effect of flavonoids on the stability of O/W emulsions, which were subjected to physicochemical and thermal stability tests. The formulations have been applied to cotton fabrics to evaluate antimicrobial properties against Staphylococcus aureus, Escherichia coli, Candida albicans and Aspergillus brasiliensis. Furthermore, prepared cotton fabrics have been tested on keratinocytes seeded in cell culture inserts for wound healing. Results of freeze thaw cycle test indicated enhanced thermo-stability with no major changes in pH and viscosity, likewise the results of centrifugation assay. However, the addition of Pycnogenol® has tremendously decreased the viscosity of the topical formulation (10,900 cp.). In terms of antimicrobial activity, 2% P. brutia treated cotton fabrics decreased the proliferation of Aspergillus brasiliensis 78.8%, which were more effective than that of Pycnogenol® formulation (62.9%). As for wound healing, 2% P. brutia treated cotton fabrics increased HaCaT keratinocyte cell proliferation and accelerated the cell-free gap closure compared to Pycnogenol® and untreated control groups. The obtained results indicate the utilization of pine bark for developing an eco-friendly natural antifungal finish for medical textiles.

Lung carcinoma spheroids embedded in a microfluidic platform.

Three-dimensional (3D) spheroid cell cultures are excellent models used in cancer biology research and drug screening. The objective of this study was to develop a lung carcinoma spheroid based microfluidic platform with perfusion function to mimic lung cancer pathology and investigate the effect of a potential drug molecule, panaxatriol. Spheroids were successfully formed on agar microtissue molds at the end of 10 days, reaching an average diameter of about 317.18 ± 4.05 µm and subsequently transferred to 3D dynamic microfluidic system with perfusion function. While the size of the 3D spheroids embedded in the Matrigel matrix in the platform had gradually increased both in the static and dynamic control groups, the size of the spheroids were reduced and fragmented in the drug treated groups. Cell viability results showed that panaxatriol exhibited higher cytotoxic effect on cancer cells than healthy cells and the IC50 value was determined as 61.55 µM. Furthermore, panaxatriol has been more effective on single cells around the spheroid structure, whereas less in 3D spheroid tissues with a compact structure in static conditions compared to dynamic systems, where a flow rate of 2 µL/min leading to a shear stress of 0.002 dyne/cm2 was applied. Application of such dynamic systems will contribute to advancing basic research and increasing the predictive accuracy of potential drug molecules, which may accelerate the translation of novel therapeutics to the clinic, possibly decreasing the use of animal models. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00470-7.

An alginate-poly(acrylamide) hydrogel with TGF-ß3 loaded nanoparticles for cartilage repair: Biodegradability, biocompatibility and protein adsorption.

Current implantable materials are limited in terms of function as native tissue, and there is still no effective clinical treatment to restore articular impairments. Hereby, a functionalized polyacrylamide (PAAm)-alginate (Alg) Double Network (DN) hydrogel acting as an articular-like tissue is developed. These hydrogels sustain their mechanical stability under different temperature (+4 °C, 25 °C, 40 °C) and humidity conditions (60% and 75%) over 3 months. As for the functionalization, transforming growth factor beta-3 (TGF-ß3) encapsulated (NPTGF-ß3) and empty poly(lactide-co-glycolide) (PLGA) nanoparticles (PLGA NPs) are synthesized by using microfluidic platform, wherein the mean particle sizes are determined as 81.44 ± 9.2 nm and 126 ± 4.52 nm with very low polydispersity indexes (PDI) of 0.194 and 0.137, respectively. Functionalization process of PAAm-Alg hydrogels with ester-end PLGA NPs is confirmed by FTIR analysis, and higher viscoelasticity is obtained for functionalized hydrogels. Moreover, cartilage regeneration capability of these hydrogels is evaluated with in vitro and in vivo experiments. Compared with the PAAm-Alg hydrogels, functionalized formulations exhibit a better cell viability. Histological staining, and score distribution confirmed that proposed hydrogels significantly enhance regeneration of cartilage in rats due to stable hydrogel matrix and controlled release of TGF-ß3. These findings demonstrated that PAAm-Alg hydrogels showed potential for cartilage repair and clinical application.

One-Step Microfluidic Coating of Phospholipid Microbubbles with Natural Alginate Polymer as a Delivery System for Human Epithelial Lung Adenocarcinoma.

In this study, the neoplastic drug frequently used in the treatment of lung cancer, carboplatin is loaded to microbubbles via a microfluidic platform. In order to increase the drug loading capacity of microbubbles, carboplatin is encapsulated into alginate polymer layer. The phospholipid microbubbles (MBs) are synthesized by MicroSphere Creator, which is connected with T-junction and micromixer for the treatment with CaCl2 solution to provide gelation of the alginate coated phospholipid microbubbles (AMBs). The carboplatin loaded alginate coated phospholipid microbubbles (CAMBs) result in 12.2 ± 0.21 µm mean size, obtained by mixing with 0.05% CaCl2 using T-junction. The cytotoxic activities of the synthesized MBs, AMBs, and CAMBs are also investigated with the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) (MTT) and live/dead fluorescent dying assays in the A549 and BEAS-2B cell lines. The one-step microfluidic coating of lipid microbubbles with natural alginate polymer appears to be a promising strategy for enhanced drug reservoir properties.

Nano-vesicular formulation of propolis and cytotoxic effects in a 3D spheroid model of lung cancer.

BACKGROUND: Propolis exhibits therapeutic properties due to the presence of phenolic acids, esters, and flavonoids. The scope of this study was to develop a nano-vesicular formulation and establish a three-dimensional (3D) spheroid model in which lung cancer is recapitulated. RESULTS: Niosome vesicles doped with galangin-rich propolis extract were synthesized by the ether injection method using a cholesterol : surfactant mass ratio of 1 : 3 at 40 °C for 1 h. Formulated niosomes were administered to 3D lung cancer spheroid model and the cytotoxicity was compared with that of a two-dimensional (2D) setting. The galangin content was determined as 86 µg mg-1 propolis extract by ultra-performance liquid chromatography (UPLC). The particle size of loaded niosome was 151 ± 2.84 nm with a polydispersity index (PDI) of about 0.232, and an encapsulation efficiency of 70% was achieved. CONCLUSION: The decrease in cell viability and the scattering in the 3D spheroids of A549 lung cancer cells treated with propolis-loaded niosomes were notable, indicating a profound cytotoxic effect and suggesting that they can be utilized as an effective nano-vesicle. © 2020 Society of Chemical Industry.

Development and verification of a three-dimensional (3D) breast cancer tumor model composed of circulating tumor cell (CTC) subsets.

Breast cancer is one of the most common cancer types among women in which early tumor invasion leads to metastases and death. EpCAM (epithelial cellular adhesion molecule) and HER2 (human epidermal growth factor receptor 2) are two main circulating tumor cell (CTC) subsets in HER2+ breast cancer patients. In this regard, the main aim of this study is to develop and characterize a three-dimensional (3D) breast cancer tumor model composed of CTC subsets to evaluate new therapeutic strategies and drugs. For this reason, EpCAM(+) and HER2(+) sub-populations were isolated from different cell lines to establish 3D tumor model that mimics in situ (in vivo) more closely than two-dimensional (2D) models. EpCAM(+)/HER2(+) cells had a high proliferation rate and low tendency to attach to the surface in comparison with parental MDA-MB-453 cells as CTC subsets. Aggressive breast cancer subpopulations cultured in 3D porous chitosan scaffold had enhanced cell-cell and cell-matrix interactions compared to 2D cultured cells and these 3D models showed more aggressive morphology and behavior, expressed higher levels of pluripotency marker genes, Nanog, Sox2 and Oct4. For the verification of the 3D model, the effects of doxorubicin which is a chemotherapeutic agent used in breast cancer treatment were examined and increased drug resistance was determined in 3D cultures. The 3D tumor model comprising EpCAM(+)/HER2(+) CTC subsets developed in this study has a promising potential to be used for investigation of an aggressive CTC microenvironment in vitro that mimics in vivo characteristics to test new drug candidates against CTCs.

The use of Toxoplasma gondii tachyzoites produced in HeLa cells adhered to Cytodex 1 microcarriers as antigen in serological assays: an application of microcarrier technology.

Toxoplasma gondii can infect nearly all warm-blooded animals, including humans. In the laboratory diagnosis of toxoplasmosis, serological tests have importance in detecting antibody response. Traditionally T. gondii tachyzoites grown in vivo are being used as an antigen source in serological assays. Currently, tachyzoites produced in vitro are being tested as an antigen source in order to decrease animal use. Microcarrier technology allowed us to grow anchorage-dependent host cells on microcarrier suspension in short time and approximately 10 times more than traditional flask technique. The ability of T. gondii tachyzoites to grow in host cells adhered to microcarriers has not been analyzed yet. In this study, we aimed to develop a novel in vitro culture method to produce T. gondii tachyzoites abundantly using HeLa cells adhered to Cytodex 1 microcarriers. Initially, the growth of HeLa cells adhered to Cytodex 1 was analyzed using RPMI 1640, DMEM, and EMEM. Next, HeLa cells with a concentration of 1 × 105 cells/ml and 2 × 105 cells/ml were adhered to Cytodex 1 and grown in spinner flasks. Then, T. gondii tachyzoites were inoculated with 1:1 and 2:1 cell:tachyzoite ratios to HeLa cells adhered to microcarriers in spinner flaks. During continuous production in spinner flasks, tachyzoites were harvested at the 2nd, 4th, and 7th day of culture and the quality of antigens produced from these tachyzoites were tested in ELISA and Western Blotting using sera of patients with toxoplasmosis. The optimization studies showed that finest HeLa inoculation value was 2 × 105 cells/ml using RPMI 1640, and the cell:tachyzoite ratio to obtain the highest tachyzoite yield (17.1 × 107) was 1:1 at the 4th day of inoculation. According to the results of ELISA comparing HeLa cell and mouse derived antigens, the highest correlation with mouse antigen was achieved at the 4th day of HeLa cell culture with 1:1 HeLa:tachyzoite ratio (P < 0.0001). The sensitivity and specificity ratios of ELISA were 100%. In addition, Western blotting banding patterns of the antigen derived at the 4th day of HeLa cell culture with 1:1 HeLa:tachyzoite ratio was comparable with mouse derived antigen. Overall, this novel methodology can be an alternative source of antigen in diagnostic assays, decrease animal use for antigen production, and contribute to the solution of ethical and economic problems.

Bioengineering-inspired three-dimensional culture systems: Organoids to create tumor microenvironment.

In recent years, in vitro three-dimensional (3D) cell culture systems that better mimic in vivo physiology and tissue microenvironment have become important. It is used for the pre-clinical evaluation of the efficacy and safety profiles of new drug candidates developed especially for the treatment of various diseases including cancer. 3D cell culture techniques overcome the inadequacy of conventional two-dimensional (2D) cell culture models as they are much more informative than the 2D systems. And they can easily replace or reduce the number of in vivo studies which arise many ethical problems in the development of novel therapeutics. This review focuses on the organoid cultures which are 3D bioengineered cell culture systems that guide cell behavior and cell order to mimic in vivo tumor microenvironment.