Dimensional Cell Culture Research Articles

An alveolus lung-on-a-chip model of Mycobacterium fortuitum lung infection.

Lung disease due to non-tuberculous mycobacteria (NTM) is rising in incidence. While both two dimensional cell culture and animal models exist for NTM infections, a major knowledge gap is the early responses of human alveolar and innate immune cells to NTM within the human alveolar microenvironment. Here we describe development of a humanized, three-dimensional, alveolus lung-on-a-chip (ALoC) model of Mycobacterium fortuitum lung infection that incorporates only primary human cells such as pulmonary vascular endothelial cells in a vascular channel, and type I and II alveolar cells and monocyte-derived macrophages in an alveolar channel along an air-liquid interface. M. fortuitum introduced into the alveolar channel primarily infected macrophages, with rare bacteria inside alveolar cells. Bulk-RNA sequencing of infected chips revealed marked upregulation of transcripts for cytokines, chemokines and secreted protease inhibitors (SERPINs). Our results demonstrate how a humanized ALoC system can identify critical early immune and epithelial responses to M. fortuitum infection. We envision potential application of the ALoC to other NTM and for studies of new antibiotics.

Actively targeted photodynamic therapy in multicellular colorectal cancer spheroids via functionalised gold nanoparticles

Photodynamic therapy (PDT) holds great potential to overcome limitations associated with common colorectal cancer (CRC) treatment approaches. Targeted photosensitiser (PS) delivery systems using nanoparticles (NPs) with targeting moieties are continually being designed, which are aimed at enhancing PS efficacy in CRC PDT. However, the optimisation of targeted PS delivery systems in most, in vitro PDT studies has been conducted on two dimensional (2D) monolayers cell cultures. In our present study, we developed a nano PS delivery system for in vitro cultured human colorectal three-dimensional multicellular spheroids (3D MCTS). PEGylated gold nanoparticles (PEG-AuNPs) were prepared and attached to ZnPcS4PS and further functionalised with specific CRC targeting anti-Guanylate Cyclase monoclonal antibodies(mAb). The ZnPcS4-AuNP-Anti-GCC Ab (BNC) nanoconjugates were successfully synthesised and their photodynamic effect investigated following exposure to laser irradiation and demonstrated enhanced anticancer effects in Caco-2 cells cultivated as 3D MCTS spheroids. Our findings suggest that targeted BNC nanoconjugates can improve the efficacy of PDT and highlight the potential of 3D MCTS tumour model for evaluating of targeted PDT.

Biomimetic Scaffolds-A Novel Approach to Three Dimensional Cell Culture Techniques for Potential Implementation in Tissue Engineering.

Biomimetic scaffolds imitate native tissue and can take a multidimensional form. They are biocompatible and can influence cellular metabolism, making them attractive bioengineering platforms. The use of biomimetic scaffolds adds complexity to traditional cell cultivation methods. The most commonly used technique involves cultivating cells on a flat surface in a two-dimensional format due to its simplicity. A three-dimensional (3D) format can provide a microenvironment for surrounding cells. There are two main techniques for obtaining 3D structures based on the presence of scaffolding. Scaffold-free techniques consist of spheroid technologies. Meanwhile, scaffold techniques contain organoids and all constructs that use various types of scaffolds, ranging from decellularized extracellular matrix (dECM) through hydrogels that are one of the most extensively studied forms of potential scaffolds for 3D culture up to 4D bioprinted biomaterials. 3D bioprinting is one of the most important techniques used to create biomimetic scaffolds. The versatility of this technique allows the use of many different types of inks, mainly hydrogels, as well as cells and inorganic substances. Increasing amounts of data provide evidence of vast potential of biomimetic scaffolds usage in tissue engineering and personalized medicine, with the main area of potential application being the regeneration of skin and musculoskeletal systems. Recent papers also indicate increasing amounts of in vivo tests of products based on biomimetic scaffolds, which further strengthen the importance of this branch of tissue engineering and emphasize the need for extensive research to provide safe for humansbiomimetic tissues and organs. In this review article, we provide a review of the recent advancements in the field of biomimetic scaffolds preceded by an overview of cell culture technologies that led to the development of biomimetic scaffold techniques as the most complex type of cell culture.

Low-molecular-weight fucoidan inhibits the proliferation of melanoma via Bcl-2 phosphorylation and PTEN/AKT pathway.

Fucoidan, a sulfate polysaccharide obtained from brown seaweed, has various bioactive properties, including anti-inflammatory, anti-cancer, anti-viral, anti-oxidant, anti-coagulant, anti-thrombotic, anti-angiogenic, and anti-Helicobacter pylori properties. However, the effects of low-molecular-weight fucoidan (LMW-F) on melanoma cell lines and three dimensional (3D) cell culture models are not well understood. This study aimed to investigate the effects of LMW-F on A375 human melanoma cells and cryopreserved biospecimens derived from patients with advanced melanoma. Ultrasonic wave was used to fragment fucoidan derived from Fucus vesiculosus into smaller LMW-F. MTT and live/dead assays showed that LMW-F inhibited cell proliferation in both A375 cells and patient-derived melanoma explants in a 3D-printed collagen scaffold. The PTEN/AKT pathway was found to be involved in the anti-melanoma effects of fucoidan. Western blot analysis revealed that LMW-F reduced the phosphorylation of Bcl-2 at Thr 56, which was associated with the prevention of anti-apoptotic activity of cancer cells. Our findings suggested that LMW-F could enhance anti-melanoma chemotherapy and improve the outcomes of patients with melanoma resistance.

The promoter effect of laminin-derived IKVAV peptide on three dimensional HUVEC microtissue

Tissue engineering research is recently a popular field but the vascularization process of existing methods limits the study area. Human Umbilical Vein Endothelial Cells (HUVEC) are essential cell models for vascularization study in vitro. Although studies about vascular biomaterial are mostly performed in traditional 2 Dimensional (D) cell culture, the system has some disadvantages. However, 3D scaffold-free microtissue can be used to overcome these disadvantages for the identification of the optimum concentration of biomaterials. IKVAV is an active unit of laminin which is an effective protein in the extracellular matrix. IKVAV may increase cell adhesion, proliferation, migration, and cellular differentiation. Since IKVAV directly affects endothelial cells, the definition of the optimum concentration of IKVAV is critically important for HUVEC growth and viability during vascularization. Thus, the study aimed identification of the optimal IKVAV peptide concentration for the production and viability of 3D HUVEC SFM. After peptide synthesis, 3D SFM was fabricated. 0.5 mM and 1 mM concentrations of IKVAV peptide were treated with SFM. The control group was incubated without any peptide concentration. Diameters and viabilities of SFMs were evaluated. 1 mM concentration showed the highest diameter and viability. The increasing concentrations may support HUVEC growth and viability so it may induce vascularization in vivo conditions.

Low-Cost ITO Microelectrodes for Bio-Sensing and Impedance Based Cellular Assays

Excellent electronic and optical properties make Indium tin oxide (ITO) an attractive electrode substrate.1 It is an alloy of indium oxide (In2O3) and tin oxide (SnO2) in a weight ratio of 90:10. It is transparent as thin film, so, it is also good to be viewed under different types of microscopes used in cell-culture studies. Despite the commercial availability of finest quality ITO and some low-cost methods for direct deposition being in use by now, definition of patterns on them is still a concern. Placing its popularity and extensive usage aside, the manufacturing of ITO electrodes so far lacks a rapid, proficient, highly reproducible, flexible, cost-effective, easy patterning process that could surpass non-trivial, time consuming techniques as lithography. A promising advancement towards achieving these limitations was the use of lasers for the fabrication of the ITO electrodes and related structure. Even though several laser techniques are explored, with the exception of CO2 lasers, none of the others provides the benefit of being widely available and comparatively inexpensive means for fabrication of microelectrode sensors. Presently, these are the highest-power continuous wave lasers available. With their high efficiency, they are usually employed in laser cutting, welding, drilling and surface treatment.2 A cost-effective method based on CO2 laser irradiation for the preparation of ITO microelectrodes to find applications in the bio-sensing, impedance-based analysis, and more, is presented. Electrodes of different sizes and shapes were examined to identify the performance of the samples. They were later optimized to suit our applications for studies in three dimensional cell-cultures. For rectangular shaped electrodes, the smallest sizes optimized were 25, 50 and 100 µm wide. The figure attached shows cyclic voltammetry results illustrating the reproducibility of 100 µm electrodes with ferrocenedimethanol as the redox probe in potassium nitrate. However, smaller electrodes are not always desirable. For the cell-culture work, electrodes with area 1.75mm2 were fabricated and characterized to get reproducible results. Initial measurements of impedance based cellular assays have been conducted first in DMEM medium and then in the cell culture medium to understand the parameters and study the adhesion of the cells on to the electrodes. Analysis of glucose sensing properties of the system have also been attempted. Further modifications of the electrode structure and characterizations for validation will be discussed.References Stadler A. et al. J.Materials, 2012, 5, 661-683Wang Y. et al. J Energy Chem. 2021, 59, 642-665 Figure 1

B12-induced reassembly of split photoreceptor protein enables photoresponsive hydrogels with tunable mechanics.

Although the tools based on split proteins have found broad applications, ranging from controlled biological signaling to advanced molecular architectures, many of them suffer from drawbacks such as background reassembly, low thermodynamic stability, and static structural features. Here, we present a chemically inducible protein assembly method enabled by the dissection of the carboxyl-terminal domain of a B12-dependent photoreceptor, CarHC. The resulting segments reassemble efficiently upon addition of cobalamin (AdoB12, MeB12, or CNB12). Photolysis of the cofactors such as AdoB12 and MeB12 further leads to stable protein adducts harboring a bis-His-ligated B12. Split CarHC enables the creation of a series of protein hydrogels, of which the mechanics can be either photostrengthened or photoweakened, depending on the type of B12. These materials are also well suited for three dimensional cell culturing. Together, this new protein chemistry, featuring negligible background autoassembly, stable conjugation, and phototunability, has opened up opportunities for designing smart materials.

A critical analysis of research methods and experimental models to study pulpitis

Pulpitis is the inflammatory response of the dental pulp to a tooth insult, whether it is microbial, chemical, or physical in origin. It is traditionally referred to as reversible or irreversible, a classification for therapeutic purposes that determines the capability of the pulp to heal. Recently, new knowledge about dental pulp physiopathology led to orientate therapeutics towards more frequent preservation of pulp vitality. However, full adoption of these vital pulp therapies by dental practitioners will be achieved only following better understanding of cell and tissue mechanisms involved in pulpitis. The current narrative review aimed to discuss the contribution of the most significant experimental models developed to study pulpitis. Traditionally, in vitro two (2D)- or three (3D)-dimensional cell cultures or in vivo animal models were used to analyse the pulp response to pulpitis inducers at cell, tissue or organ level. In vitro, 2D cell cultures were mainly used to decipher the specific roles of key actors of pulp inflammation such as bacterial by-products, pro-inflammatory cytokines, odontoblasts or pulp stem cells. However, these simple models did not reproduce the 3D organisation of the pulp tissue and, with rare exceptions, did not consider interactions between resident cell types. In vitro, tissue/organ-based models were developed to better reflect the complexity of the pulp structure. Their major disadvantage is that they did not allow the analysis of blood supply and innervation participation. On the contrary, in vivo models have allowed researchers to identify key immune, vascular and nervous actors of pulpitis and to understand their function and interplay in the inflamed pulp. However, inflammation was mainly induced by iatrogenic dentine drilling associated with simple pulp exposure to the oral environment or stimulation by individual bacterial by-products for short periods. Clearly, these models did not reflect the long and progressive development of dental caries. Lastly, the substantial diversity of the existing models makes experimental data extrapolation to the clinical situation complicated. Therefore, improvement in the design and standardisation of future models, for example by using novel molecular biomarkers, databased models and artificial intelligence, will be an essential step in building an incremental knowledge of pulpitis in the future.

Enabling high throughput drug discovery in 3D cell cultures through a novel bioprinting workflow.

Advanced three dimensional cell culture techniques have been adopted in many laboratories to better model in vivo tissue by recapitulating multi-cellular architecture and the presence of extracellular matrix features. We describe here a 3D cell culture platform in a small molecule screening workflow that uses traditional biomarker and intracellular kinase end point assay readouts. By combining the high throughput bioprinter RASTRUM with the high throughput screening assay AlphaLISA, we demonstrate the utility of the protocol in 3D synthetic hydrogel cultures with breast cancer (MDA-MB-231 and MCF-7) and fibroblast cells. To establish and validate the workflow, we treated the breast cancer cultures with doxorubicin, while fibroblast cultures were stimulated with the pro-inflammatory lipopolysaccharide. 3D and 2D MDA-MB-231 cultures were equally susceptible to doxorubicin treatment, while showing opposite ERK phosphorylation changes. Doxorubicin readily entered embedded MCF-7 spheroids and markedly reduced intracellular GSK3β phosphorylation. Furthermore, quantifying extracellular interleukin 6 levels showed a very similar activation profile for fibroblasts in 2D and 3D cultures, with 3D fibroblast networks being more resistant against the immune challenge. Through these validation experiments we demonstrate the full compatibility of the bioprinted 3D cell cultures with several widely-used 2D culture assays. The efficiency of the workflow, minimal culture handling, and applicability of traditional screening assays, demonstrates that advanced encapsulated 3D cell cultures can be used in 2D cell culture screening workflows, while providing a more holistic view on cell biology to increase the predictability to in vivo drug response.

In 2D and 3D Cell Culture Models, Effects of Endothelial Cells on E-cadherin / β-catenin Expression Levels and Spheroid Sizes in Ishikawa Cells.

Objective:Increasing evidence shows that three dimensional cell culture models better reflect the in vivo tumor microenvironment than two dimensional cell culture models. Co-culture models are ideal cell culture models for understanding the communication between cells and the in vivo microenvironment. Changes in expression levels of E-cadherin are closely related to cancer metastasis and progression. β-catenin mediates cell adhesion of E-cadherin. Endothelial cells are stromal cell components in the tumor microenvironment. It is known that there is little or no expression of E-cadherin in endothelial cells. Methods:In our study, both two-dimensional and three dimensional mono-culture and co-culture models were created using Huvec and Ishikawa cells (endometrial cancer cell lines) to better reflect cell interactions. Spheroids were followed for three days in the three-dimensional cell culture model. E-cadherin and β-catenin expression levels of two-dimensional and three dimensional mono-culture and co-culture models were measured by immunofluorescence staining. Spheroid images were recorded using a Z-stack. Intensity measurements in both two-dimensional and three-dimensional mono-culture and co-culture models were made using the Image J software. Study groups were evaluated by one-way analysis of variance (One-Way ANOVA). Values of p <0.05 were considered statistically significant. Results:The size of the co-culture spheroids was recorded significantly larger than the mono-culture spheroids (p <0.0001). In two (p = 0.0175) and three dimensional models (p <0.0001), expression levels of E-cadherin in the mono-culture of Ishikawa cells were recorded significantly higher than in Huvec and co-culture cells. Likewise, while the expression levels of β-catenin were higher in the mono-culture of Ishikawa cells in two-dimensional models (p <0.05), no significant difference was observed in three dimensional models (p> 0.05). Conclusion:In summary, it has been noted that the expression levels of E-cadherin are significantly reduced in co-cultures of Ishikawa cells with Huvec cells in both two and three dimensions . These results support the idea that endothelial cells may cause changes in endometrial cancer progression by suppressing E-cadherin expression in Ishikawa cells.

Identification and evaluation of an appropriate housekeeping gene for real time gene profiling of hepatocellular carcinoma cells cultured in three dimensional scaffold.

Assessing an optimal reference gene as an internal control for target gene normalization is important during quantitative real time polymerase chain reaction (RT-qPCR) of three dimensional (3D) cell culture. Especially, gene profiling of cancer cells under a complex 3D microenvironment in a polymer scaffold provides a deeper understanding of tumor functioning in vivo. Expression of six housekeeping genes (HKG's): Glyceraldehyde-3-phosphodehydrogenase (GAPDH), β-actin (ACTB), beta-2-microglobulin (B2M), 18S ribosomal RNA (18S rRNA), peptidyl-propyl-isomerase A (PPIA), and ribosomal protein L13 (RPL-13) during two dimensional (2D) culture, and alginate-carboxymethylcellulose scaffold based 3D culture conditioned up to 21days was analysed for hepatocellular carcinoma (Huh-7) cells. The gene expression studies were performed by determining primer efficiency, melting curve and threshold cycle analysis. Further, RT-qPCR data was validated statistically using geNorm and NormFinder softwares. The study indicated RPL-13, 18S rRNA and B2M to be stable among selected referral HKG candidates. An exploration of a reliable HKG is necessary for normalization of gene expression in RT-qPCR during varying cell culture conditions.

Erlotinib Promotes Ligand-Induced EGFR Degradation in 3D but Not 2D Cultures of Pancreatic Ductal Adenocarcinoma Cells.

Simple SummaryThe EGFR is a tyrosine kinase receptor that responds to different stresses such as UV irradiation, hypoxia and drug treatment by internalizing into endosomal compartments. Receptor trafficking and degradation due to tyrosine kinase inhibitors has been widely studied in two- dimensional (2D) cell culture systems, but little is known about how cells respond to these types of drugs in more physiologically relevant models such as three-dimensional (3D) cultures, whose nanostructured properties allow cells to grow, proliferate, migrate and extend cellular processes in their 3D space. In this study, we show that EGFR suffers degradation in response to erlotinib treatment in 3D-cultured cancer cells but not in classic 2D culture systems, demonstrating that dimensionality strongly affects cell drug response. This 3D model may pave the way for the development of more physiological culture platforms to obtain mechanistic insights into how cells respond to chemotherapy.The epithelial growth factor receptor (EGFR) is a tyrosine kinase receptor that participates in many biological processes such as cell proliferation. In addition, EGFR is overexpressed in many epithelial cancers and therefore is a target for cancer therapy. Moreover, EGFR responds to lots of stimuli by internalizing into endosomes from where it can be recycled to the membrane or further sorted into lysosomes where it undergoes degradation. Two-dimensional cell cultures have been classically used to study EGFR trafficking mechanisms in cancer cells. However, it has been widely demonstrated that in 2D cultures cells are exposed to a non-physiological environment as compared to 3D cultures that provide the normal cellular conformation, matrix dimensionality and stiffness, as well as molecular gradients. Therefore, the microenvironment of solid tumors is better recreated in 3D culture models, and this is why they are becoming a more physiological alternative to study cancer physiology. Here, we develop a new model of EGFR internalization and degradation upon erlotinib treatment in pancreatic ductal adenocarcinoma (PDAC) cells cultured in a 3D self-assembling peptide scaffold. In this work, we show that treatment with the tyrosine kinase inhibitor erlotinib promotes EGFR degradation in 3D cultures of PDAC cell lines but not in 2D cultures. We also show that this receptor degradation does not occur in normal fibroblast cells, regardless of culture dimensionality. In conclusion, we demonstrate not only that erlotinib has a distinct effect on tumor and normal cells but also that pancreatic ductal adenocarcinoma cells respond differently to drug treatment when cultured in a 3D microenvironment. This study highlights the importance of culture systems that can more accurately mimic the in vivo tumor physiology.

Effects of Gallic Acid on Endometrial Cancer Cells in Two and Three Dimensional Cell Culture Models.

Background and Aim:Cell culture studies are an indispensable tools used to understand basic physiological, biophysical and biomolecular mechanisms. Although traditional two-dimensional (2D) cell culture models are more preferred in experimental studies, three-dimensional (3D) cell culture models, attract more attention due to several advantages including mimicking tumor physiology, biochemistry and biomechanics. We aimed to investigate the effects of Gallic Acid, an antimutagenic, antioxidant and anticarcinogenic agent, on both 2D and 3D cultured endometrial cancer cells for the first time. Methods:IC50 values were determined in 2D and 3D cultured endometrial cancer cells exposed to different doses of GA. In the 2D culture model exposed to GA, Caspase 3 expression levels were analyzed. In addition, the effect of GA on the migration of 2D cultured endometrium cancer cells was investigated. Results:IC50 value in the 3D model was found significantly higher than the 2D model. In 2D culture model, Caspase 3 expression and apoptosis was increased significantly in cells of GA exposed group compared to the control group. GA did not have a significant effect on the migration profile of cells. Conclusion:Gallic Acid is shown to induce apoptosis in Ishikawa cells via Caspase 3 activation. We demonstrated a significantly higher IC50 level in 3D model which provide evidence to prefer 3D models in drug-test trials. The data obtained in the current study will provide a basis for further experiments to analyze the effects of GA on endometrial cancer and to develop strategies for clinical treatment.

Propagation of Dental and Respiratory Cells and Organs in Microgravity.

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

Three Dimensional Cell Culturing for Modeling Adrenal and Pituitary Tumors.

In vitro monolayer conditions are not able to reproduce the complexity of solid tumors, still, there is scarce information about the 3D cell culture models of endocrine tumor types. Therefore, our aim was to develop in vitro 3D tumor models by different methodologies for adrenocortical carcinoma (H295R), pituitary neuroendocrine tumor (RC-4B/C and GH3) and pheochromocytoma (PC-12). Various methodologies were tested. Cell biological assays (cell viability, proliferation and live cell ratio) and steroid hormone production by HPLC-MS/MS method were applied to monitor cellular well-being. Cells in hanging drops and embedded in matrigel formed multicellular aggregates but they were difficult to handle and propagate for further experiments. The most widely used methods: ultra-low attachment plate (ULA) and spheroid inducing media (SFDM) were not the most viable 3D model of RC-4B/C and GH3 cells that would be suitable for further experiments. Combining spheroid generation with matrigel scaffold H295R 3D models were viable for 7 days, RC-4B/C and GH3 3D models for 7–10 days. ULA and SFDM 3D models of PC-12 cells could be used for further experiments up to 4 days. Higher steroid production in 3D models compared to conventional monolayer culture was detected. Endocrine tumor cells require extracellular matrix as scaffold for viable 3D models that can be one reason behind the lack of the usage of endocrine 3D cultures. Our models help understanding the pathogenesis of endocrine tumors and revealing potential biomarkers and therapeutic targets. They could also serve as an excellent platform for preclinical drug test screening.

Impact of high-energy electron irradiation on mechanical, structural and chemical properties of agarose hydrogels

Due to their excellent biocompatibility and biodegradability, natural hydrogels are highly demanded biomaterials for biomedical applications such as wound dressing, tissue engineering, drug delivery or three dimensional cell culture. Highly energetic electron irradiation up to 10 MeV is a powerful and fast tool to sterilize and tailor the material's properties. In this study, electron radiation treatment of agarose hydrogels was investigated to evaluate radiation effects on physical, structural and chemical properties. The viscoelastic behavior, surface hydrophilicity and swelling behavior in a range of typical sterilization doses of 0 kGy to 30 kGy was analyzed. The mechanical properties were determined by rheology measurements and decreased by more than 20% compared to the initial moduli. The number average molecular weight between crosslinks was estimated based on rubber elasticity theory to judge on the radiation degradation. In this dose range, the number average molecular weight between crosslinks increased by more than 6%. Chemical structure was investigated by FTIR spectroscopy to evaluate the radiation resistance of agarose hydrogels. With increasing electron dose, an increasing amount of carbonyl containing species was observed. In addition, irradiation was accompanied by formation of gas cavities in the hydrogels. The gas products were specified for CO2, CO and H2O. Based on the radiolytic products, a radiolysis mechanism was proposed. Electron beam treatment under high pressure conditions was found to reduce gas cavity formation in the hydrogels.

Effects of Three-Dimensional Sodium Alginate Scaffold on Maturation and Developmental Gene Expressions in Fresh and Vitrified Preantral Follicles of Mice.

Background:Prior to chemotherapy interventions, in vitro maturation (IVM) of follicles through vitrification can be used to help young people conserve their fertility. The aim of study was to investigate effect of sodium alginat scaffold on follicles development and improvement of the culture medium.Materials and Methods:This experimental study was conducted on immature female BALB/c mice (12-14 days). Follicles were gathered mechanically and placed in α-Minimal Essential Medium (α-MEM) containing 5% fetal bovine serum (FBS). Some pre-antral follicles were frozen. The fresh and vitrified follicles were cultured in different concentrations of sodium alginate (0.25%, 0.5%, and 1%) and two dimensional (2D) medium for 12 days. The samples were evaluated for viability percentage, the number of MII-phase oocytes and reactive oxygen specious (ROS) level. Additionally, Gdf9, Bmp15, Bmp7, Bmp4, Gpx, mnSOD and Gcs gene expressions were assessed in the samples.Results:The highest and lowest percentages of follicle viability and maturation in the fresh and vitrified groups were respectively 0.5% concentration and 2D culture. There was no significant difference among the concentrations of 0.25% and 1%. Viability and maturation of follicles showed a significant increase in the fresh groups in comparison with the vitrified groups. ROS levels in the both fresh and vitrified groups with different concentrations of alginate showed a significant decrease compared to the control group. ROS levels in follicles showed a significant decrease in the fresh groups in comparison with the vitrified groups (P≤0.0001). The highest gene expression levels were observed in the 0.5% alginate (P≤0.0001). Moreover, the viability percentage, follicle maturation, and gene expression levels were higher in the fresh groups than the vitrified groups (P≤0.0001).Conclusion:Alginate hydrogel at a proper concentration of 5%, not only helps follicle get mature, but also promotes the expression of developmental genes and reduces the level of intracellular ROS. Follicular vitrification decreases quality of the follicles, which are partially compensated using a three dimensional (3D)

Bio-inspired plant leaf skeleton based three dimensional scaffold for three dimensional cell culture

Large quantities of natural fibers are available in the plant biomass that can be utilized for various purposes including three dimensional cell culture and tissue engineering due to their biocompatibility, ecofriendly, easy availability and cost effective. Especially, leaf skeletons (venation architecture) have a complex hierarchical architecture with novel properties. In this present invention describes about developing three dimensional scaffold from plant leaf skeletons for three dimensional cell culture. Plant leaf skeleton is prepared by simple and rapid method which using sodium hydroxide pretreatment under pressurized condition at 120°C for 1 h. The prepared plant skeleton has microporous surface topography. Also, the plant leaf skeleton is mainly composed by hemicellulose, cellulose and lignin. The microscopic analysis clearly indicates that human mesenchymal stem cells (hMSCs) attached and proliferated on plant leaf skeleton. Interestingly, cell density of hMSCs is increased on plant leaf skeleton by incubation time-dependent manner. Our study confirmed that sodium hydroxide via surface modified Ficus religiosa leaf skeleton enhances the attachment and proliferation of the human mesenchymal stem cells because of their biocompatibility and porous nature. The chemical nature and venation architecture of leaf skeleton has facilitated nutrient and oxygen absorption to promote cell-cell interaction, long term cell culture, and possible scope for induction of cell differentiation. Thus, leaf venation architecture can be applied as three dimensional (3D) cell-culture platform for multi-layer cell culture, cell-based assay model, high-throughput drug screening, cell-replacement therapy, wound healing and substitute for skin. Moreover, this scaffold could also be well-suited to co-culture screening strategies and stem cell differentiation for tissue engineering. Our present invention highlighted agro-wastes as precursor for novel scaffold materials to construct the 3D cell culture platform.

Low magnitude high frequency vibrations expedite the osteogenesis of bone marrow stem cells on paper based 3D scaffolds.

Anabolic effects of low magnitude high frequency (LMHF) vibrations on bone tissue were consistently shown in the literature in vivo, however in vitro efforts to elucidate underlying mechanisms are generally limited to 2D cell culture studies. Three dimensional cell culture platforms better mimic the natural microenvironment and biological processes usually differ in 3D compared to 2D culture. In this study, we used laboratory grade filter paper as a scaffold material for studying the effects of LHMF vibrations on osteogenesis of bone marrow mesenchymal stem cells in a 3D system. LMHF vibrations were applied 15 min/day at 0.1 g acceleration and 90 Hz frequency for 21 days to residing cells under quiescent and osteogenic conditions. mRNA expression analysis was performed for alkaline phosphatase (ALP) and osteocalcin (OCN) genes, Alizarin red S staining was performed for mineral nodule formation and infrared spectroscopy was performed for determination of extracellular matrix composition. The highest osteocalcin expression, mineral nodule formation and the phosphate bands arising from the inorganic phase was observed for the cells incubated in osteogenic induction medium with vibration. Our results showed that filter paper can be used as a model scaffold system for studying the effects of mechanical loads on cells, and LMHF vibrations induced the osteogenic differentiation of stem cells.

Effect of cell imprinting on viability and drug susceptibility of breast cancer cells to doxorubicin

This study demonstrates the effect of substrate's geometrical cues on viability and the efficacy of an anti-cancer drug, doxorubicin (DOX), on breast cancer cells. It is hypothesized that the surface topographical properties can mediate the cellular drug intake. Pseudo-three dimensional (3D) platforms were fabricated using imprinting technique from polydimethylsiloxane (PDMS) and gelatin methacryloyl (GelMA) hydrogel to recapitulate topography of cells’ membranes. The cells exhibited higher viability on the cell-imprinted platforms for both PDMS and GelMA materials compared to the plain/flat counterparts. For instance, MCF7 cells showed a higher metabolic activity (11.9%) on MCF7-imprinted PDMS substrate than plain PDMS. The increased metabolic activity for the imprinted GelMA was about 44.2% compared to plain hydrogel. The DOX response of cells was monitored for 24 h. Although imprinted substrates demonstrated enhanced biocompatibility, the cultured cells were more susceptible to the drug compared to the plain substrates. In particular, MCF7 cells on imprinted PDMS and GelMA substrates showed 37% and 50% higher in cell death compared to the corresponding plain PDMS and GelMA, respectively. Interestingly, the drug susceptibility of the cells on the imprinted hydrogel was about 70% higher than the cells cultured on imprinted PDMS substrates. Having MCF7 cell-imprinted substrates, DOX responses of two other breast cancer cell lines, SKBR3 and ZR-75-1, were also evaluated. The results support that cell membrane curvature developed by multiscale topography is able to mediate intracellular signaling and drug intake. Statement of SignificanceResearch in biological sciences and drug discovery mostly rely on two dimensional (2D) cell culture techniques which cannot provide a reliable physiologically relevant environment. Lack of extracellular matrix and a large shift in physicochemical properties of conventional 2D substrates can induce aberrant cellular behaviors. While chemical composition, topographical, and mechanical properties of substrates have remarkable impacts on drug susceptibility, gene expression, and protein synthesis, the most cell culture plates are from rigid and plain substrates. A number of (bio)polymeric 3D-platforms have been introduced to resemble innate cell microenvironment. However, their intricate culture protocols restrain their applications in demanding high-throughput drug screening. To address the above concerns, in the present study, a hydrogel-based pseudo-3D substrate with imprinted cell features has been introduced.