Cell Culture Environment Research Articles

Abstract 6834: Periostin involvement in cellular senescence in ovarian cancer

Abstract Purpose: Despite improvements in care and advancements in the understanding of ovarian cancer (OC) pathobiology and genetics, survival rates remain disappointingly low compared to other types of cancer. This is due to a lack of diagnosis strategies, high recurrence rates, and the development of chemo-resistant diseases, which require synergy between cancer cells and the tumor microenvironment (TME). We have demonstrated the functional importance of a TME gene, periostin (POSTN), in OC and recurrent OC, using in vitro and in vivo models. Our objectives were to identify differentially expressed genes by POSTN expression in the TME that contribute to the progression of ovarian cancers. Procedures: Gene expression data was generated from an ovarian cancer cell line, HEYA8, cultured in a high POSTN conditioned 3D cell culture environment, using the NovaSeq 6000 system to generate RNA-seq data. The data was analyzed to determine the genes expressed at different levels between the HEYA8 cells grown with or without POSTN-high conditioned medium (CM). The gene functional annotation and KEGG pathway enrichment were further analyzed from 10,115 significant differentially expressed genes between the HEYA8 cells grown in high levels of POSTN (CMPOSTNhigh) or low levels of POSTN (CMCTL) culture conditions, using DAVID bioinformatics. Results: Using a cell culture and co-culture model, we found that OC cells cultured under conditioned media containing CMPOSTNhigh exhibited faster cell migration, more invasiveness (p=0.006), more chemo-resistance (p<0.05), and increased resistance to paclitaxel-induced apoptosis, compared to OC cells cultured with control medium (CMCTL). OC cell lines cultured with CMPOSTNhigh showed increased side population cells relative to CMCTL -cultured cells, suggesting POSTN plays important roles in cancer stem cell enrichment. POSTN-transfected 3T3-L1 cells exhibited more intracellular and extracellular lipids and were linked to increased cancer cell expression of the oncogene fatty acid synthetase. In correspondence to the functions of POSTN in OC cells, the pathways identified to be significantly impacted by POSTN expression included, but were not limited to, the cell cycle, apoptosis, p53 signaling, MAPK signaling, and PI3K-Akt signaling pathways. Interestingly, we also found cellular senescence pathways was one of the most significantly impacted by POSTN suggesting that POSTN may play roles in ovarian cancer via the regulation of cellular senescence, which has not yet been explored. Conclusions: These data emphasize the importance of TME factors in tumor progression and prognosis via various ways in ovarian cancer, establishing promising potential therapeutic targets of OC. Citation Format: Jihua Feng, Ethan Nguyen, Jocelyn Reyes, Zhiqing Huang. Periostin involvement in cellular senescence in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6834.

Directed differentiation of human embryonic stem cells into parathyroid cells and establishment of parathyroid organoids.

Differentiation of human embryonic stem cells (hESCs) into human embryonic stem cells-derived parathyroid-like cells (hESC-PT) has clinical significance in providing new therapies for congenital and acquired parathyroid insufficiency conditions. However, a highly reproducible, well-documented method for parathyroid differentiation remains unavailable. By imitating the natural process of parathyroid embryonic development, we proposed a new hypothesis about the in vitro differentiation of parathyroid-like cells. Transcriptome, differentiation marker protein detection and parathyroid hormone (PTH) secretion assays were performed after the completion of differentiation. To optimize the differentiation protocol and further improve the differentiation rate, we designed glial cells missing transcription factor 2 (GCM2) overexpression lentivirus transfection assays and constructed hESCs-derived parathyroid organoids. The new protocol enabled hESCs to differentiate into hESC-PT. HESC-PT cells expressed PTH, GCM2 and CaSR proteins, low extracellular calcium culture could stimulate hESC-PT cells to secrete PTH. hESC-PT cells overexpressing GCM2 protein secreted PTH earlier than their counterpart hESC-PT cells. Compared with the two-dimensional cell culture environment, hESCs-derived parathyroid organoids secreted more PTH. Both GCM2 lentiviral transfection and three-dimensional cultures could make hESC-PT cells functionally close to human parathyroid cells. Our study demonstrated that hESCs could differentiate into hESC-PT in vitro, which paves the road for applying the technology to treat hypoparathyroidism and introduces new approaches in the field of regenerative medicine.

Directing the Encapsulation of Single Cells with DNA Framework Nucleator-Based Hydrogel Growth.

Encapsulating individual mammalian cells with biomimetic materials holds potential in ex vivo cell culture and engineering. However, current methodologies often present tradeoffs between homogeneity, stability, and cell compatibility. Here, inspired by bacteria that use proteins stably anchored on their outer membranes to nucleate biofilm growth, we develop a single-cell encapsulation strategy by using a DNA framework structure as a nucleator (DFN) to initiate the growth of DNA hydrogels under cell-friendly conditions. We find that among the tested structures, the tetrahedral DFN can evenly and stably reside on cell membranes, effectively initiating hybridization chain reactions which generate homogeneously dense yet flexible single-cell encapsulation for diverse cell lines. The encapsulation persists for up to 72 hours in a serum-containing cell culture environment, representing a ~70-fold improvement compared to encapsulations mediated by single-stranded DNA nucleators. The metabolism and proliferation of the encapsulated cells are suppressed, but can be restored to the original efficiencies upon release, suggesting the superior cell compatibility of the encapsulation. We also find that compared to naked cells, the encapsulated cells exhibit a lower autophagy level after undergoing mechanical stress, suggesting the protective effect of the DNA encapsulation. This method may provide a new tool for ex vivo cell engineering.

Elucidating uptake and metabolic fate of dipeptides in CHO cell cultures using 13C labeling experiments and kinetic modeling

The rapidly growing market of biologics including monoclonal antibodies has stimulated the need to improve biomanufacturing processes including mammalian host systems such as Chinese Hamster Ovary (CHO) cells. Cell culture media formulations continue to be enhanced to enable intensified cell culture processes and optimize cell culture performance. Amino acids, major components of cell culture media, are consumed in large amounts by CHO cells. Due to their low solubility and poor stability, certain amino acids including tyrosine, leucine, and phenylalanine can pose major challenges leading to suboptimal bioprocess performance. Dipeptides have the potential to replace amino acids in culture media. However, very little is known about the cleavage, uptake, and utilization kinetics of dipeptides in CHO cell cultures. In this study, replacing amino acids, including leucine and tyrosine by their respective dipeptides including but not limited to Ala-Leu and Gly-Tyr, supported similar cell growth, antibody production, and lactate profiles. Using 13C labeling techniques and spent media studies, dipeptides were shown to undergo both intracellular and extracellular cleavage in cultures. Extracellular cleavage increased with the culture duration, indicating cleavage by host cell proteins that are likely secreted and accumulate in cell culture over time. A kinetic model was built and for the first time, integrated with 13C labeling experiments to estimate dipeptide utilization rates, in CHO cell cultures. Dipeptides with alanine at the N-terminus had a higher utilization rate than dipeptides with alanine at the C-terminus and dipeptides with glycine instead of alanine at N-terminus. Simultaneous supplementation of more than one dipeptide in culture led to reduction in individual dipeptide utilization rates indicating that dipeptides compete for the same cleavage enzymes, transporters, or both. Dipeptide utilization rates in culture and cleavage rates in cell-free experiments appeared to follow Michaelis-Menten kinetics, reaching a maximum at higher dipeptide concentrations. Dipeptide utilization behavior was found to be similar in cell-free and cell culture environments, paving the way for future testing approaches for dipeptides in cell-free environments prior to use in large-scale bioreactors. Thus, this study provides a deeper understanding of the fate of dipeptides in CHO cell cultures through an integration of cell culture, 13C labeling, and kinetic modeling approaches providing insights in how to best use dipeptides in media formulations for robust and optimal mammalian cell culture performance.

Ionic Liquid Interface as a Cell Scaffold.

In sharp contrast to conventional solid/hydrogel platforms, water-immiscible liquids, such as perfluorocarbons and silicones, allow the adhesion of mammalian cells via protein nanolayers (PNLs) formed at the interface. However, fluorocarbons and silicones, which are typically used for liquid cell culture, possess only narrow ranges of physicochemical parameters and have not allowed for a wide variety of cell culturing environments. In this paper, it is proposed that water-immiscible ionic liquids (ILs) are a new family of liquid substrates with tunable physicochemical properties and high solvation capabilities. Tetraalkylphosphonium-based ILs are identified as non-cytotoxic ILs, whereon human mesenchymal stem cells are successfully cultured. By reducing the cation charge distribution, or ionicity, via alkyl chain elongation, the interface allows cell spreading with matured focal contacts. High-speed atomic force microscopy observations of the PNL formation process suggest that the cation charge distribution significantly altered the protein adsorption dynamics, which are associated with the degree of protein denaturation and the PNL mechanics. Moreover, by exploiting dissolution capability of ILs, an ion-gel cell scaffold is fabricated. This enables to further identify the significant contribution of bulk subphase mechanics to cellular mechanosensing in liquid-based culture scaffolds.

Facilitating long-term cell examinations and time-lapse recordings in cell biology research with CO2 mini-incubators

In recent years, microscopy has revolutionized the study of dynamic living cells. However, performing long-term live cell imaging requires stable environmental conditions such as temperature, pH, and humidity. While standard incubators have traditionally provided these conditions, other solutions, like stagetop incubators are available. To further enhance the accessibility of stable cell culture environments for live cell imaging, we developed a portable CO2 cell culture mini-incubator that can be easily adapted to any x–y inverted microscope stage, enabling long-term live cell imaging. This mini-incubator provides and maintains stable environmental conditions and supports cell viability comparable to standard incubators. Moreover, it allows for parallel experiments in the same environment, saving both time and resources. To demonstrate its functionality, different cell lines (VERO and MDA-MB-231) were cultured and evaluated using various assays, including crystal violet staining, MTT, and flow cytometry tests to assess cell adhesion, viability, and apoptosis, respectively. Time-lapse imaging was performed over an 85-h period with MDA-MB-231 cells cultured in the mini-incubator. The results indicate that this device is a viable solution for long-term imaging and can be applied in developmental biology, cell biology, and cancer biology research where long-term time-lapse recording is required.

The Antimelanogenic Activity of the Extract of Heukharang Lettuce (Lactuca sativa L.) Leaf

Heukharang (Lactuca sativa L.), the artificially developed lettuce cultivar for enhancement of lactucin content, exhibits antioxidant activities and sleep-promoting effects. However, potential of Heukharang as a raw material for the skin-whitening agent has not been investigated yet. This study evaluated the effects of Heukharang extract (HHE) on α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis in vitro and in vivo models. Our findings revealed that HHE (25–100 μg/mL) effectively inhibited α-MSH-induced melanin synthesis in both 2D and 3D cell culture environments without compromising the viability of the B16F10 murine melanoma cell line. Furthermore, HHE suppressed intracellular tyrosinase activity, along with both gene and protein expression of tyrosinase (TYR) and tyrosinase-related protein (TRP)-1 and TRP-2. HHE downregulated the mRNA level of microphthalmia-associated transcription factor (MITF) and the phosphorylation of cAMP response element-binding (CREB) protein. The antimelanogenic activity of HHE (50 and 100 μg/mL) was also confirmed using a zebrafish embryo model, highlighting its efficacy in inhibiting α-MSH-induced melanogenesis through the downregulating CREB/MITF/TYR signaling pathways. Moreover, our study demonstrated that a corresponding amount of lactucin (5–20 μM) to the HHE inhibited and regulated melanogenesis. These results collectively suggest that HHE, enriched with lactucin, holds promise as a potential skin-whitening agent for nutricosmetic industries.

Abstract A063: CancerTools.org: Research Tools Supporting Breast Cancer Studies

Abstract CancerTools.org is the latest, non-profit cancer-focused biorepository. Driven by a mission to accelerate cancer research discoveries, the platform allows scientists to deposit and access tangible laboratory research materials. Till date, researchers from over 175 academic institutes globally have already deposited their tools, generating a cutting-edge portfolio of over 5,000 reagents, including a range of cell-based models and antibodies. Breast cancer is one of the most frequent malignancies in women worldwide. A comprehensive collection of tools has been curated at CancerTools.org to support this important area of research. Cell-based models include the RMAM007-3 breast cancer organoid which recapitulates in vivo tumour characteristics and is suitable for disease modelling, 3D imaging, and anticancer drug discovery. Cell lines such as human anti-oestrogen resistant lines developed from MCF-7 and T47D cells, demonstrate resistance to hormone-dependent treatments and are useful for studying therapy resistance mechanisms and biomarker development for breast cancer detection. NCI-HPN-F murine cell lines with recurrent chromosomal aneuploidies, have enabled investigation and identification of tumorigenesis related cancer-specific genes and signalling pathways. Further models include gene-edited cell lines to explore BRCA1 hypermethylation pathways; reporter breast cancer cell lines to assess apoptosis, necrosis, and autophagy; and immortalised human breast epithelial cell lines to study differentiation and phenotypic transformation. To provide a physiologically relevant cell culture environment, PlasmaxTM, a defined media that mirrors the concentration of over 50 components to human plasma, is also part of the repertoire. It has been used to culture triple-negative breast cancer cell lines to recapitulate the tumour metabolic environment. Antibodies to study breast cancer biology, including tumour suppressors, angiogenesis and metastasis, form part of the portfolio. Antibodies targeting tumour suppressors include RB1, which is often lost by mutation, deletion, transcriptional silencing and hyper-phosphorylation in malignancies- including breast cancer. Deletion and rearrangement of RB1 is estimated in ~20-25% of breast cancer cell lines. Tools identifying specific RB1 phosphorylation sites, include Anti-Phospho-Rb for Ser608 and Thr356. Additional insights into breast cancer prognosis are offered by Anti-Maspin [16F7] which recognises wildtype and denaturated forms of the protein. Maspin is an inhibitor of serine proteases with tumour suppressor activity in breast cancer. Its nuclear staining associates with favourable outcomes in contrast to cytoplasmic staining. MUC1 is also aberrantly overexpressed in 90% of human breast cancer, thus, representing a promising marker for breast cancer diagnosis and prognosis. Further research support is offered by an extensive range of antibodies for MUC1’s different isoforms. Visit our stand to explore more about depositing and accessing tools and technologies that support breast cancer research. Citation Format: Vera Moiseeva, Sara Omenetti, Abby Henney, Katie Valencia. CancerTools.org: Research Tools Supporting Breast Cancer Studies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A063.

Effect of laser surface texturing on the corrosion behaviour and biocompatibility of zinc hydroxyapatite implant material

The demand to optimize pure zinc with adjustable rates of corrosion and biocompatibility has recently increased, and metal matrix composites offer a promising solution. In this study, an optimized Zinc-Hydroxyapatite combination was prepared through a powder metallurgy process, and surface modifications were performed using Laser Surface Texturing. The feasibility of the composite in corrosive and cell culture environments was systematically evaluated through physical and mechanical tests, revealing properties matching those of bone tissue. Three different textures were tested, and textured samples showed better resistance to corrosion and higher cell viability than non-textured ones, with hexagonal texture being the most effective. Contact angle measurements were performed to analyse the sample's hydrophilic nature, which improved with the texturing process. These results suggest that texturing increased the surface area and surface wettability, leading to better biocompatibility results. This study highlights the potential of metal matrix composites for in-vivo conditions and calls for further investigation.

Universal Biomaterial-on-Chip: a versatile platform for evaluating cellular responses on diverse biomaterial substrates

Microfluidics has emerged as a promising approach for assessing cellular behavior in vitro, providing more physiologically relevant cell culture environments with dynamic flow and shear stresses. This study introduces the Universal Biomaterial-on-Chip (UBoC) device, which enables the evaluation of cell response on diverse biomaterial substrates in a 3D-printed microfluidic device. The UBoC platform offers mechanical stimulation of the cells and monitoring of their response on diverse biomaterials, enabling qualitative and quantitative in vitro analysis both on- and off-chip. Cell adhesion and proliferation were assessed to evaluate the biocompatibility of materials with different physical properties, while mechanical stimulation was performed to investigate shear-dependent calcium signaling in pre-osteoblasts. Moreover, the applicability of the UBoC platform in creating more complex in vitro models by culturing multiple cell types was demonstrated, establishing a dynamic multicellular environment to investigate cellular interfaces and their significance in biological processes. Overall, the UBoC presents an adaptable tool for in vitro evaluation of cellular behavior, offering opportunities for studying various biomaterials and cell interactions in microfluidic environments.Graphical

Adaptation of SIVmac to baboon primary cells results in complete absence of in vivo baboon infectivity.

While simian immunodeficiency virus (SIV) infection is non-pathogenic in naturally infected African nonhuman primate hosts, experimental or accidental infection in rhesus macaques often leads to AIDS. Baboons, widely distributed throughout Africa, do not naturally harbor SIV, and experimental infection of baboons with SIVmac results in transient low-level viral replication. Elucidation of mechanisms of natural immunity in baboons could uncover new targets of antiviral intervention. We tested the hypothesis that an SIVmac adapted to replicate in baboon primary cells will gain the capacity to establish chronic infections in vivo. Here, we generated SIVmac variants in baboon cells through serial passage in PBMC from different donors (SIVbn-PBMC s1), in PBMC from the same donors (SIVbn-PBMC s2), or in isolated CD4 cells from the same donors used for series 2 (SIVbn-CD4). While SIVbn-PBMC s1 and SIVbn-CD4 demonstrated increased replication capacity, SIVbn-PBMC s2 did not. Pharmacological blockade of CCR5 revealed SIVbn-PBMC s1 could more efficiently use available CCR5 than SIVmac, a trait we hypothesize arose to circumvent receptor occupation by chemokines. Sequencing analysis showed that all three viruses accumulated different types of mutations, and that more non-synonymous mutations became fixed in SIVbn-PBMC s1 than SIVbn-PBMC s2 and SIVbn-CD4, supporting the notion of stronger fitness pressure in PBMC from different genetic backgrounds. Testing the individual contribution of several newly fixed SIV mutations suggested that is the additive effect of these mutations in SIVbn-PBMC s1 that contributed to its enhanced fitness, as recombinant single mutant viruses showed no difference in replication capacity over the parental SIVmac239 strain. The replicative capacity of SIVbn-PBMC passage 4 (P4) s1 was tested in vivo by infecting baboons intravenously with SIVbn-PBMC P4 s1 or SIVmac251. While animals infected with SIVmac251 showed the known pattern of transient low-level viremia, animals infected with SIVbn-PBMC P4 s1 had undetectable viremia or viral DNA in lymphoid tissue. These studies suggest that adaptation of SIV to grow in baboon primary cells results in mutations that confer increased replicative capacity in the artificial environment of cell culture but make the virus unable to avoid the restrictive factors generated by a complex multicellular organism.

Differential curcumin absorption and curcumin-induced STAT3 inhibition during 3T3-L1 cell adipogenesis in 2D and 3D cultures

It is increasingly considered that 3D cell culture systems are superior to 2D in evaluating health promoting effects of candidate molecules because they better mimic tissue-like structures, thus testing in such systems will increase their in vivo applicability. The present study sought to examine the potential usefulness of spheroid models for assessing the antiadipogenic effect of polyphenols. The major food polyphenols were compared for their antiadipogenic effects in 3T3-L1 cells grown in conventional 2D conditions and as 3D spheroids. Further, the study evaluated the effect of the cell culture environment on the inhibition of the adipogenesis-related signaling pathway mediated by curcumin (CUR), the most effective antiadipogenic polyphenol identified in 2D and 3D, and its cellular absorption rate. CUR treatment inhibited the activity of signal transducer and activator of transcription 3 (STAT3) in cells cultured in both conditions; however, it was more effective in 2D. Notably, 3T3-L1 preadipocytes in a 3D cell culture system maintained high STAT3 activity and CCAAT/enhancer-binding protein alpha (C/EBPα) expression compared with a 2D system. Additionally, the cellular absorption rate was lower in 3D cultures. Thus, different cellular absorption rates, innate STAT3 activity, and C/EBPα expression levels may contribute to the difference in CUR efficacy. This study demonstrated that the STAT3 inhibition at least partly contributes to antiadipogenic effect of CUR. Moreover, it showed that the presentation of cells in 3D significantly alters activation of intracellular signaling pathways and absorption rate of CUR. Therefore, 3D spheroid models are valuable tools to evaluate the anti-adipogenicity of candidate molecules.

A comparative analysis of stem cell differentiation on 2D and 3D substrates using Raman microspectroscopy.

Chondrogenesis is a complex cellular process that involves the transformation of mesenchymal stem cells (MSCs) into chondrocytes, the specialised cells that form cartilage. In recent years, three-dimensional (3D) culture systems have emerged as a promising approach to studying cell behaviour and development in a more physiologically relevant environment compared to traditional two-dimensional (2D) cell culture. The use of these systems provided insights into the molecular mechanisms that regulate chondrogenesis and has the potential to revolutionise the development of new therapies for cartilage repair and regeneration. This study demonstrates the successful application of Raman microspectroscopy (RMS) as a label-free, non-destructive, and sensitive method to monitor the chondrogenic differentiation of bone marrow-derived rat mesenchymal stem cells (rMSCs) in a collagen type I hydrogel, and explores the potential benefits of 3D hydrogels compared to conventional 2D cell culture environments. rMSCs were cultured on 3D substrates for 3 weeks and their differentiation was monitored by measuring the spectral signatures of their subcellular compartments. Additionally, the evolution of high-density micromass cultures was investigated to provide a comprehensive understanding of the process and complex interactions between cells and their surrounding extracellular matrix. For comparison, rMSCs were induced into chondrogenesis in identical medium conditions for 21 days in monolayer culture. Raman spectra showed that rMSCs cultured in a collagen type I hydrogel are able to undergo a distinct chondrogenic differentiation pathway at a significantly higher rate than the 2D culture cells. 3D cultures expressed stronger and more homogeneous chondrogenesis-associated peaks such as collagens, glycosaminoglycans (GAGs), and aggrecan while manifesting changes in proteins and lipidic content. These results suggest that 3D type I collagen hydrogel substrates are promising for in vitro chondrogenesis studies, and that RMS is a valuable tool for monitoring chondrogenesis in 3D environments.

Isolation and culture of chickenembryonic synovial fibroblasts.

Cells obtained from chicken embryos are often preferred for in vitro studies. These cells, which easily adapt to rapid and continuous growth in the appropriate cell culture environment, are thought to be one of the effective methods in the investigation of leg diseases that are frequently observed in poultry. Leg diseases, especially affecting the joints in chickens, cause locomotor problems and adversely affect animal welfare. In addition, they cause significant health problems and increase mortality. It is known that synovial fibroblasts play an important role in joint diseases. In this study, chicken embryonic synovial fibroblasts were isolated from tissue explants taken from the tibio-metatarsal joint region of brown layer chicken embryos. Characterization of cells was evaluated by immunocytochemistry and hemacolor staining. chicken embryonic synovial fibroblasts showed a strong positive reaction to the vimentin antibody. As a result of hemacolor staining, it was noted that the cell morphology was spindle-shaped. The absence of macrophages in chicken embryonic synovial fibroblast culture was confirmed by the carbon powder uptake. In this present study, we aim to present a useful cell culture protocol such as primary culture, passage, and characterization suitable for chicken embryonic synovial fibroblast to be used in the new scientific research.

Vasculature-on-a-Chip with a Porous Membrane Electrode for In Situ Electrochemical Detection of Nitric Oxide Released from Endothelial Cells.

Vasculature-on-a-chip is a microfluidic cell culture device used for modeling vascular functions by culturing endothelial cells. Porous membranes are widely used to create cell culture environments. However, in situ real-time measurements of cellular metabolites in microchannels are challenging. In this study, a novel microfluidic device with a porous membrane electrode was developed for the in situ monitoring of nitric oxide (NO) released by endothelial cells in real time. In this system, a porous Au membrane electrode was placed directly beneath the cells for in situ and real-time measurements of NO, a biomarker of endothelial cells. First, the device was electrochemically characterized to construct a calibration plot for NO. Next, NO released by human umbilical vein endothelial cells under l-arginine stimulation was successfully quantified. Furthermore, the changes in NO release with culture time (in days) using the same sample were successfully recorded by exploiting minimally invasive measurements. This is the first report on the combination of a microfluidic device and porous membrane electrode for the electrochemical analysis of endothelial cells. This device will contribute to the development of organ-on-a-chip technology for real-time in situ cell analyses.

Proof-of-Concept and Characterization of Photosynthetic Bacteria as Radiation Enhancers

Molecular O2 is a potent chemical radiosensitizer. Re-sensitizing hypoxic tumor tissues with O2 can enhance the therapeutic efficacy of ionizing radiation. Photosynthetic cyanobacteria (S. elongatus) or cyanos are currently being explored for various bioengineering applications due to their ability to produce oxygen when exposed to light. Here, we explore the use of these bacteria to replenish molecular O2 content inside hypoxic tumors, which in turn will enhance the effect of radiation. To assess the feasibility of this approach, we used A549 lungs cancer cell spheroids as models, which are three-dimensional (3D) in vitro cell aggregates that mimic tumors in terms of oxygen and nutrient gradients. The cyanos were grown in 12:12h light /dark conditions in a bacterial shaker incubator (37ºC and 150 rpm). Biocompatibility of cyanos co-cultured withA549 monolayers and spheroids was estimated using the MTT assay and Calcein AM staining. The level of hypoxia content in the spheroids were measured using the Image IT green hypoxia dye. Spheroids co-cultured with cyanos were subjected to full spectrum lights for different time periods. After light incubation, different dosages of radiation (4, 6 and 12 Gy) were delivered to the spheroids using an X-ray irradiator (225 kBp, 13 mA). Post irradiation, the spheroids were harvested and analyzed based on Nuc green dye staining to estimate the dead cell population and clonogenic survival to estimate the post-therapy survival fraction. Under exposure to full spectrum LED light, cyanos increased the concentration of dissolved O2 in the medium, eventually reaching fourfold higher levels (i.e., ∼240% of dissolved O2). Addition of cyanos to mammalian cell cultures did not result in detectable toxicity for either cellular monolayers or 3D spheroids. Cell viability was 100% even at the highest tested concentration of 1 × 107 number of cyanos cultured with 10,000 A549 cells. This confirmed the biocompatibility of the cyanos in cell culture environment. Post irradiation, as depicted from the Nuc green dye staining, was twofold higher (p<0.01) in spheroids co-cultured with cyanos and exposed to light. Surprisingly, in the clonogenic assay, this group of spheroids exhibited a higher survival fraction compared to the control group. This unexpected outcome warrants further investigation of the potentially radioprotecting effect of cyanos. Altogether, this supports the potential therapeutic efficacy of the cyanos in hypoxic tumors. Cyanos in the presence of light are effective in enhancing radiation-induced cell death. While clinical translation is still far down the road, such a method could be employed in the context of brachytherapy or superficial radiotherapy to overcome tumor hypoxia.

Whole Liver Derived Acellular Extracellular Matrix for Bioengineering of Liver Constructs: An Updated Review.

Biomaterial templates play a critical role in establishing and bioinstructing three-dimensional cellular growth, proliferation and spatial morphogenetic processes that culminate in the development of physiologically relevant in vitro liver models. Various natural and synthetic polymeric biomaterials are currently available to construct biomimetic cell culture environments to investigate hepatic cell-matrix interactions, drug response assessment, toxicity, and disease mechanisms. One specific class of natural biomaterials consists of the decellularized liver extracellular matrix (dECM) derived from xenogeneic or allogeneic sources, which is rich in bioconstituents essential for the ultrastructural stability, function, repair, and regeneration of tissues/organs. Considering the significance of the key design blueprints of organ-specific acellular substrates for physiologically active graft reconstruction, herein we showcased the latest updates in the field of liver decellularization-recellularization technologies. Overall, this review highlights the potential of acellular matrix as a promising biomaterial in light of recent advances in the preparation of liver-specific whole organ scaffolds. The review concludes with a discussion of the challenges and future prospects of liver-specific decellularized materials in the direction of translational research.

Characterization of Bioinks Prepared via Gelifying Extracellular Matrix from Decellularized Porcine Myocardia.

Since the emergence of 3D bioprinting technology, both synthetic and natural materials have been used to develop bioinks for producing cell-laden cardiac grafts. To this end, extracellular-matrix (ECM)-derived hydrogels can be used to develop scaffolds that closely mimic the complex 3D environments for cell culture. This study presents a novel cardiac bioink based on hydrogels exclusively derived from decellularized porcine myocardium loaded with human-bone-marrow-derived mesenchymal stromal cells. Hence, the hydrogel can be used to develop cell-laden cardiac patches without the need to add other biomaterials or use additional crosslinkers. The scaffold ultrastructure and mechanical properties of the bioink were characterized to optimize its production, specifically focusing on the matrix enzymatic digestion time. The cells were cultured in 3D within the developed hydrogels to assess their response. The results indicate that the hydrogels fostered inter-cell and cell-matrix crosstalk after 1 week of culture. In conclusion, the bioink developed and presented in this study holds great potential for developing cell-laden customized patches for cardiac repair.

Carrageenan-Based Crowding and Confinement Combination Approach to Increase Collagen Deposition for In Vitro Tissue Development.

Connective tissue models grown from cell monolayers can be instrumental in a variety of biomedical fields such as drug screening, wound healing, and regenerative engineering. However, while connective tissues contain abundant fibrillar collagen, achieving a sufficient assembly and retention of fibrillar collagen in vitro is challenging. Unlike the dilute cell culture environment, the body's environment is characterized by a high density of soluble macromolecules (crowding) and macromolecular networks (confinement), which contribute to extracellular matrix (ECM) assembly in vivo. Consequently, macromolecular crowding (MMC) has been successfully used to enhance the processing of type I procollagen, leading to significant increases in fibrillar collagen assembly and accumulation during in vitro culture of a variety of cell types. In this study, we developed a combination approach using a carrageenan hydrogel, which released soluble macromolecules and served as a confinement barrier. We first evaluated the local carrageenan release and then confirmed the effectiveness of this combination approach on collagen accumulation by the human MG-63 bone cell line. Additionally, computational modeling of oxygen and glucose transport within the culture system showed no negative effects of the hydrogel and its releasates on cell viability.

Structurally decoupled stiffness and solute transport in multi-arm poly(ethylene glycol) hydrogels

Synthetic hydrogels are widely used as artificial 3D environments for cell culture, facilitating the controlled study of cell-environment interactions. However, most hydrogels are limited in their ability to represent the physical properties of biological tissues because stiffness and solute transport properties in hydrogels are closely correlated. Resultingly, experimental investigations of cell-environment interactions in hydrogels are confounded by simultaneous changes in multiple physical properties. Here, we overcame this limitation by simultaneously manipulating four structural parameters to synthesize a library of multi-arm poly (ethylene glycol) (PEG) hydrogel formulations with robustly decoupled stiffness and solute transport. This structural design approach avoids chemical alterations or additions to the network that might have unanticipated effects on encapsulated cells. An algorithm created to statistically evaluate stiffness-transport decoupling within the dataset identified 46 of the 73 synthesized formulations as robustly decoupled. We show that the swollen polymer network model accurately predicts 11 out of 12 structure-property relationships, suggesting that this approach to decoupling stiffness and solute transport in hydrogels is fundamentally validated and potentially broadly applicable. Furthermore, the unprecedented control of hydrogel network structure provided by multi-arm PEG hydrogels confirmed several fundamental modeling assumptions. This study enables nuanced hydrogel design for uncompromised investigation of cell-environment interactions.