Two-dimensional Tissue Culture Research Articles

Microspheres of stem cells from human exfoliated deciduous teeth exhibit superior pulp regeneration capacity

ObjectivesEngineering spheroids to create three-dimensional (3D) cell cultures has gained increasing attention in recent years due to their potential advantages over traditional two-dimensional (2D) tissue culture methods. Stem cells derived from human exfoliated deciduous teeth (SHEDs) demonstrate significant potential for pulpal regeneration applications. Nevertheless, the feasibility of microsphere formation of SHEDs and its impact on pulpal regeneration remain unclear. MethodsIn this study, SHEDs were isolated, identified, and cultured in ultra-low attachment six-well plates to produce SHED microspheres. The biological properties of SHED microspheres were compared to those of traditional 2D culture using live-dead staining, Alizarin red staining, Oil-red O staining, scratch experiments, Immunofluorescence, Transmission electron microscopy scan, Western blotting, RNA sequencing, and a nude mice subcutaneous transplantation model. ResultsWe found SHED cells can form microspheres with a dense internal structure. SHED microspheres exhibited notable advantages over SHED cells in terms of biological properties, maintaining cell activity and enhancing cell differentiation, migration, and stemness in vitro. RNA-seq revealed that the SHED microspheres potentially influenced cell development, regulation of neurogenesis, skeletal system development, tissue morphogenesis singling pathway. In vivo, SHED microspheres promoted the generation of pulp tissue in dental pulp compared to traditional 2D culture. ConclusionsMicrosphereization of SHED through 3D cell culture enhances its pulp regeneration capacity, presenting a novel strategy for dental pulp regeneration and the clinical treatment of dental pulp diseases.

Resveralogues protect HepG2 cells against cellular senescence induced by hepatotoxic metabolites

Progressive liver disease and dysfunction cause toxic metabolites including ammonia and unconjugated bilirubin to accumulate in plasma. As the population ages alternatives to liver transplantation become increasingly important. One approach for use as a bridge to transplant or recovery is the use of bioartificial liver systems (BALS) containing primary or immortalised hepatocytes as ex-vivo replacements or supports for endogenous liver function. However, exposure to the hepatotoxic metabolites present in plasma causes the rapid failure of these cells to carry out their primary metabolic functions despite remaining viable. Hypothesizing that this loss of core hepatocyte phenotypes was caused by cell senescence we exposed HepG2 cell populations, grown in both standard two-dimensional tissue culture systems and in three dimensional cultures on novel alginate modified HEMA-MBA cryogels, to physiologically reflective concentrations of hepatotoxic metabolites and cytokines. HepG2 cells are forced into senescence by the toxic metabolites in under six hours (as measured by loss of thymidine analog incorporation or detectable Ki67 staining) which is associated with a ten to twenty-fold reduction in the capacity of the cultures to synthesise albumin or urea. This state of senescence induced by liver toxins (SILT) can be prevented by preincubation with either 2–5 µM resveratrol, its major in vivo metabolite dihydroresveratrol or a series of novel resveralogues with differential capacities to scavenge radicals and activate SIRT1 (including V29 which does not interact with the protein). SILT appears to be a previously unrecognised barrier to the development of BALS which can now be overcome using small molecules that are safe for human use at concentrations readily achievable in vivo.

Differentiation, maturation, and collection of THP-1-derived dendritic cells based on a PEG hydrogel culture platform

PurposeDendritic cell (DC) is a spearhead responsible for immune response and surrounded by extracellular matrix in three-dimensional (3D) tissue. Nevertheless, conventional DC culture has relied on suspension or two-dimensional (2D) tissue culture plate (TCP)-based culture system. This culture condition often fails to recapitulate the physiological behavior of DC in real tissue. In this work, the effect of culture condition on DC physiology was explored with varying 3D hydrogel property (i.e., degradability, adhesion, and stiffness). In particular, DC differentiation and maturation in 3D were evaluated comparing the conventional TCP-based culture condition.MethodTHP-1 cells were encapsulated in poly(ethylene glycol) (PEG) hydrogel via thiol-ene photocrosslinking with non-degradable or proteolytically degradable peptide crosslinker. Hydrogel stiffness was manipulated by controlling the concentration of crosslinker. The metabolic activities and cytotoxicity of the encapsulated cells were measured by resazurin and Live/Dead assays, respectively. Cell harvesting was conducted via enzymatic degradation using α-chymotrypsin, and differentiation and maturation of the liberated DCs were evaluated by quantitative polymerase chain reaction and flow cytometry.ResultsTHP-1 cells well proliferated in the soft degradable hydrogel with a higher metabolic activity. However, the stiff matrix inhibited cell growth in 3D. The gene expression assay indicated that the 3D hydrogel condition was superior to 2D culture in terms of differentiation and maturation of DC. Interestingly, the stiffness of matrix was important factor in DC function. In the stiff hydrogel, the expression levels of differentiation and maturation markers were higher compared to the low stiffness hydrogel. The mature DCs caged in the hydrogel matrix were harvested after short enzymatic digestion of hydrogel and the liberated cells had over 90% viability. The flow cytometric result revealed that the proportion of CD80 + /CD86 + cells from the stiff hydrogel was relatively higher than cells either from 2D or soft hydrogel in 3D.ConclusionThe collected evidence indicated that the proteolytically degradable PEG hydrogel matrix promoted DC differentiation and maturation. In addition, the matrix stiffness control could manipulate the marker expressions of differentiation and maturation. Particularly, the mature DC was successfully collected from the hydrogel matrix. These results highlighted the PEG hydrogel-based DC culture might be a useful tool for potential DC-based immunotherapies.

Modeling immunity in microphysiological systems.

There is a need for better predictive models of the human immune system to evaluate safety and efficacy of immunomodulatory drugs and biologics for successful product development and regulatory approvals. Current in vitro models, which are often tested in two-dimensional (2D) tissue culture polystyrene, and preclinical animal models fail to fully recapitulate the function and physiology of the human immune system. Microphysiological systems (MPSs) that can model key microenvironment cues of the human immune system, as well as of specific organs and tissues, may be able to recapitulate specific features of the in vivo inflammatory response. This minireview provides an overview of MPS for modeling lymphatic tissues, immunity at tissue interfaces, inflammatory diseases, and the inflammatory tumor microenvironment in vitro and ex vivo. Broadly, these systems have utility in modeling how certain immunotherapies function in vivo, how dysfunctional immune responses can propagate diseases, and how our immune system can combat pathogens.

Patientenabgeleitete Tumororganoide – ein Therapiemodell für die Präzisionsonkologie

Organoids are three-dimensional, multicellular and stem-cell-derived in vitro tissue cultures with self-organizing and self-renewing capabilities. In the field of personalized treatment approaches, organoids can serve as a suitable treatment model for molecular and functional analyses, due to their high similarities to the primary tissue that they were derived from. Especially in the field of precision oncology, where a high demand for patient-specific therapy models remains, organoids can serve as an alternative to conventional two-dimensional tissue culture and patient-derived xenografts.

Studying T Cell Development in Neonatal and Adult Thymic Slices.

T cell development occurs in the thymus and is coordinated temporally and spatially within the highly complex thymic microenvironment. Therefore, T cell selection and maturation events cannot be fully recapitulated using traditional two-dimensional tissue culture in vitro. The thymic slice system provides a highly versatile system for studying T cell development ex vivo while preserving three-dimensional thymic architecture. Using the thymic slice system, T cell selection and maturation events can be visualized by live imaging and quantified by flow cytometry. Here we describe the process for generating slices from neonatal and adult mice.

A Novel 3D Helical Microelectrode Array for In Vitro Extracellular Action Potential Recording.

Recent advances in cell and tissue engineering have enabled long-term three-dimensional (3D) in vitro cultures of human-derived neuronal tissues. Analogous two-dimensional (2D) tissue cultures have been used for decades in combination with substrate integrated microelectrode arrays (MEA) for pharmacological and toxicological assessments. While the phenotypic and cytoarchitectural arguments for 3D culture are clear, 3D MEA technologies are presently inadequate. This is mostly due to the technical challenge of creating vertical electrical conduction paths (or ‘traces’) using standardized biocompatible materials and fabrication techniques. Here, we have circumvented that challenge by designing and fabricating a novel helical 3D MEA comprised of polyimide, amorphous silicon carbide (a-SiC), gold/titanium, and sputtered iridium oxide films (SIROF). Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) testing confirmed fully-fabricated MEAs should be capable of recording extracellular action potentials (EAPs) with high signal-to-noise ratios (SNR). We then seeded induced pluripotent stems cell (iPSC) sensory neurons (SNs) in a 3D collagen-based hydrogel integrated with the helical MEAs and recorded EAPs for up to 28 days in vitro from across the MEA volume. Importantly, this highly adaptable design does not intrinsically limit cell/tissue type, channel count, height, or total volume.

Electrodynamic assisted self-assembled fibrous hydrogel microcapsules: a novel 3D in vitro platform for assessment of nanoparticle toxicity.

Nanoparticle (NP) toxicity assessment is a critical step in assessing the health impacts of NP exposure to both consumers and occupational workers. In vitro assessment models comprising cells cultured in a two-dimensional tissue culture plate (2D-TCP) are an efficient and cost-effective choice for estimating the safety risks of NPs. However, in vitro culture of cells in 2D-TCPs distorts cell–integrin and cell–cell interactions and is not able to replicate an in vivo phenotype. Three-dimensional (3D) in vitro platforms provide a unique alternative to bridge the gap between traditional 2D in vitro and in vivo models. In this study, novel microcapsules of alginate hydrogel incorporated with natural polymeric nanofibers (chitin nanofibrils) and synthetic polymeric nanofibers poly(lactide-co-glycolide) are designed as a 3D in vitro platform. This study demonstrates for the first time that electrodynamic assisted self-assembled fibrous 3D hydrogel (3D-SAF hydrogel) microcapsules with a size in the range of 300–500 μm in diameter with a Young's modulus of 12.7–42 kPa can be obtained by varying the amount of nanofibers in the hydrogel precursor solutions. The 3D-SAF microcapsules were found to mimic the in vivo cellular microenvironment for cells to grow, as evaluated using A549 cells. Higher cellular spreading and prolonged proliferation of A549 cells were observed in 3D-SAF microcapsules compared to control microcapsules without the nanofibers. The 3D-SAF microcapsule integrated well plate was used to assess the toxicity of model NPs, e.g., Al2O3 and ZnO. The toxicity levels of the model NPs were found to be dependent on the chemistry of the NPs and their physical agglomeration in the test media. Our results demonstrate that 3D-SAF microcapsules with an in vivo mimicking microenvironment can be developed as a physiologically relevant platform for high-throughput toxicity screening of NPs or pharmaceutical drugs.

Biomimetic 3D Models for Investigating the Role of Monocytes and Macrophages in Atherosclerosis.

Atherosclerosis, the inflammation of artery walls due to the accumulation of lipids, is the most common underlying cause for cardiovascular diseases. Monocytes and macrophages are major cells that contribute to the initiation and progression of atherosclerotic plaques. During this process, an accumulation of LDL-laden macrophages (foam cells) and an alteration in the extracellular matrix (ECM) organization leads to a local vessel stiffening. Current in vitro models are carried out onto two-dimensional tissue culture plastic and cannot replicate the relevant microenvironments. To bridge the gap between in vitro and in vivo conditions, we utilized three-dimensional (3D) collagen matrices that allowed us to mimic the ECM stiffening during atherosclerosis by increasing collagen density. First, human monocytic THP-1 cells were embedded into 3D collagen matrices reconstituted at low and high density. Cells were subsequently differentiated into uncommitted macrophages (M0) and further activated into pro- (M1) and anti-inflammatory (M2) phenotypes. In order to mimic atherosclerotic conditions, cells were cultured in the presence of oxidized LDL (oxLDL) and analyzed in terms of oxLDL uptake capability and relevant receptors along with their cytokine secretomes. Although oxLDL uptake and larger lipid size could be observed in macrophages in a matrix dependent manner, monocytes showed higher numbers of oxLDL uptake cells. By analyzing major oxLDL uptake receptors, both monocytes and macrophages expressed lectin-like oxidized low-density lipoprotein receptor-1 (LOX1), while enhanced expression of scavenger receptor CD36 could be observed only in M2. Notably, by analyzing the secretome of macrophages exposed to oxLDL, we demonstrated that the cells could, in fact, secrete adipokines and growth factors in distinct patterns. Besides, oxLDL appeared to up-regulate MHCII expression in all cells, while an up-regulation of CD68, a pan-macrophage marker, was found only in monocytes, suggesting a possible differentiation of monocytes into a pro-inflammatory macrophage. Overall, our work demonstrated that collagen density in the plaque could be one of the major factors driving atherosclerotic progression via modulation of monocyte and macrophages behaviors.

Multifunctional biomimetic hydrogel systems to boost the immunomodulatory potential of mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) hold great therapeutic potential, in part because of their immunomodulatory properties. However, these properties can be transient and depend on multiple factors. Here, we developed a multifunctional hydrogel system to synergistically enhance the immunomodulatory properties of MSCs, using a combination of sustained inflammatory licensing and three-dimensional (3D) encapsulation in hydrogels with tunable mechanical properties. The immunomodulatory extracellular matrix hydrogels (iECM) consist of an interpenetrating network of click functionalized-alginate and fibrillar collagen, in which interferon γ (IFN-γ) loaded heparin-coated beads are incorporated. The 3D microenvironment significantly enhanced the expression of a wide panel of pivotal immunomodulatory genes in bone marrow-derived primary human MSCs (hMSCs), compared to two-dimensional (2D) tissue culture. Moreover, the inclusion of IFN-γ loaded heparin-coated beads prolonged the expression of key regulatory genes upregulated upon licensing, including indoleamine 2,3-dioxygenase 1 (IDO1) and galectin-9 (GAL9). At a protein level, iECM hydrogels enhanced the secretion of the licensing responsive factor Gal-9 by hMSCs. Its presence in hydrogel conditioned media confirmed the correct release and diffusion of the factors secreted by hMSCs from the system. Furthermore, co-culture of iECM-encapsulated hMSCs and activated human T cells resulted in suppressed proliferation, demonstrating direct regulation on immune cells. These data highlight the potential of iECM hydrogels to enhance the immunomodulatory properties of hMSCs in cell therapies.

Three-dimensional culture models to study drug resistance in breast cancer.

Despite recent advances in breast cancer treatment, drug resistance frequently presents as a challenge, contributing to a higher risk of relapse and decreased overall survival rate. It is now generally recognized that the extracellular matrix and cellular heterogeneity of the tumor microenvironment influences the cancer cells' ultimate fate. Therefore, strategies employed to examine mechanisms of drug resistance must take microenvironmental influences, as well as genetic mutations, into account. This review discusses three-dimensional (3D) in vitro model systems which incorporate microenvironmental influences to study mechanisms of drug resistance in breast cancer. These bioengineered models include spheroid-based models, biomaterial-based models such as polymeric scaffolds and hydrogels, and microfluidic chip-based models. The advantages of these model systems over traditionally studied two-dimensional tissue culture polystyrene are examined. Additionally, the applicability of such 3D models for studying the impact of tumor microenvironment signals on drug response and/or resistance is discussed. Finally, the potential of such models for use in the development of strategies to combat drug resistance and determine the most promising treatment regimen is explored.

Modeling the Efficacy of Oncolytic Adenoviruses In Vitro and In Vivo: Current and Future Perspectives

Oncolytic adenoviruses (OAd) selectively target and lyse tumor cells and enhance anti- tumor immune responses. OAds have been used as promising cancer gene therapies for many years and there are a multitude of encouraging pre-clinical studies. However, translating OAd therapies to the clinic has had limited success, in part due to the lack of realistic pre-clinical models to rigorously test the efficacy of OAds. Solid tumors have a heterogenous and hostile microenvironment that provides many barriers to OAd treatment, including structural and immunosuppressive components that cannot be modeled in two-dimensional tissue culture. To replicate these characteristics and bridge the gap between pre-clinical and clinical success, studies must test OAd therapy in three-dimensional culture and animal models. This review focuses on current methods to test OAd efficacy in vitro and in vivo and the development of new model systems to test both oncolysis and immune stimulatory components of oncolytic adenovirotherapy.

Engineering of Primary Pancreatic Islet Cell Spheroids for Three-dimensional Culture or Transplantation: A Methodological Comparative Study

Three-dimensional (3D) cell culture by engineering spheroids has gained increasing attention in recent years because of the potential advantages of such systems over conventional two-dimensional (2D) tissue culture. Benefits include the ability of 3D to provide a more physiologically relevant environment, for the generation of uniform, size-controlled spheroids with organ-like microarchitecture and morphology. In recent years, different techniques have been described for the generation of cellular spheroids. Here, we have compared the efficiency of four different methods of islet cell aggregation. Rat pancreatic islets were dissociated into single cells before reaggregation. Spheroids were generated either by (i) self-aggregation in nonadherent petri dishes, (ii) in 3D hanging drop culture, (iii) in agarose microwell plates or (iv) using the Sphericalplate 5D™. Generated spheroids consisted of 250 cells, except for the self-aggregation method, where the number of cells per spheroid cannot be controlled. Cell function and morphology were assessed by glucose stimulated insulin secretion (GSIS) test and histology, respectively. The quantity of material, labor intensity, and time necessary for spheroid production were compared between the different techniques. Results were also compared with native islets. Native islets and self-aggregated spheroids showed an important heterogeneity in terms of size and shape and were larger than spheroids generated with the other methods. Spheroids generated in hanging drops, in the Sphericalplate 5D™, and in agarose microwell plates were homogeneous, with well-defined round shape and a mean diameter of 90 µm. GSIS results showed improved insulin secretion in response to glucose in comparison with native islets and self-aggregated spheroids. Spheroids can be generated using different techniques and each of them present advantages and inconveniences. For islet cell aggregation, we recommend, based on our results, to use the hanging drop technique, the agarose microwell plates, or the Sphericalplate 5D™ depending on the experiments, the latter being the only option available for large-scale spheroids production.

End-to-End Platform for Human Pluripotent Stem Cell Manufacturing.

Industrialization of stem-cell based therapies requires innovative solutions to close the gap between research and commercialization. Scalable cell production platforms are needed to reliably deliver the cell quantities needed during the various stages of development and commercial supply. Human pluripotent stem cells (hPSCs) are a key source material for generating therapeutic cell types. We have developed a closed, automated and scalable stirred tank bioreactor platform, capable of sustaining high fold expansion of hPSCs. Such a platform could facilitate the in-process monitoring and integration of online monitoring systems, leading to significantly reduced labor requirements and contamination risk. hPSCs are expanded in a controlled bioreactor using perfused xeno-free media. Cell harvest and concentration are performed in closed steps. The hPSCs can be cryopreserved to generate a bank of cells, or further processed as needed. Cryopreserved cells can be thawed into a two-dimensional (2D) tissue culture platform or a three-dimensional (3D) bioreactor to initiate a new expansion phase, or be differentiated to the clinically relevant cell type. The expanded hPSCs express hPSC-specific markers, have a normal karyotype and the ability to differentiate to the cells of the three germ layers. This end-to-end platform allows a large scale expansion of high quality hPSCs that can support the required cell demand for various clinical indications.

The Mini-Organo: A rapid high-throughput 3D coculture organotypic assay for oncology screening and drug development.

BackgroundThe use of in vitro cell cultures is a powerful tool for obtaining key insights into the behaviour and response of cells to interventions in normal and disease situations. Unlike in vivo settings, in vitro experiments allow a fine‐tuned control of a range of microenvironmental elements independently within an isolated setting. The recent expansion in the use of three‐dimensional (3D) in vitro assays has created a number of representative tools to study cell behaviour in a more physiologically 3D relevant microenvironment. Complex 3D in vitro models that can recapitulate human tissue biology are essential for understanding the pathophysiology of disease.AimThe development of the 3D coculture collagen contraction and invasion assay, the “organotypic assay,” has been widely adopted as a powerful approach to bridge the gap between standard two‐dimensional tissue culture and in vivo mouse models. In the cancer setting, these assays can then be used to dissect how stromal cells, such as cancer‐associated fibroblasts (CAFs), drive extracellular matrix (ECM) remodelling to alter cancer cell behaviour and response to intervention. However, to date, many of the published organotypic protocols are low‐throughput, time‐consuming (up to several weeks), and work‐intensive with often limited scalability. Our aim was to develop a fast, high‐throughput, scalable 3D organotypic assay for use in oncology screening and drug development.Methods and resultsHere, we describe a modified 96‐well organotypic assay, the “Mini‐Organo,” which can be easily completed within 5 days. We demonstrate its application in a wide range of mouse and human cancer biology approaches including evaluation of stromal cell 3D ECM remodelling, 3D cancer cell invasion, and the assessment of efficacy of potential anticancer therapeutic targets. Furthermore, the organotypic assay described is highly amenable to customisation using different cell types under diverse experimental conditions.ConclusionsThe Mini‐Organo high‐throughput 3D organotypic assay allows the rapid screening of potential cancer therapeutics in human and mouse models in a time‐efficient manner.

Abstract 5173: Development of a novel tunable synthetic 3D hydrogel platform for the study of tumor and stromal cell interactions

Abstract Numerous cancer cell models exist used to investigate disease mechanisms and to screen potential cancer therapeutics. Roughly 90% of promising preclinical drugs fail to result in efficacious human treatments. Traditional two-dimensional (2D) tissue culture models lack realistic complexity, while animal models are expensive, time consuming, and too frequently fail to reflect human tumor biology. Recently, three-dimensional (3D) cell culture models are a new method to generate new drug candidates before moving to expensive and time-consuming animal models. We have developed a biochemically defined hydrogel platform formed by mixing various polymers with chemical crosslinkers with enhanced functionality. The hydrogel system employs one of two types of backbone polymers: a synthetic non-degradable polyvinyl alcohol (PVA) or an enzymatically-degradable dextran. Both polymers are functionalized either with fast or slow thiol-reactive groups. Crosslinkers consist of either PEG non-cell-degradable or a CD cell-degradable crosslinkers containing peptide sequences that create cleavage sites for matrix metalloproteases (MMPs) that allow cell migration. The technology provides mechanical and biochemical cues to investigate both morphological and physiological properties of cells in a 3D environment. The hydrogel allows precise control over hydrogel stiffness, gelation speed, cell migration and allows cell recovery for downstream applications. Here, we demonstrate the utility of this hydrogel platform to grow epithelial, fibroblast and tumor cells in 3D cell cultures with high cell viabilities and functionalities. Furthermore, we have constructed a 3D tumor/stromal cell co-culture using the hydrogel to model the dynamic tumor cell microenvironment. This technology will allow the creation of more accurate 3D cancer cell models for basic research and drug discovery applications. Note: This abstract was not presented at the meeting. Citation Format: Hsu-Kun Wang, Vi Chu, Nick Asbrock. Development of a novel tunable synthetic 3D hydrogel platform for the study of tumor and stromal cell interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5173.

A Melanoma Patient-Derived Xenograft Model.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Peptide-mediated delivery of therapeutic mRNA in ovarian cancer

Ovarian cancer is the most lethal gynecological malignancy in the developed world. In spite of intensive research, the mortality has hardly decreased over the past twenty years. This necessitates the exploration of novel therapeutic modalities. Transient protein expression through delivery of mRNA is emerging as a highly promising option. In contrast to gene therapy there is no risk of integration into the genome. Here, we explore the expression of mRNA in models of ovarian cancer of increasing complexity. The cell-penetrating peptide (CPP) PepFect 14 (PF14) was used to formulate CPP-mRNA nanoparticles. Efficient expression of a reporter protein was achieved in two-dimensional tissue cultures and in three-dimensional cancer cell spheroids. PF14 nanoparticles greatly outperformed a lipid-based transfection agent in vivo, leading to expression in various cell types of tumor associated tissue. Protein expression was restricted to the peritoneal cavity. Messenger RNA expression across different cell types was confirmed in primary ovarian cancer explants. As ovarian cancer is confined to the peritoneal cavity in most cases, the results create the basis for applications in which the tumor microenvironment is transiently modified through protein expression.

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D.

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

3D matrix-embedding inhibits cycloheximide-mediated sensitization to TNF-alpha-induced apoptosis of human endothelial cells.

The programmed form of cell death (apoptosis) is essential for normal development of multicellular organisms. Dysregulation of apoptosis has been linked with embryonal death and is involved in the pathophysiology of various diseases. Others and we previously demonstrated endothelial biology being intertwined with biochemical and structural composition of the subendothelial basement membrane. We now demonstrate that a three-dimensional growing environment significantly shields endothelial cells from cytokine-induced apoptosis. Detailed analysis reveals differences in intracellular signaling pathways in naive endothelial cells and cytokine-stimulated endothelial cells when cells are grown within a three-dimensional collagen-based matrix compared to cells grown on two-dimensional tissue culture plates. Main findings are significantly reduced p53 expression and level of p38-phosphorylation in three-dimensional grown endothelial cells. Despite similar concentrations of focal adhesion kinase, three-dimensional matrix-embedded endothelial cells express significantly less tyrosine-phosphorylated focal adhesion kinase. Pretreatment with antibodies against integrin αv β3 partially reversed the protective effect of three-dimensional matrix-embedding on endothelial apoptosis. Our findings provide detailed insights into the mechanisms of endothelial apoptosis with respect to the spatial matrix environment. These results enhance our understanding of endothelial biology and may otherwise help in the design of tissue-engineered materials. Furthermore, findings on focal adhesion kinase phosphorylation might enhance our understanding of clinical studies with tyrosine kinase inhibitors.