Three-dimensional Co-culture Research Articles

Host-Pathogen Interaction Reconstituted in Three-Dimensional Cocultures of Mucosa and Candida albicans.

Candida albicans frequently causes recurrent intimal infectious disease (ID). This demands the treatment of multiple phases of the infection. The objective of this study was to uncover the host-pathogen interaction using two-dimensional (2D) epithelium cell-barrier and three-dimensional (3D) subepithelium tissue cells of human mucosa. The 2D cell cultures assessed C. albicans adhesion. Addition of the antifungal drug Fluconazol did not inhibit the adhesion, despite its pathogen growth inhibition (minimal inhibitory concentration value 0.08 μg/mL). A 3D tissue was engineered in multitranswells by placing human fibroblast cultures on a thick porous scaffold. This contained the yeast placed in the top compartment and prevented passive penetration. After 28 h, the pathogen transmigrated the barrier and was collected in the bottom compartment. A change in pathogen morphology was observed where hypha formed and grew to be 231 μm long after 28 h. The hypha was thus long enough to cross the 200 μm thick 3D tissue. The 3D infection was inhibited by addition of Fluconazol (0.08 μg/mL), confirming that penetration is dependent on pathogen growth. In conclusion, ID was reconstituted step-by-step on 2D epithelium surface and in 3D connective tissue of human mucosa. Fluconazol growth-inhibition of the pathogen C. albicans was confirmed in the 3D tissue. We thus propose that this ID in vitro test is suitable for the identification and characterization of new treatments against C. albicans.

PO-304 Three-dimensional co-culture of colorectal cancer spheroid with cancer-associated fibroblast as a model to study immune cell modulation

IntroductionColorectal cancer (CRC) is a complex disease in which inflammation is often a prominent feature. Hence, it serves as an appropriate paradigm to unveil the clinical importance of tumour microenvironment especially the infiltrating immune and inflammatory cells in the pathology of the disease. A 3-dimensional (3D) in vitro spheroid co-culture model that allows the interaction with stromal components is considered an essential element in developing a more clinically relevant tumour model. Hence, in this project, we aim to establish a model consisting of different subtypes of CRC and cancer-associated fibroblast (CAF), a major component of the tumour microenvironment, to study the role of immune cells, for example, macrophages, in CRC biology.Material and methodsCRC cell lines, HCT116 and DLD-1, and cancer-associated fibroblast (CAF) isolated from CRC patient tumour tissue were seeded together at the total number 2000 cells/well on 96-well round bottom ultra-low cluster/attachment plates at different ratios (1:1 and 1:2) to form tumour spheroids. Then, different numbers of monocytes isolated from a healthy human volunteer were added (100, 250 and 500 cells) into the CRC-CAF co-culture system. The CRC cells and CAF were stably transduced with TdTomato and GFP fluorescent proteins respectively for microscopic visualisation and monitoring of growth. Spheroid growth was also monitored by volumetric measurement using image analysis software (Fiji).Results and discussionsCo-culture of HCT116 showed that the presence of CAF increased CRC spheroid growth compared to monoculture of the cancer cells. Meanwhile, no growth stimulation of CAF-enriched culture of DLD-1 cells was observed despite the smaller size of the spheroids. Similarly, no enhancement of HCT116 growth was observed when monocytes were added to the co-cultures yet this resulted in the more disrupted morphology of spheroids.ConclusionThis preliminary study highlights the differential enhancement of tumour growth by CAF stimuli on different types of CRC. Further studies on characterisation of the spheroids containing monocytes and the monocyte differentiation are required to investigate their effect on tumour biology. The findings will be correlated to the observation found in our cohort of patient tissues.

Advanced in vitro models of vascular biology: Human induced pluripotent stem cells and organ-on-chip technology.

The vascular system is one of the first to develop during embryogenesis and is essential for all organs and tissues in our body to develop and function. It has many essential roles including controlling the absorption, distribution and excretion of compounds and therefore determines the pharmacokinetics of drugs and therapeutics. Vascular homeostasis is under tight physiological control which is essential for maintaining tissues in a healthy state. Consequently, disruption of vascular homeostasis plays an integral role in many disease processes, making cells of the vessel wall attractive targets for therapeutic intervention. Experimental models of blood vessels can therefore contribute significantly to drug development and aid in predicting the biological effects of new drug entities. The increasing availability of human induced pluripotent stem cells (hiPSC) derived from healthy individuals and patients have accelerated advances in developing experimental in vitro models of the vasculature: human endothelial cells (ECs), pericytes and vascular smooth muscle cells (VSMCs), can now be generated with high efficiency from hiPSC and used in 'microfluidic chips' (also known as 'organ-on-chip' technology) as a basis for in vitro models of blood vessels. These near physiological scaffolds allow the controlled integration of fluid flow and three-dimensional (3D) co-cultures with perivascular cells to mimic tissue- or organ-level physiology and dysfunction in vitro. Here, we review recent multidisciplinary developments in these advanced experimental models of blood vessels that combine hiPSC with microfluidic organ-on-chip technology. We provide examples of their utility in various research areas and discuss steps necessary for further integration in biomedical applications so that they can be contribute effectively to the evaluation and development of new drugs and other therapeutics as well as personalized (patient-specific) treatments.

Comparative effectiveness of three-dimensional scaffold, differentiation media and co-culture with native cardiomyocytes to trigger in vitro cardiogenic differentiation of menstrual blood and bone marrow stem cells

The main purpose of this study was to find effectiveness of 3D silk fibroin scaffold in comparison with co-culturing in presence of native cardiomyocytes on cardiac differentiation propensity of menstural blood(MenSCs)-versus bone marrow-derived stem-cells (BMSCs). We showed that both 3D fibroin scaffold and co-culture system supported efficient cardiomyogenic differentiation of MenSCs and BMSCs, as judged by the expression of cardiac-specific genes and proteins, Connexin-43, Connexin-40, alpha Actinin (ACTN-2), Tropomyosin1 (TPM1) and Cardiac Troponin T (TNNT2). No significant difference (except for higher expression of ACTN-2 in co-cultured MenSCs) was found between differentiation potential of the cells cultured in 3D fibroin scaffold and co-culture system. Collectively, our results imply that inductive signals served by biological factors of native cardiomyocytes to trigger cardiogenic differentiation of stem-cells may be efficiently provided by natural and biocompatible 3D fibroin scaffold suggesting the usefulness of this construct for cardiac tissue engineering.

Stratification of mouse vaginal epithelium. 1. Development of three-dimensional models in vitro with clonal cell lines.

The mouse vagina consists of stratified squamous epithelium and stroma and is regulated by ovarian hormones. Vaginal epithelial cells do not stratify, but rather form a monolayer and show an inconsistent responsiveness to ovarian hormones when cultured on plastic dish or matrix. To address the discrepancy between in vivo and in vitro observations, three-dimensional (3D) co-culture models are developed with clonal vaginal epithelial and stromal cell lines; stromal cells are embedded in collagen gel and epithelial cells are seeded on the gel. In the 3D models, epithelial cells express Transformation related protein 63 (Trp63) and begin to stratify when they are co-cultured with two out of three stromal cell lines, but not with the other stromal cell line. Stroma may consist of various types of cells with distinct functions.

Biomimetic models to examine microenvironmental regulation of glioblastoma stem cells

Glioblastoma multiforme (GBM), a malignant brain tumor, is the deadliest form of human cancer with low survival rates because of its highly invasive nature. In recent years, there has been a growing appreciation for the role that glioblastoma stem cells (GSCs) play during tumorigenesis and tumor recurrence of GBM. GSCs are a specialized subset of GBM cells with stem cell-like features that contribute to tumor initiation and therapeutic resistance. Thus, to enhance therapeutic efficiency and improve survival, targeting GSCs and their microenvironmental niche appears to be a promising approach. To develop this approach, understanding GSC-microenvironment interactions is crucial. This review discusses various biomimetic model systems to understand the impact of biophysical, biochemical, and cellular microenvironmental cues on GSC behaviors. These models include two-dimensional or matrix-free environment models, engineered biomaterial-based three-dimensional models, co-culture models, and mouse and rat in vivo models. These systems have been used to study the effects of biophysical factors, modulation of signaling pathways, extracellular matrix components, and culture conditions on the GSC phenotype. The advantages and disadvantages of these model systems and their impact in the field of GSC research are discussed.

RIP1 regulates TNF-α-mediated lymphangiogenesis and lymphatic metastasis in gallbladder cancer by modulating the NF-κB-VEGF-C pathway.

BackgroundTumor necrosis factor alpha (TNF-α) enhances lymphangiogenesis in gallbladder carcinoma (GBC) via activation of nuclear factor (NF-κB)-dependent vascular endothelial growth factor-C (VEGF-C). Receptor-interacting protein 1 (RIP1) is a multifunctional protein in the TNF-α signaling pathway and is highly expressed in GBC. However, whether RIP1 participates in the signaling pathway of TNF-α-mediated VEGF-C expression that enhances lymphangiogenesis in GBC remains unclear.MethodsThe RIP1 protein levels in the GBC-SD and NOZ cells upon stimulation with increasing concentrations of TNF-α as indicated was examined using Western blot. Lentiviral RIP1 shRNA and siIκBα were constructed and transduced respectively them into NOZ and GBC-SD cells, and then PcDNA3.1-RIP1 vectors was transduced into siRIP1 cell lines to reverse RIP1 expression. The protein expression of RIP1, inhibitor of NF-κB alpha (IκBα), p-IκBα, TAK1, NF-κB essential modulator were examined through immunoblotting or immunoprecipitation. Moreover, VEGF-C mRNA levels were measured by quantitative real-time polymerase chain reaction, VEGF-C protein levels were measured by immunoblotting and enzyme-linked immunosorbent assay, and VEGF-C promoter and NF-κB activities were quantified using a dual luciferase reporter assay. The association of NF-κB with the VEGF-C promoter was analysed by chromatin immunoprecipitation assay. A three-dimensional coculture method and orthotopic transplantation nude mice model were used to evaluate lymphatic tube-forming and metastasis ability in GBC cells. The expression of RIP1 protein, TNF-α protein and lymphatic vessels in human GBC tissues was examined by immunohistochemistry, and the dependence between RIP1 protein with TNF-α protein and lymphatic vessel density was analysed.ResultsTNF-α dose- and time-dependently increased RIP1 protein expression in the GBC-SD and NOZ cells of GBC, and the strongest effect was observed with a concentration of 50 ng/ml. RIP1 is fundamental for TNF-α-mediated NF-κB activation in GBC cells and can regulate TNF-α-mediated VEGF-C expression at the protein and transcriptional levels through the NF-κB pathway. RIP1 can regulate TNF-α-mediated lymphatic tube formation and metastasis in GBC cells both in vitro and vivo. The average optical density of RIP1 was linearly related to that of TNF-α protein and the lymphatic vessel density in GBC tissues.ConclusionWe conclude that RIP1 regulates TNF-α-mediated lymphangiogenesis and lymph node metastasis in GBC by modulating the NF-κB-VEGF-C pathway.

The application of the improved 3D rat testicular cells co-culture model on the in vitro toxicity research of HZ1006

The aims of the present research are to further validate the application of the improved three-dimensional (3 D) rat testicular cell co-culture model to evaluate the effects of various reprotoxic chemicals on the function of the main somatic cells, as well as on spermatogonial cell differentiation and even spermatogenesis, and to investigate the specific toxicant mechanisms in testes treated with HZ1006, a hydroxamate-based a hydroxamate-based histone deacetylase inhibitor (HDACI). Based on the characteristics of HZ1006, the appropriate exposure duration (8, 16, or 24 days), dosage (0, 3.125, 6.25, 12.5, or 25 μM) and toxic endpoints suitable for detection were selected in the experiments. The results showed inhibition of cell proliferation, reduced testosterone levels, and decreased spermatogonial cell meiosis-specific gene expression, as well as decreased protein levels of androgen receptor (AR) and decreased expression of the AR target gene PSA, accompanied by inhibition of Hdac6 expression after HZ1006 exposure in the 3 D rat testicular cell co-culture model. These findings indicate that the improved 3 D rat testicular cell co-culture model we have established has the potential to become a new testicular toxicity test system that can be used to test toxic characteristics and mechanisms of new compounds and has good application prospects, although more research on the model is required.

A microfluidic device for studying chemotaxis mechanism of bacterial cancer targeting

Bacterial cancer targeting may become an efficacious cancer therapy, but the mechanisms underlying bacterial specificity for cancer cells need to be explored prior to adopting it as a new clinical application. To characterize the mechanism of bacterial chemotactic preference towards cancer cells, we developed a microfluidic device for in vitro study. The device consists of a cell culture chamber on both sides of a central bacteria channel, with micro-channels used as barriers between them. The device, when used as model for lung cancer, was able to provide simultaneous three-dimensional co-culture of multiple cell lines in separate culture chambers, and when used as model for bacterial chemotaxis, established constant concentration gradients of biochemical compounds in a central channel by diffusion through micro-channels. Fluorescence intensity of green fluorescence protein (GFP)-encoding bacteria was used to measure bacterial taxis behavior due to established chemotactic gradients. Using this platform, we found that Escherichia coli (E. coli) clearly illustrated the preference for lung cancer cells (NCI-H460) which was attributed to biochemical factors secreted by carcinoma cells. Furthermore, by secretome analysis and validation experiments, clusterin (CLU) was found as a key regulator for the chemotaxis of E. coli in targeting lung cancer.

Development and Evaluation of a Tissue-Engineered Fibrin-based Canine Mitral Valve Three-dimensional Cell Culture System

Myxomatous mitral valve disease is the most common cardiac disease of the dog, but examination of the associated cellular and molecular events has relied on the use of cadaveric valve tissue, in which functional studies cannot be undertaken. The aim of this study was to develop a three-dimensional (3D) cell co-culture model as an experimental platform to examine disease pathogenesis. Mitral valve interstitial (VIC) and endothelial (VEC) cells were cultured from normal and diseased canine (VIC only) valves. VICs were embedded in a fibrin-based hydrogel matrix and one surface was lined with VECs. The 3D static cultures (constructs) were examined qualitatively and semiquantitatively by light microscopy, immunofluorescence microscopy and protein immunoblotting. Some constructs were manipulated and the endothelium damaged, and the response examined. The construct gross morphology and histology demonstrated native tissue-like features and comparable expression patterns of cellular (α-smooth muscle actin [SMA] and embryonic smooth muscle myosin heavy chain [SMemb]) and extracellular matrix associated markers (matrix metalloproteinase [MMP]-1 and MMP-3), reminiscent of diseased valves. There were no differences between constructs containing normal valve VICs and VECs (type 1) and those containing diseased valve VICs and normal valve VECs (type 2). Mechanical manipulation and endothelial damage (type 3) tended to decrease α-SMA and SMemb expression, suggesting reversal of VIC activation, but with retention of SMemb+ cells adjacent to the wounded endothelium consistent with response to injury. Fibrin-based 3D mitral valve constructs can be produced using primary cell cultures derived from canine mitral valves, and show a phenotype reminiscent of diseased valves. The constructs demonstrate a response to endothelial damage indicating their utility as experimental platforms.

Kinin-B1 Receptor Stimulation Promotes Invasion and is Involved in Cell-Cell Interaction of Co-Cultured Glioblastoma and Mesenchymal Stem Cells

Glioblastoma multiforme (GBM) represents the most lethal brain tumour, and these tumours have very limited treatment options. Mesenchymal stem cells (MSC) are considered as candidates for advanced cell therapies, due to their tropism towards GBM, possibly affecting their malignancy, thus also representing a potential therapeutic vector. Therefore, we aimed to compare the effects of bone-marrow-derived versus adipose-tissue-derived MSC (BM-/AT-MSC) on heterogeneous populations of tumour cells. This cells’ interplay was addressed by the in-vitro two-dimensional (monolayer) and three-dimensional (spheroid) co-culture models, using U87 and U373 GBM cell lines, expressing genotypically different mesenchymal transcriptome profiles. U87 cell low mesenchymal profile expressed high levels of kinin receptor 1 (B1R) and their invasion was greatly enhanced by the B1R agonist des-Arg9-bradykinin upon BM-MSC co-culturing in 3D co-cultures. This correlated to significantly higher cell-cell interactions in U87/BM-MSC mixed spheroids. This was not observed with the U373 cells and not in AT-MSC co-cultures. Altogether, these data support the on-going exploration of B1R as target for adjuvant approach in GBM therapy. Secondly, the results emphasize the need for further careful exploration of the selectivity regarding the origin of MSC as potential candidates for cell therapies, particular in cancer, where they may adversely affect heterogeneous tumour cell populations.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System.

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1β) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Investigating the Vascular Niche: Three-Dimensional Co-culture of Human Skeletal Muscle Stem Cells and Endothelial Cells.

Angiogenesis, the growth of new blood vessels, is crucial for efficient skeletal muscle regeneration. Myogenesis and angiogenesis take place concomitantly during muscle regeneration. Myogenic precursor cells (MPCs) are in close proximity to vessels and interact with neighboring endothelial cells (ECs) to expand and differentiate. To demonstrate functional interplay between the two cell types, we established a robust and predictive ex vivo assay to evaluate activity of MPCs on angiogenesis and vice-et-versa, of ECs on myogenesis. Here, we describe an optimized three-dimensional co-culture protocol for the assessment of biological interactions between MPCs and ECs during skeletal muscle regeneration.

3D cellular structures and co-cultures formed through the contactless magnetic manipulation of cells on adherent surfaces.

A magnet array is employed to manipulate diamagnetic cells that are contained in paramagnetic medium to demonstrate for the first time the contactless bioprinting of three-dimensional (3D) cellular structures and co-cultures of breast cancer MCF-7 and endothelial HUVEC at prescribed locations on tissue culture treated well plates. Sequential seeding of different cell lines and the spatial displacement of the magnet array creates co-cultured cellular structures within a well without using physically intrusive well inserts. Both monotypic and co-culture experiments produce morphologically rich 3D cell structures that are otherwise absent in regular monolayer cell cultures. The magnetic contactless bioprinting of cells provides further insight into cell behaviour, invasion strategies and transformations that are useful for potential applications in drug screening, 3D cell culture formation and tissue engineering.

Long Non-Coding RNA H19 Acts as an Estrogen Receptor Modulator that is Required for Endocrine Therapy Resistance in ER+ Breast Cancer Cells

Background/Aims: Blocking estrogen signaling with endocrine therapies (Tamoxifen or Fulverstrant) is an effective treatment for Estrogen Receptor-α positive (ER⁺) breast cancer tumours. Unfortunately, development of endocrine therapy resistance (ETR) is a frequent event resulting in disease relapse and decreased overall patient survival. The long noncoding RNA, H19, was previously shown to play a significant role in estrogen-induced proliferation of both normal and malignant ER⁺ breast epithelial cells. We hypothesized that H19 expression is also important for the proliferation and survival of ETR cells. Methods: Here we utilized established ETR cell models; the Tamoxifen (Tam)-resistant LCC2 and the Fulvestrant and Tam cross-resistant LCC9 cells. Gain and loss of H19 function were achieved through lentiviral transduction as well as pharmacological inhibitors of the Notch and c-Met receptor signaling pathways. The effects of altered H19 expression on cell viability and ETR were assessed using three-dimensional (3D) organoid cultures and 2D co-cultures with low passage tumour-associated fbroblasts (TAFs). Results: Here we report that treating ETR cells with Tam or Fulvestrant increases H19 expression and that it’s decreased expression overcomes resistance to Tam and Fulvestrant in these cells. Interestingly, H19 expression is regulated by Notch and HGF signaling in the ETR cells and pharmacological inhibitors of Notch and c-MET signaling together significantly reverse resistance to Tam and Fulvestrant in an H19-dependent manner in these cells. Lastly, we demonstrate that H19 regulates ERα expression at the transcript and protein levels in the ETR cells and that H19 protects ERα against Fulvestrant-mediated downregulation of ERα protein. We also observed that blocking Notch and the c-MET receptor signaling also overcomes Fulvestrant and Tam resistance in 3D organoid cultures by decreasing ERα and H19 expression in the ETR cells. Conclusion: In endocrine therapy resistant breast cancer cells Fulvestrant is ineffective in decreasing ERα levels. Our data suggest that in the ETR cells, H19 expression acts as an ER modulator and that its levels and subsequently ERα levels can be substantially decreased by blocking Notch and c-MET receptor signaling. Consequently, treating ETR cells with these pharmacological inhibitors helps overcome resistance to Fulvestrant and Tamoxifen.

Three-Dimensional Co-culture of Human Hematopoietic Stem/Progenitor Cells and Mesenchymal Stem/Stromal Cells in a Biomimetic Hematopoietic Niche Microenvironment.

The development of cellular therapies to treat hematological malignancies has motivated researchers to investigate ex vivo culture systems capable of expanding the number of hematopoietic stem/progenitor cells (HSPC) before transplantation. The strategies exploited to achieve relevant cell numbers have relied on culture systems that lack biomimetic niche cues thought to be essential to promote HSPC maintenance and proliferation. Although stromal cells adhered to 2-D surfaces can be used to support the expansion of HSPC ex vivo, culture systems aiming to incorporate cell-cell interactions in a more intricate 3-D environment can better contribute to recapitulate the bone marrow (BM) hematopoietic niche in vitro.Herein, we describe the development of a 3-D co-culture system of human umbilical cord blood (UCB)-derived CD34+ cells and BM mesenchymal stem/stromal cell (MSC) spheroids in a microwell-based platform that allows to attain large numbers of spheroids with uniform sizes. Further comparison with a traditional 2-D co-culture system exploiting the supportive features of feeder layers of MSC is provided, while functional in vitro assays to assess the features of HSPC expanded in the 2-D vs. 3-D MSC co-culture systems are suggested.

Effect of adipocyte on invasion process of breast tumor cells in three-dimensional co-culture model

Effect of adipocyte on invasion process of breast tumor cells in three-dimensional co-culture model

Three-dimensional organotypic matrices from alternative collagen sources as pre-clinical models for cell biology

Organotypic co-cultures bridge the gap between standard two-dimensional culture and mouse models. Such assays increase the fidelity of pre-clinical studies, to better inform lead compound development and address the increasing attrition rates of lead compounds within the pharmaceutical industry, which are often a result of screening in less faithful two-dimensional models. Using large-scale acid-extraction techniques, we demonstrate a step-by-step process to isolate collagen I from commercially available animal byproducts. Using the well-established rat tail tendon collagen as a benchmark, we apply our novel kangaroo tail tendon collagen as an alternative collagen source for our screening-ready three-dimensional organotypic co-culture platform. Both collagen sources showed equal applicability for invasive, proliferative or survival assessment of well-established cancer models and clinically relevant patient-derived cancer cell lines. Additional readouts were also demonstrated when comparing these alternative collagen sources for stromal contributions to stiffness, organization and ultrastructure via atomic force microscopy, second harmonic generation imaging and scanning electron microscopy, among other vital biological readouts, where only minor differences were found between the preparations. Organotypic co-cultures represent an easy, affordable and scalable model to investigate drug responses within a physiologically relevant 3D platform.

Subcellular Interactions during Vascular Morphogenesis in 3D Cocultures between Endothelial Cells and Fibroblasts.

Background: Increasing the complexity of in vitro systems to mimic three-dimensional tissues and the cellular interactions within them will increase the reliability of data that were previously collected with in vitro systems. In vivo vascularization is based on complex and clearly defined cell–matrix and cell–cell interactions, where the extracellular matrix (ECM) seems to play a very important role. The aim of this study was to monitor and visualize the subcellular and molecular interactions between endothelial cells (ECs), fibroblasts, and their surrounding microenvironment during vascular morphogenesis in a three-dimensional coculture model. Methods: Quantitative and qualitative analyses during the generation of a coculture tissue construct consisting of endothelial cells and fibroblasts were done using transmission electron microscopy and immunohistochemistry. Results: Dynamic interactions were found in cocultures between ECs, between fibroblasts (FBs), between ECs and FBs, and between the cells and the ECM. Microvesicles were involved in intercellular information transfer. FBs took an active and physical part in the angiogenesis process. The ECM deposited by the cells triggered endothelial angiogenic activity. Capillary-like tubular structures developed and matured. Moreover, some ECM assembled into a basement membrane (BM) having three different layers equivalent to those seen in vivo. Finally, the three-dimensional in vitro construct mirrored the topography of histological tissue sections. Conclusion: Our results visualize the importance of the physical contact between all cellular and acellular components of the cocultures.

Three-Dimensional Co-Culture Bioassay for Screening of Retinal Gene Delivery Systems.

Herein we describe a three-dimensional co-culture bioassay protocol designed to assess the therapeutic potential of the proteins expressed from gene delivery transfected cells through the evaluation of expressed protein bioavailability and bioactivity. Using a combination of enzyme-linked immunosorbent assay (ELISA) and immunofluorescent-based neurite length profiling methodologies, the bioavailability of the secreted therapeutic protein in the medium can be quantitated, and the bioactivity of the secreted therapeutic protein can also be evaluated through neurite length profiling, respectively. The versatility and rationale of this bioassay could serve as a useful screening tool in the development of retinal gene delivery systems.