Matrigel Scaffold Research Articles

Abstract 1475: Investigating the carcinogenic effects and the mechanism of cadmium exposure on WPMY-1 prostate myofibroblast cell line

Abstract Prostate cancer (PCa) is the second most common cancer among males with an incidence rate of PCa is 288,300 new cases annually. Risk factors include age, genetics, and lifestyle, but the underlying stimuli remain largely uncertain. One potential emerging cause of PCa is exposure to metal carcinogens as cadmium (Cd) which is now classified as a group I human carcinogen. Chronic exposure to Cd on epithelial cells has been extensively shown to induce carcinogenesis, making it a likely contributor to PCa development. In this study, we expand this information by examining how chronic Cd exposure affects prostate-derived myofibroblast (WPMY-1) cells transforming into a cancer-associated fibroblast like phenotype. To study the transformation, WPMY-1 cells were treated with 500nM of Cd for a period of 11 months establishing the WPMY-1 Cd model. Fibroblasts are a crucial tumor microenvironment (TME) component and potentially contribute to PCa progression when transformed and activated into cancer-associated fibroblasts (CAFs). To begin, we used an MTT assay to measure the effect of Cd toxicity on normal prostate myofibroblasts versus WPMY-1 Cd. The data showed that 10µM Cd solution was toxic to WPMY-1 cells, but WPMY-Cd cells depicted high cell viability at 50 µM dose, conferring to chronic Cd exposure improved resiliency to Cd toxicity. We analyzed the expression of CAF-specific markers in WPMY-Cd cells, such as vimentin, FAP, α-SMA, and PDGFR-β. WPMY-Cd cells showed a distinct upregulation for these markers when compared to untreated WPMY-1 cells, suggesting that chronic exposure to Cd promotes CAF-like phenotypic transformation in WPMY-Cd. To further ascertain the cancer-promoting properties of WPMY-Cd, an in vitro 3-D spheroid assay was performed by growing the cells on a Matrigel scaffold. The WPMY-Cd cells formed many more tumor-like spheroid structures than WPMY-1 and exhibited sprouting. The sprouting spheroids displayed lamellipodium and filopodium-like structures, demonstrating the initiation of migratory phenotype in WPMY-Cd. Additionally, previous research suggests that CAFs may play a role in PCa progression by promoting tumor growth via the secretion of cytokines and growth factors. We tested this theory by exposing Cd-treated epithelial (BPH-Cd) cells to conditioned media containing secretions from CAFs and WPMY-Cd. WPMY-1 Cd conditioned media in conferring cell migration on epithelial BPH-Cd cells was confirmed by a wound healing assay wherein BPH-Cd cells treated with WPMY-1 Cd conditioned media led to complete wound closure in 24 hours as compared to WPMY-1 and CAF conditioned media. Overall, there is considerable evidence that Cd exposure in fibroblasts may play a crucial role in PCa disease progression and is a promising area for researching PCa treatment strategies against a crucial component of the tumor microenvironment. Citation Format: Sakshi V. Bansod, Gnanasekar Munarithinam. Investigating the carcinogenic effects and the mechanism of cadmium exposure on WPMY-1 prostate myofibroblast cell line [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 1475.

Bioactive and chemically defined hydrogels with tunable stiffness guide cerebral organoid formation and modulate multi-omics plasticity in cerebral organoids.

Organoids are an emerging technology with great potential in human disease modelling, drug development, diagnosis, tissue engineering, and regenerative medicine. Organoids as 3D-tissue culture systems have gained special attention in the past decades due to their ability to faithfully recapitulate the complexity of organ-specific tissues. Despite considerable successes in culturing physiologically relevant organoids, their real-life applications are currently limited by challenges such as scarcity of an appropriate biomimetic matrix. Peptide amphiphiles (PAs) due to their well-defined chemistry, tunable bioactivity, and extracellular matrix (ECM)-like nanofibrous architecture represent an attractive material scaffold for organoids development. Using cerebral organoids (COs) as exemplar, we demonstrate the possibility to create bio-instructive hydrogels with tunable stiffness ranging from 0.69kPa to 2.24kPa to culture and induce COs growth. We used orthogonal chemistry involving oxidative coupling and supramolecular interactions to create two-component hydrogels integrating the bio-instructive activity and ECM-like nanofibrous architecture of a laminin-mimetic PAs (IKVAV-PA) and tunable crosslinking density of hyaluronic acid functionalized with tyramine (HA-Try). Multi-omics technology including transcriptomics, proteomics, and metabolomics reveals the induction and growth of COs in soft HA-Tyr hydrogels containing PA-IKVAV such that the COs display morphology and biomolecular signatures similar to those grown in Matrigel scaffolds. Our materials hold great promise as a safe synthetic ECM for COs induction and growth. Our approach represents a well-defined alternative to animal-derived matrices for the culture of COs and might expand the applicability of organoids in basic and clinical research. STATEMENT OF SIGNIFICANCE: Synthetic bio-instructive materials which display tissue-specific functionality and nanoscale architecture of the native extracellular matrix are attractive matrices for organoids development. These synthetic matrices are chemically defined and animal-free compared to current gold standard matrices such as Matrigel. Here, we developed hydrogel matrices with tunable stiffness, which incorporate laminin-mimetic peptide amphiphiles to grow and expand cerebral organoids. Using multi-omics tools, the present study provides exciting data on the effects of neuro-inductive cues on the biomolecular profiles of brain organoids.

Abstract 184: Multiple Myeloma 3D model: A platform for testing drug effects on myeloma in conjunction with the bone marrow niche

Abstract Background: Multiple myeloma is a cancer that remains mostly incurable despite of the wide array of treatment options. In part, this may be due to its ability to develop resistance to therapy with time. To evaluate key aspects of the disease such as the protective effects of the tumor niche, the immune repertoire that could influence immunotherapies and the ability to study primary tumor cells, developing a new platform is necessary. In order to overcome the obstacles of growing primary myeloma cells and having a model that better replicates the tumor microenvironment, we used a 3D organoid model with patient-derived cells to evaluate different conditions to observe how current therapies affects cancer cells and the tumor niche in which they live. Methods: We used a 3D organoid model using patient bone marrow aspirates containing myeloma cells and stroma in a Matrigel scaffold and incubated over 21 days with partial media change containing key growth factors found in the bone marrow space to maintain cells alive. Established agents used to treat myeloma were used to expose the cultures and then monitored at different timepoints to study its immediate and delayed effect in the tumor cells, immune repertoire, and stroma. Bortezomib, Lenalidomide, Selinexor, and Dexamethasone, which are commonly used to treat myeloma patients and influence the immune compartment, were used at different concentrations. ATP, alamar blue, histology, and flow cytometry were performed at key timepoints of the experiment. Results: H&E sections demonstrated hematopoietic elements with good cellular preservation suggesting that the model may be a functional way to recapitulate the tumor environment. Patient-derived Multiple Myeloma organoids treated with Bortezomib 10nM and Selinexor 10uM and 30uM showed significant inhibition in viability compared to control. Lenalidomide 100 uM had variable effect in viability between organoids. Flow Cytometry analysis captured the immunomodulatory effect of Lenalidomide on the immune cells. Conclusions: The 3D organoid model was successful at maintaining viability for 21 days and having a heterogeneous representation of the tumor niche when using bone marrow aspirates of myeloma patients. The experiments performed allowed us to explore not just the effect of drugs on myeloma cells, but we can see the impact they have on the other cells in the bone marrow niche. A unique interest in the ability to evaluate changes in the immune compartment may help better understand immunotherapy and optimize personalized medicine by helping select the most effective treatments for a particular patient. Citation Format: Hanadi M. Rashad, Giovanni Insuasti, Graca Almeida-Porada, Cesar Rodriguez. Multiple Myeloma 3D model: A platform for testing drug effects on myeloma in conjunction with the bone marrow niche [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 184.

Quantifying the Morphology and Mechanisms of Cancer Progression in 3D In-Vitro Environments: Integrating Experiments and Multiscale Models.

Mathematical models of cancer growth have become increasingly more accurate both in the space and time domains. However, the limited amount of data typically available has resulted in a larger number of qualitative rather than quantitative studies. In the present study, we provide an integrated experimental-computational framework for the quantification of the morphological characteristics and the mechanistic modelling of cancer progression in 3D environments. The proposed framework allows for the calibration of multiscale, spatiotemporal models of cancer growth using state-of-the-art 3D cell culture data, and their validation based on the resulting experimental morphological patterns using spatial point-pattern analysis techniques. We applied this framework to the study of the development of Triple Negative Breast Cancer cells cultured in Matrigel scaffolds, and validated the hypothesis of chemotactic migration using a multiscale, hybrid Keller-Segel model. The results revealed transient, non-random spatial distributions of cancer cells that consist of clustered, and dispersion patterns. The proposed model was able to describe the general characteristics of the experimental observations and suggests that chemotactic migration together with random motion was found to be a plausible mechanism leading to accumulation, during the examined time period of development. The developed framework enabled us to pursue two goals; first, the quantitative description of the morphology of cancer growth in 3D cultures using point-pattern analysis, and second, the relation of tumour morphology with underlying biophysical mechanisms that govern cancer growth and migration.

Extended longevity geometrically-inverted proximal tubule organoids

This study reports the cellular self-organization of primary human renal proximal tubule epithelial cells (RPTECs) around a minimal Matrigel scaffold to produce basal-in and apical-out proximal tubule organoids (tubuloids). These tubuloids are produced and maintained in hanging drop cultures for 90+ days, the longest such culture of any kind reported to date. The tubuloids upregulate maturity markers, such as aquaporin-1 (AQP1) and megalin (LRP2), and exhibit less mesenchymal and proliferation markers, such as vimentin and Ki67, compared to 2D cultures. They also experience changes over time as revealed by a comparison of gene expression patterns of cells in 2D culture and in day 31 and day 67 tubuloids. Gene expression analysis and immunohistochemistry reveal an increase in the expression of megalin, an endocytic receptor that can directly bind and uptake protein or potentially assist protein uptake. The tubuloids, including day 90 tubuloids, uptake fluorescent albumin and reveal punctate fluorescent patterns, suggesting functional endocytic uptake through these receptors. Furthermore, the tubuloids release kidney injury molecule-1 (KIM-1), a common biomarker for kidney injury, when exposed to albumin in both dose- and time-dependent manners. While this study focuses on potential applications for modeling proteinuric kidney disease, the tubuloids may have broad utility for studies where apical proximal tubule cell access is required.

Modulation of designer biomimetic matrices for optimized differentiated intestinal epithelial cultures

The field of intestinal biology is thirstily searching for different culture methods that complement the limitations of organoids, particularly the lack of a differentiated intestinal compartment. While being recognized as an important milestone for basic and translational biological studies, many primary cultures of intestinal epithelium (IE) rely on empirical trials using hydrogels of various stiffness, whose mechanical impact on epithelial organization remains vague until now. Here, we report the development of hydrogel scaffolds with a range of elasticities and their influence on IE expansion, organization, and differentiation. On stiff substrates (>5 kPa), mouse IE cells adopt a flat cell shape and detach in the short-term. In contrast, on soft substrates (80–500 Pa), they sustain for a long-term, pack into high density, develop columnar shape with improved apical-basal polarity and differentiation marker expression, a phenotype reminiscent of features in vivo mouse IE. We then developed a soft gel molding process to produce 3D Matrigel scaffolds of close-to-nature stiffness, which support and maintain a culture of mouse IE into crypt-villus architecture. Thus, the present work is up-to-date informative for the design of biomaterials for ex vivo intestinal models, offering self-renewal in vitro culture that emulates the mouse IE.

Abstract PO-080: Patient-derived organoids and cancer associated fibroblasts as a co-culture model to explore cell type interactions in pancreatic cancer

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) remains challenging to treat in part due to the dense desmoplastic stroma characteristic of the tumor microenvironment (TME). Often, less than 50% of the tumor mass is composed of malignant ductal epithelial cells. The relationship between the TME and the ductal tumor is important in tumorigenesis, metastasis, and treatment resistance. Cancer associated fibroblasts (CAFs) are an abundant cell type in the TME and have a dynamic role in both promoting and inhibiting tumor progression. Efforts to target fibroblast function therapeutically have yielded mixed results. While pre-clinical data have generated some optimism that CAF inhibition may improve clinical outcomes, this has not translated to the clinical setting. One likely reason for these inconsistent findings is the evolving understanding of CAF heterogeneity. Expanding our understanding of the interactions between the epithelial tumor and CAFs may identify new modalities for modulating the TME to augment treatment efficacy and improve patient outcomes. This requires the generation of human model systems to better interrogate intercellular interactions. Methods: We have developed an ex vivo model system to investigate interactions between ductal epithelial tumor cells and CAFs using matched patient-derived organoids (PDOs) and CAFs extracted following dissociation of surgical specimens. Flow cytometry was used to evaluate the viability of both cellular components as well as associated proliferation and cell identity. Moreover, we have used immunohistochemistry to further characterize this system while preserving the spatial relationship of the cell populations. Most recently, we employed the MULTI-seq multiplex single cell RNA sequencing (scRNA-seq) pipeline to explore the transcriptional dynamics of this system at intervals over 96 hours by examining 4 conditions with 1,500 cells sequenced per condition per time point. Results: Flow cytometry demonstrated that both PDOs and CAFs remain viable in this co-culture construct and that both cell types can be reliably identified using epithelial markers to identify organoid cells and an exclusion panel to identify the CAFs. We have further validated these findings using immunohistochemistry and demonstrated that in co-culture both PDOs and CAFs remain viable and continue to proliferate. We have also demonstrated successful extraction of both cell types from the Matrigel scaffold in which they are grown for use in downstream high dimensional genomics assays. Conclusions: We have previously demonstrated the utility of a PDO model to explore clinically relevant biological mechanisms in PDAC. However, the addition of CAFs to this model provides a more comprehensive representation of the in vivo tumor. This ex vivo system is viable and flexible for multiple end point assays and can be used to explore relevant cellular interactions and mechanisms. Our ongoing investigation will specifically focus on PDO-CAF interactions and transcriptional dynamics over time as we interrogate the scRNA-seq data. Citation Format: Jacquelyn W. Zimmerman, Genevieve Stein-O'Brien, Richard A. Burkhart, Elana J. Fertig, Elizabeth M. Jaffee. Patient-derived organoids and cancer associated fibroblasts as a co-culture model to explore cell type interactions in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-080.

Epithelial–stromal cell interactions and extracellular matrix mechanics drive the formation of airway-mimetic tubular morphology in lung organoids

SummaryComplex human airway cellular organization where extracellular matrix (ECM) and epithelial and stromal lineages interact present challenges for organ study in vitro. Current in vitro lung models that focus on the lung epithelium do not represent complex airway morphology and cell-ECM interactions seen in vivo. Models including stromal populations often separate them via a semipermeable barrier precluding cell–cell interaction or the effect of ECM mechanics. We investigated the effect of stromal cells on basal epithelial cell-derived bronchosphere structure and function through a triple culture of human bronchial epithelial, lung fibroblast, and airway smooth muscle cells. Epithelial–stromal cross-talk resulted in epithelial cell-driven branching tubules with stromal cells surrounding epithelial cells termed bronchotubules. Agarose– Matrigel scaffold (Agrigel) formed a mechanically tuneable ECM, with adjustable viscoelasticity and stiffness enabling long-term tubule survival. Bronchotubule models may enable research into how epithelial–stromal cell and cell–ECM communication drive tissue patterning, repair, and development of disease.

Matrigel/Umbilical Cord-Derived Mesenchymal Stem Cells Promote Granulosa Cell Proliferation and Ovarian Vascularization in a Mouse Model of Premature Ovarian Failure.

In women of reproductive age, severe injuries to the ovary are often accompanied by premature ovarian failure (POF), which can result in amenorrhea or infertility. Hormone replacement therapy has been used to treat POF; however, it has limited therapeutic efficiency and may cause several side effects. In this study, we aimed to fabricate a Matrigel scaffold loaded with human umbilical cord-derived mesenchymal stem cells (MSCs) and explore its potential to restore ovarian function and repair ovarian structures in vitro and in vivo. POF mouse models were established by injecting mice with cyclophosphamide for 15 consecutive days. Then, MSC/Matrigel was transplanted into the ovaries of the mice. Five weeks later, the morphology of the ovaries and follicles was observed by hematoxylin/eosin staining, and the tissue fibrosis ratio was measured using Masson's trichrome staining. The number of blood vessels was evaluated by α-smooth muscle actin and CD31 immunofluorescence, and Ki67 expression was used to determine the proliferation of granulosa cells. The expression of vascular endothelial growth factor (VEGF)-A was assessed by western blotting. The Matrigel scaffold regulated the expression of VEGF-A in vitro. Moreover, it promoted MSC survival and proliferation and prevented MSC apoptosis in vivo. After the transplantation of the MSC/Matrigel, the number of follicles was significantly increased in the mice with POF, and the tissue fibrosis ratio was reduced. Furthermore, the MSC/Matrigel significantly improved the proliferation rate of granulosa cells, increased the number of blood vessels, and upregulated the expression of VEGF-A. These findings demonstrate that MSC/Matrigel may support follicular development and help restore ovarian structures in vivo.

Abstract 2966: Novel ex vivo model for ERα positive breast cancer

Abstract Three-dimensional ex vivo cultures from patient-derived tumor tissue offer new opportunities for drug development and personalized treatment of cancer. However, one of the main challenges in establishing a tumor type faithful ex vivo tissue culture system is in optimization of biochemical and physical microenvironment to preserve desired phenotypic features. A collaboration with the Helsinki University Hospital enables us to receive live patient-derived breast cancer tissue samples from most breast cancer surgeries performed in the Southern Finland, typically samples arriving twice a week. Together with a group of materials scientists from Aalto Technical University we have explored seven different 3D culture matrices and defined their biomechanical properties and stiffness values with rheological measurements. Four investigated matrices preserved the luminal CK8+ cell phenotype of the luminal cancers they derived from, whereas rest of the matrices that included commonly used Matrigel and collagen scaffolds promoted a switch in the cell identity from luminal CK8+ to basal CK14+ phenotype. Furthermore, the genetic expression profiles examined with NGS corresponded to observed phenotypic switch. We noticed significant species-specific differences between mouse mammary and human breast samples in their morphology, phenotypic responses and ERα+ preservation to microenvironmental matrix stiffness. Furthermore, a combination of scaffold stiffness and composition of biochemical matrix constituency is required for long-term maintenance of ERα+ luminal tumour phenotype. Therefore, both physical properties and composition of the microenvironment need to be optimized for maintenance of breast tumor type specific phenotype and genetic profiles of ex vivo tumor explant cultures. Citation Format: Pauliina M. Munne, Lahja Martikainen, Iiris Räty, Kia Bertula, Nonappa Nonappa, Aino Peura, Minna Mutka, Marjut Leidenius, Tuomo Meretoja, Panu Kovanen, Johanna Mattson, Paivi Heikkila, Heikki Joensuu, Olli Ikkala, Juha T. Klefstrom. Novel ex vivo model for ERα positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2966.

Bioprinting of Matrigel Scaffolds for Cancer Research.

Cancer is one of the most life-threatening diseases worldwide. Despite the huge efforts, the failure rate of therapies remains high due to cells heterogeneity, so physiologically relevant models are strictly necessary. Bioprinting is a technology able to form highly complex 3D tissue models and enables the creation of large-scale constructs. In cancer research, Matrigel® is the most widely used matrix, but it is hardly bioprinted pure, without the use of any other bioink as reinforcement. Its complex rheological behavior makes the control with a standard bioprinting process nearly impossible. In this work, we present a customized bioprinting strategy to produce pure Matrigel® scaffolds with good shape fidelity. To this aim, we realized a custom-made volumetric dispensing system and performed printability evaluations. To determine optimal printing parameters, we analyzed fibers spreading ratio on simple serpentines. After identifying an optimal flow rate of 86.68 ± 5.77 µL/min and a printing speed of 10 mm/min, we moved forward to evaluate printing accuracy, structural integrity and other key parameters on single and multi-layer grids. Results demonstrated that Matrigel® was able to maintain its structure in both simple and complex designs, as well as in single and multilayer structures, even if it does not possess high mechanical strength. In conclusion, the use of volumetric dispensing allowed printing pure Matrigel® constructs with a certain degree of shape fidelity on both single and multiple layers.

Three Dimensional Cell Culturing for Modeling Adrenal and Pituitary Tumors.

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

Epithelial-Stromal Cell Interactions and ECM Mechanics Drive the Formation of Airway-Mimetic Tubular Morphology in Lung Organoids

The complex cellular organisation of the human airway tract where interaction between epithelial and stromal lineages and the extracellular matrix (ECM) make it a difficult organ to study in vitro. Current in vitro lung models focus on modelling the lung epithelium such as air-liquid interface (ALI) cultures and bronchospheres, do not model the complex morphology and the cell-ECM interaction seen in vivo. Models that include stromal populations often separate them via a semipermeable barrier, which precludes the effect of cell-cell interaction or do not include the ECM or the effect of ECM mechanics such as viscoelasticity and stiffness. Here we investigated the effect of stromal cells on basal epithelial cell-derived bronchosphere structure and function through a triple culture of bronchial epithelial, lung fibroblast and airway smooth muscle cells. Epithelial-stromal cross talk enabled formation of epithelial cell-driven branching tubules consisting of luminal epithelial cells surrounded by stromal cells termed bronchotubules. Addition of agarose to the Matrigel scaffold (Agrigel) created a mechanically tunable ECM, where viscoelasticity and stiffness could be altered to enable long term tubule survival. Bronchotubule models enable the investigation of how epithelial-stromal cell and cell-ECM communication drive tissue patterning, repair and development of disease.

Abstract A27: Engineering a 3D melanoma microenvironment and identifying novel therapeutic targets

Abstract Resistance to targeted therapies remains an obstacle in curing cancers. A reason for this is testing of therapeutic agents in 2D culture conditions that do not recapitulate growth conditions of tumors in vivo. A critical component for defining the cell survival in vivo is the extracellular matrix (ECM). The ECM not only modulates cell-matrix and cell-cell interactions, but also determines drug accessibility to the tumor tissue. Many labs thus evaluate drug perturbations in 3D cell culture platforms using Matrigel and collagen scaffolds. A caveat with these models is the simplistic use of only basement membrane proteins or individual ECM proteins that, again, do not efficiently mimic the tumor microenvironment (TME). Although in vivo mouse models can be a better alternative, the time to conduct experiments, the associated cost, and inability to recapitulate patient TME remain a crucial challenge. Thus, we aim to regenerate an in vitro melanoma TME by engineering a melanoma extracellular matrix. We aim to do this by first identifying the patient melanoma ECM. On conducting proteomics for ECM proteins on patient tissues we identified collagen VI to be the most dominant ECM protein. However, prior work from other labs reports collagen I as the most abundant protein. This information was used to construct a platform through which we can recapitulate the melanoma ECM. We did this by generating cell-derived matrices (CDM) using human foreskin fibroblasts. On comparing the CDM from HFF1 to patient ECM, we could detect ~80 of ~120 core ECM proteins. Upon performing proteomic studies on CDMs, we again identified collagen VI as the dominant ECM protein. We also tested CDM deposition with cancer-associated fibroblasts, yielding similar results. This brought us to the question of whether collagen VI plays a role in drug resistance in melanoma. To address the role of cell-derived matrices and collagen VI, we created collagen VI KO fibroblasts through CRISPR/Cas9, which were then used to generate these matrices and will be used to evaluate its effect on drug treatments. While building this in vitro melanoma TME, we understood the need for a 3D assay to have a robust readout on effect of drugs in 3D compared to 2D. Although many viability and proliferation assays are used by various labs, these assays do not look at the effect of treatments at a single-cell resolution and neither distinguishes if the drug is cytostatic or cytotoxic. Towards that goal, we established a 3D assay by culturing melanoma cells in collagen and tested for Raf-Mek combination therapies. We made comparisons of cells either cultured in 3D as single cells or spheroids. Imaging of these platforms was performed on epifluorescence and light-sheet microscopes. We tested the platform for both viability and proliferation and the images were analyzed through individual Z planes of the 3D image. While we observed differences in viability and proliferation in 2D vs. 3D upon drug treatments, there was no difference in 3D single cells vs. spheroids. Citation Format: Vasanth Siruvallur Murali, Justin Cillay, Erik Welf, Gaudenz Danuser, Murat Can Cobanoglu. Engineering a 3D melanoma microenvironment and identifying novel therapeutic targets [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A27.

Matrigel Scaffolding Enhances BMP9-induced Bone Formation in Dental Follicle Stem/Precursor Cells.

Bone tissue engineering requires a combination of cells, efficient biochemical and physicochemical factors, and biocompatible scaffolds. In this study, we evaluated the potential use of injectable Matrigel as a scaffold for the delivery of rat dental follicle stem/precursor cells (rDFSCs) transduced by bone morphogenetic protein (BMP) 9 to enhance osteogenic differentiation in vitro and promote ectopic bone formation in vivo. Recombinant adenovirus was used to overexpress BMP9 in rDFSCs. Alkaline phosphatase activity was measured using a histochemical staining assay and a chemiluminescence assay kit. Quantitative real-time polymerase chain reaction was used to determine mRNA expression levels of bone-related genes including distal-less homeobox 5 (DLX5), osteopontin (OPN), osterix (Osx), and runt-related transcription factor 2 (Runx2). Matrix mineralization was examined by Alizarin Red S staining. rDFSCs proliferation was analyzed using the Cell Counting Kit-8 assay. Subcutaneous implantation of rDFSCs-containing Matrigel scaffolds was used, and micro-computed tomography analysis, histological evaluation, and trichrome staining of implants extracted at 6 weeks were performed. We found that BMP9 enhanced alkaline phosphatase activity and mineralization in rDFSCs. The expression of bone-related genes (DLX5, OPN, Osx, and Runx2) was also increased as a result of BMP9 stimulation. Micro-computed tomography analysis and histological evaluation revealed that the bone masses retrieved from BMP9-overexpressing rDFSCs were significantly more pronounced in those with than in those without Matrigel. Our results suggest that BMP9 effectively promote osteogenic differentiation of rDFSCs, and Matrigel facilitate BMP9-induced osteogenesis of rDFSCs in vivo.

Fabrication and characterization of 3D microtubular collagen scaffolds for peripheral nerve repair.

Understanding the structure-function relationship in biomaterial constructs is critical in optimizing biological outcomes. For ensheathed structures such as peripheral nerve, engineering implantable tissue substitutes has been challenging. This is due to a unique geometry of thin-walled microtube arrays composed mostly of basement membrane. In this work, we propose a sacrificial templating method to create Matrigel scaffolds that resemble endogenous peripheral nerve. These paralleled microtube constructs possess high void space and membrane-like walls. Additionally, we investigated the effect of chemical crosslinking in altering the physical, mechanical, and biologic properties of Matrigel. Results show that both glutaraldehyde and genipin increased the modulus and failure stress of Matrigel while also improving degradation resistance. However, glutaraldehyde crosslinking induced some cytotoxicity whereas genipin showed good biocompatibility. PC-12 cells, Schwann cells, and primary chick dorsal root ganglia cultured onto microtube scaffolds demonstrated viability up to 10 days. Strong cellular alignment along the channels was observed in Schwann cells whereas neurite outgrowth in primary chick dorsal root ganglia was also biased along the major axis of the microtubes. This suggests that the microtubes may mediate cell orientation and axon pathfinding. This proof of concept study provides a tunable workflow that may be adapted to other collagen types.

3D cell-based biosensor for cell viability and drug assessment by 3D electric cell/matrigel-substrate impedance sensing

Preclinical efficacy and toxicity assessment of drug candidates plays a significant role in drug discovery and development. Traditional planar cell culture is a common way to perform the preclinical drug test, but it is difficult to correctly predict the drug efficacy and toxicity due to the simple two-dimensional (2D) extracellular microenvironment. Compared to the planar cell culture, three-dimensional (3D) cell culture system can better mimic the complex extracellular microenvironment where cells reside in the 3D tissues/organs in vivo. However, the conventional imaging techniques are difficult to achieve the dynamic and label-free monitoring of cellular behavior in thick sample by 3D cell culture. Here, 3D electric cell/matrigel-substrate impedance sensing (3D-ECMIS) is developed for real-time and non-invasive monitoring of 3D cell viability and drug susceptibility. In this study, human hepatoma cells (HepG2) are encapsulated in the matrigel scaffold and cultured in a 3D ECMIS chip which involves a pair of vertical golden electrodes on the opposite sidewalls of the culture chamber for the in-situ impedance measurement. Moreover, a portable multichannel system is developed to monitor the 3D cell/matrigel construct. The number of 3D-cultured cells was inversely proportional to the impedance magnitude of the entire cell/matrigel construct. Furthermore, anti-cancer drug screening will be conducted on the 3D-cultured cells when the cell proliferation reaches to a plateau phase. To validate the performance of 3D-ECMIS for the cell viability and drug susceptibility, the cell live/dead staining are utilized to confirm the results of drug susceptibility by this 3D-cell-based biosensor system. It is demonstrated that the 3D cell-based biosensor and 3D-ECMIS system will be a promising platform to improve the accuracy of cell-based anti-cancer drug screening in vitro.

Gametocytes of the Malaria Parasite Plasmodium falciparum Interact With and Stimulate Bone Marrow Mesenchymal Cells to Secrete Angiogenetic Factors.

The gametocytes of Plasmodium falciparum, responsible for the transmission of this malaria parasite from humans to mosquitoes, accumulate and mature preferentially in the human bone marrow. In the 10 day long sexual development of P. falciparum, the immature gametocytes reach and localize in the extravascular compartment of this organ, in contact with several bone marrow stroma cell types, prior to traversing the endothelial lining and re-entering in circulation at maturity. To investigate the host parasite interplay underlying this still obscure process, we developed an in vitro tridimensional co-culture system in a Matrigel scaffold with P. falciparum gametocytes and self-assembling spheroids of human bone marrow mesenchymal cells (hBM-MSCs). Here we show that this co-culture system sustains the full maturation of the gametocytes and that the immature, but not the mature, gametocytes adhere to hBM-MSCs via trypsin-sensitive parasite ligands exposed on the erythrocyte surface. Analysis of a time course of gametocytogenesis in the co-culture system revealed that gametocyte maturation is accompanied by the parasite induced stimulation of hBM-MSCs to secrete a panel of 14 cytokines and growth factors, 13 of which have been described to play a role in angiogenesis. Functional in vitro assays on human bone marrow endothelial cells showed that supernatants from the gametocyte mesenchymal cell co-culture system enhance ability of endothelial cells to form vascular tubes. These results altogether suggest that the interplay between immature gametocytes and hBM-MSCs may induce functional and structural alterations in the endothelial lining of the human bone marrow hosting the P. falciparum transmission stages.

Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections

ObjectiveNewly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses. MethodsA platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction (PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA. ResultsProperties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-1, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system. ConclusionOur data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

Abstract 202: Engineering Artificial Sinus Node by Reprogrammed Cardiomyocytes

The lack of clinically relevant sinoatrial node (SAN) disease model makes the pathophysiological investigation and therapeutic development stagnant. We hypothesize that engineering SAN by TBX18 somatic-reprogrammed cardiomyocytes on the three-dimension (3D) scaffold could create an in vitro SAN model, sharing similar features with a native SAN. Methods: In addition to neonatal rat ventricular cardiomyocytes (NRVMs) alone, we chose cardiosphere-derived cells (CDCs), or fibroblasts as supportive cells with different mixing ratios to construct engineered SAN. Hydrogel scaffolds including matrigels or platelet gels were used and compared. The engineered tissue was reprogrammed by TBX18 over-expression. Results: The over-expression of TBX18 increased HCN4 and CX45 transcriptions in cardiomyocytes. A stable spontaneous beating rate could be created in TBX18-reprogrammed engineered tissue, made of NRVMs and fibroblasts with matrigel scaffold (beating rate, TBX18 vs. control: 105.0 ± 10.7 bpm vs. 35.5±7.1 bpm, n=12, P<0.001). Although spontaneous beating could be observed in reprogrammed engineered tissues by NRVM alone, NRVM with CDCs, or NRVMs with CDCs and fibroblasts, the beating rates were not stable and slower. The beating rate in engineered tissue did not differ between scaffolds of matrigel and platelet gel. However, inter-experimental variation is higher in platelet gels, compared to matrigels. By immunofluorescent staining, an unique spatial distribution of NRVMs and fibroblasts was identified. NRVMs formed the central core of engineered tissues, encapsulated by fibroblasts, which was similar to a native SAN. The application of a sympathomimetic drug (epinephrine) doubled the beating rate of reprogrammed engineered tissue (P=0.02, n=6-8). Conclusions: A pilot model of engineered SAN was established by TBX18-reprogrammed cardiomyocytes. The supportive cells such as fibroblasts played an important role in tissue engineering of SAN.