Cell-extracellular Matrix Interactions Research Articles

Cell-Extracellular Matrix Interactions Play Multiple Essential Roles in Aortic Arch Development.

Defects in the morphogenesis of the fourth pharyngeal arch arteries (PAAs) give rise to lethal birth defects. Understanding genes and mechanisms regulating PAA formation will provide important insights into the etiology and treatments for congenital heart disease. Cell-ECM (extracellular matrix) interactions play essential roles in the morphogenesis of PAAs and their derivatives, the aortic arch artery and its major branches; however, their specific functions are not well-understood. Previously, we demonstrated that integrin α5β1 and Fn1 (fibronectin) expressed in the Isl1 lineages regulate PAA formation. The objective of the current studies was to investigate cellular mechanisms by which integrin α5β1 and Fn1 regulate aortic arch artery morphogenesis. Using temporal lineage tracing, whole-mount confocal imaging, and quantitative analysis of the second heart field (SHF) and endothelial cell (EC) dynamics, we show that the majority of PAA EC progenitors arise by E7.5 in the SHF and contribute to pharyngeal arch endothelium between E7.5 and E9.5. Consequently, SHF-derived ECs in the pharyngeal arches form a plexus of small blood vessels, which remodels into the PAAs by 35 somites. The remodeling of the vascular plexus is orchestrated by signals dependent on the pharyngeal ECM microenvironment, extrinsic to the endothelium. Conditional ablation of integrin α5β1 or Fn1 in the Isl1 lineages showed that signaling by the ECM regulates aortic arch artery morphogenesis at multiple steps: (1) accumulation of SHF-derived ECs in the pharyngeal arches, (2) remodeling of the EC plexus in the fourth arches into the PAAs, and (3) differentiation of neural crest-derived cells adjacent to the PAA endothelium into vascular smooth muscle cells. PAA formation is a multistep process entailing dynamic contribution of SHF-derived ECs to pharyngeal arches, the remodeling of endothelial plexus into the PAAs, and the remodeling of the PAAs into the aortic arch artery and its major branches. Cell-ECM interactions regulated by integrin α5β1 and Fn1 play essential roles at each of these developmental stages.

An in situ hydrogel-forming scaffold loaded by PLGA microspheres containing carbon nanotube as a suitable niche for neural differentiation

The cell-extracellular matrix (ECM) interactions are known to have a strong impact on cell behaviors in neural tissues. Due to complex physiology system and limited regenerative capacity of nervous system, neural tissue engineering has attracted attention as a promising strategy. In this study, we designed a hydrogel loaded by poly (lactic-co-glycolic acid) (PLGA) microspheres containing carbon nanotubes (CNT) and the biochemical differentiation factors, as a scaffold, in order to replicate the neural niche for stem cell growth (and/or differentiation). Different formulations from Hyaluronic acid (H), Poloxamer (P), Ethoxy-silane-capped poloxamer (PE), and cross-linked Alginate (Alg) were utilized as an in situ gel structure matrix to mirror the mechanical properties of the ECM of CNS. Subsequently, conductivity, surface morphology, size of microspheres, and CNT dispersion in microsphere were measured using two probes electrical conductometer, scanning electron microscopy (SEM), dynamic light scattering (DLS), and Raman spectroscopy, respectively. According to SEM and fluorescent microscopy images, CNTs increased the porosity of polymeric structure, which, in turn, facilitated the adhesion of stem cells on the surface of microspheres compared with control. Microstructure and rheological behaviors of different gel compositions were investigated using SEM and parallel-plate oscillatory rheometer, respectively. The MTT assay showed the toxicity profile of hydrogels was appropriate for cell transplantation. The confocal images illustrated the 3D platform of P15%H10% and P20%H5% gel formulations containing the PLGA-CNT microspheres, which allows the proliferation of neural stem cells (NSCs) derived from MSC. The results of real-time PCR and immunocytochemistry showed neuronal differentiation capacity of cultured NSCs derived from MSC in the alginate gel that contained PLGA-CNT microspheres as well as other control groups. The dispersion of the CNT-PLGA microspheres, covered by NSCs, into alginate gel in the presence of induction factors was found to notably enhance the expression of Sox2-SYP and β-Tubulin III neuronal markers.

Functional Integrin Regulation Through Interactions with Tetraspanin CD9.

Integrins are adhesion receptors that mediate many intercellular and cell-extracellular matrix interactions with relevance in physiology and pathology. Unlike other cellular receptors, integrins critically require activation for ligand binding. Through interaction in cis with other molecules and the formation of tetraspanin-enriched membrane microdomains (TEMs), the tetraspanin CD9 regulates integrin activity and avidity. Here we present three techniques used to study CD9-integrin interactions and integrin activation.

Proximity-Dependent Biotinylation (BioID) of Integrin Interaction Partners.

Integrins are heterodimeric adhesion receptors that maintain cell-extracellular matrix (ECM) interactions in diverse tissue microenvironments. They mediate cell adhesion and signaling through the assembly of large cytoplasmic multiprotein complexes that focally connect with the cytoskeleton. Integrin adhesion complexes (IAC) are specialized by the type of integrin-ECM contact and are sensitive to mechanical forces. Thus, they encrypt context-dependent information about the microenvironment in their composition. Signals mediated through IACs modulate many aspects of cell behavior, which allows cells to adapt to their surroundings. To gain insights into their function, IACs have been isolated from cultured cells and explored by proteomics. IACs are insoluble by nature and held together by transient/weak interactions, which makes it challenging to isolate intact IACs. Usually all IACs coupled to a specified ECM, which may employ different integrins, are isolated. Here we describe an alternative method based on proximity-dependent biotin identification (BioID), where specific integrin interaction partners are labeled in live cells and isolated without the need to isolate intact IACs.

Maternal obesity persistently alters cardiac progenitor gene expression and programs adult-onset heart disease susceptibility

ObjectiveHeart disease risk can be programmed by intrauterine exposure to obesity. Dysregulating key transcription factors in cardiac progenitors can cause subsequent adult-onset heart disease. In this study, we investigated the transcriptional pathways that are altered in the embryonic heart and linked to heart disease risk in offspring exposed to obesity during pregnancy. MethodsFemale mice were fed an obesogenic diet and mated with males fed a control diet. Heart function and genome-wide gene expression were analyzed in adult offspring born to obese and lean mice at baseline and in response to stress. Cross-referencing with genes dysregulated genome-wide in cardiac progenitors from embryos of obese mice and human fetal hearts revealed the transcriptional events associated with adult-onset heart disease susceptibility. ResultsWe found that adult mice born to obese mothers develop mild heart dysfunction consistent with early stages of disease. Accordingly, hearts of these mice dysregulated genes controlling extracellular matrix remodeling, metabolism, and TGF-β signaling, known to control heart disease progression. These pathways were already dysregulated in cardiac progenitors in embryos of obese mice. Moreover, in response to cardiovascular stress, the heart of adults born to obese dams developed exacerbated myocardial remodeling and excessively activated regulators of cell-extracellular matrix interactions but failed to activate metabolic regulators. Expression of developmentally regulated genes was altered in cardiac progenitors of embryos of obese mice and human hearts of fetuses of obese donors. Accordingly, the levels of Nkx2-5, a key regulator of heart development, inversely correlated with maternal body weight in mice. Furthermore, Nkx2-5 target genes were dysregulated in cardiac progenitors and persistently in adult hearts born to obese mice and human hearts from pregnancies affected by obesity. ConclusionsObesity during pregnancy alters Nkx2-5-controlled transcription in differentiating cardiac progenitors and persistently in the adult heart, making the adult heart vulnerable to dysregulated stress responses.

Current Trends in In Vitro Modeling to Mimic Cellular Crosstalk in Periodontal Tissue.

Clinical evidence indicates that in physiological and therapeutic conditions a continuous remodeling of the tooth root cementum and the periodontal apparatus is required to maintain tissue strength, to prevent damage, and to secure teeth anchorage. Within the tooth's surrounding tissues, tooth root cementum and the periodontal ligament are the key regulators of a functional tissue homeostasis. While the root cementum anchors the periodontal fibers to the tooth root, the periodontal ligament itself is the key regulator of tissue resorption, the remodeling process, and mechanical signal transduction. Thus, a balanced crosstalk of both tissues is mandatory for maintaining the homeostasis of this complex system. However, the mechanobiological mechanisms that shape the remodeling process and the interaction between the tissues are largely unknown. In recent years, numerous 2D and 3D in vitro models have sought to mimic the physiological and pathophysiological conditions of periodontal tissue. They have been proposed to unravel the underlying nature of the cell-cell and the cell-extracellular matrix interactions. The present review provides an overview of recent in vitro models and relevant biomaterials used to enhance the understanding of periodontal crosstalk and aims to provide a scientific basis for advanced regenerative strategies.

Controlled 3D co-culture of beta cells and endothelial cells in a micropatterned collagen sheet for reproducible construction of an improved pancreatic pseudo-tissue.

The co-culture of beta cells and endothelial cells in constructing a pancreatic pseudo-tissue can provide a functional advancement for in vitro diabetic-related drug testing and biological studies or in vivo transplantation. In order to mimic the pancreatic tissue more similar to in vivo, it is necessary to control the microenvironment, including cell–cell and cell–extracellular matrix interactions. In this study, we report a geometrically controlled three-dimensional (3D) pancreatic model where MIN6 and MS1 cells are co-cultured within a micropatterned collagen sheet. In 4–10 days, depending on the cell seeding concentration, the MIN6 cells formed islet-like clusters surrounded by an endothelial MS1 cell monolayer. The MS1 cells also formed monolayers at the edge of the micropatterns connecting between the clusters, resulting in a blood vessel-like structure in which no cells were found. It was confirmed that the 3D co-culture structure was not formed in a non-patterned sheet and the structure also helped insulin secretion of MIN6 cells. By simply embedding the cell mixture and the hexagonal micropattern into the collagen sheet, we were also able to achieve the highly reproducible fabrication of a 3D pancreatic pseudo-tissue construct for in vivo and in vitro applications.

Proteomic Characterization of Ubiquitin Receptor ADRM1/Rpn13

Background and Rationale 20S proteasome-based therapies are the mainstay of treatment of patients with multiple myeloma (MM); however, resistance to proteasome inhibitor (PI) therapies commonly develops underlying relapse of disease. We showed that inhibition of 19S-associated ubiquitin receptor (UbR) ADRM1/Rpn13, upstream of 20S proteasome, inhibits MM cell growth and overcomes PI-resistance in MM. hRpn13 recognizes polyubiquitinated proteins and facilitates their disassembly via 19S-associated deubiquitinating enzyme UCHL5, allowing for protein degradation via 20S proteasomal catalytic activities. To date, however, hRpn13-modulated protein substrates and downstream signaling remains unclear. Here, we utilized multiplexed proteomics with tandem mass spectrometry; GeneOntology (GO) enrichment; as well as pathway database KEGG and Reactome to identify hRpn13-associated signaling molecules and delineate functionally significant biological pathways. Materials and Methods CRISPR-Cas9 genome editing was performed to generate hRpn13-knockout (hRpn13-KO) HCT116 cells which were subjected to multiplexed proteomics. Functionally relevant proteins were categorized using GO, as well as KEGG- and Reactome-pathway analysis. The prognostic relevance of identified proteins was derived using Gene Expression Profiling (GEP) databases on uniformly treated MM patients. Protein expression level was determined by western blotting. CRISPR-Cas9-mediated hRpn13 deletion was confirmed using PCR and sequencing, as well as immunoblot analysis. Statistical significance was assessed with Student's t test Results Among 8766 proteins analyzed, 206 proteins were down-regulated, whereas 65 proteins were significantly up-regulated in hRpn13-KO cells compared to hRpn13-WT cells. Targeted hRpn13-deletion upregulated physiological pathways related to amino acid biosynthesis and transport, metabolism, proliferation, and apoptosis. We validated 5 such upregulated proteins (SLC1A3, THBS1, GLYR1, GCLM, and TIMM8A) using immunoblotting. As for hRpn13 deletion, treatment of MM.1S cells with Rpn13 inhibitor RA190 triggered a similar increase in SLC1A3, THBS1, GLYR1, GCLM, and TIMM8A levels. These data indicate that SLC1A3, THBS1, GLYR1, GCLM, and TIMM8A proteins are direct substrates of UbR hRpn13. On the other hand, hRpn13-deletion decreased levels of proteins involved in cell adhesion, biological regulation, antigen recognition, and extracellular matrix interactions. Clinical relevance in MM: We next screened 271 proteins against GEP datasets on MM patients (GSE6477; GSE13591; GSE6691). Among the 65 molecules upregulated in hRpn13-KO cells, 18 were expressed at significantly lower levels in MM patients vs normal plasma cells; and importantly, 2 (SLC1A3, THBS1) of these 18 proteins correlated with poor survival. In a functional validation study, blockade of hRpn13 with RA190 induced increased SLC1A3 and THBS1 levels, suggesting that targeting SLC1A3/THBS1 may restore abnormal amino acid metabolism in MM. We next screened 206 of 271 proteins that were downregulated in Rpn13-KO cells in GEP database of MM patients. Results show that 32 of these 206 molecules were highly expressed in MM patient samples vsnormal plasma cells, and that 2 (SLC16A7 and DBF4) of 32 correlated with poor survival. SLC16A7 encodes for monocarboxylate transporter 2 (MCT2), a key component of glycolysis, and DBF4 regulates DNA replication/cell proliferation. Importantly, RA190 decreased SLC16A and DB4 levels. Together, these data suggest that: 1) SLC16A, DBF4, SLC1A3, and THBS1 are downstream signaling targets of hRpn13; and 2) hRpn13-inhibition triggered MM cell death involves blockade of elevated glycolysis and DNA replication/cell growth via SLC16A and DBF4, respectively, as well as restoration of normal amino acid synthesis via SLC1A3. Conclusion Collectively, our study utilized CRISPR gene-editing, biochemical, and molecular strategies to identify UbR hRpn13-mediated proteomic alterations. We identified novel targets including SLC16A7, DBF4, SLC1A3, and Thrombospondin-1 which may serve as prognostic biomarkers in MM. Importantly, our findings further support hRpn13-directed therapeutics, as well as preclinical evaluation of novel strategies targeting SLC16A, DBF4, SLC1A3, and Thrombospondin-1, to enhance cytotoxicity and improve patient outcome in MM. Disclosures Chauhan: Oncopeptide AB: Consultancy; consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees.

Computational models of migration modes improve our understanding of metastasis.

Tumor cells migrate through changing microenvironments of diseased and healthy tissue, making their migration particularly challenging to describe. To better understand this process, computational models have been developed for both the ameboid and mesenchymal modes of cell migration. Here, we review various approaches that have been used to account for the physical environment's effect on cell migration in computational models, with a focus on their application to understanding cancer metastasis and the related phenomenon of durotaxis. We then discuss how mesenchymal migration models typically simulate complex cell–extracellular matrix (ECM) interactions, while ameboid migration models use a cell-focused approach that largely ignores ECM when not acting as a physical barrier. This approach greatly simplifies or ignores the mechanosensing ability of ameboid migrating cells and should be reevaluated in future models. We conclude by describing future model elements that have not been included to date but would enhance model accuracy.

Multiple Beam Laser Guidance for Patterning Irregularly Shaped Cells

Laser cell patterning is a distinctly effective technique for creating cell arrangements in a culture that replicates in vivo tissue structure for studying contact-mediated cell-cell interactions. Conventional laser-based single-cell-manipulation techniques are limited by their inability to pattern irregularly shaped cells, such as rod-shaped cardiomyocytes. We report the use of a spatial light modulator loaded with a computer-generated phase map to shape a single laser source into multiple laser-guidance beams distributed around the outer contour of an irregularly shaped cell to achieve accurate cell patterning. In addition to describing the principle and practice of the system design, we present what is to our knowledge the first achievement of patterning large, irregularly (rod) shaped adult rat cardiomyocytes in an end-to-end connected alignment to replicate the in vivo heart muscle cell connection without the use of substrate surface modifications, which can interfere with in vivo-like cell-cell and cell-extracellular matrix interactions. Our research demonstrates that two-stage multiple-beam laser guidance is effective: 1) A dual-beam configuration horizontally translates a cell from the outlet of the microfluidic cell delivery channel to a position above the cell deposition site and 2) A quad-beam configuration rapidly propels the cell axially through the suspension medium to the culture substrate with the vertical movement of the cell patterning chamber. Our study reveals that 90% of the patterned cells maintained end-to-end connection 30 minutes after patterning, and mechanical junctions could be reinstalled between laser connected cells after overnight incubation. This demonstrates that multiple-beam laser patterning is an outstanding tool for in vitro studying contact-mediated cell-cell interactions among irregularly shaped cells.

Early uterine development in pigs

The capacity of pig uterine tissues to recognize and respond to maternal and conceptus signals determines whether pregnancy can be established and defines the environment in which embryonic and fetal growth occur. Limits of uterine capacity may be defined genetically. However, the extent to which functional uterine capacity approaches genetic potential may be determined, in part, by the success of organizational events associated with growth, morphogenesis and cytodifferentiation of uterine tissues. It is important, therefore, that these events be identified and evaluated with respect to their potential effect on adult uterine function. Histogenesis of the pig uterus begins prenatally, but is completed postnatally. Transformation of the uterine wall from histoarchitectural infancy to maturity occurs between birth and day 120. Morphogenetic events characteristic of the first 60 days of neonatal life proceed normally in gilts ovariectomized at birth. These events include appearance and proliferation of uterine glands, development of endometrial folds, and growth of the myometrium. Endometrial development during this period involves alterations in patterns of epithelial and stromal DNA synthesis, coordinated changes in the distribution and biosynthesis of extracellular matrix glycosaminoglycans and cell surface glycoconjugates, and specific alterations in patterns of uterine protein secretion. The ovary-independent, spatially coordinated nature of these events suggests that neonatal uterine development is regulated locally via dynamic cell-cell and cell-extracellular matrix interactions. The extent to which such potentially critical interactions must be preserved to ensure developmental success remains unknown. However, the normal pattern of ovary-independent cellular and molecular events associated with development of the uterine wall was disrupted by treating neonatal gilts with oestradiol valerate, and daily administration of oestrogen to gilts from birth to day 13 did not affect ovulation rate, but did reduce embryonic survival by 22% on day 45 of gestation in adults that were exposed to oestrogen neonatally. These observations support the idea that some organizational events associated with development of the neonatal uterine wall must be allowed to proceed without interruption to ensure that adult uterine function is not compromised. Efforts to identify specific developmental determinants of uterine capacity may be facilitated by examining the consequences of xenobiotically induced interruption of uterine development on adult uterine function. Such studies may also contribute to identification of uterine factors affecting embryonic survival and fetal growth.

MSC spheroids-loaded collagen hydrogels simultaneously promote neuronal differentiation and suppress inflammatory reaction through PI3K-Akt signaling pathway

It is critical for the clinical success to take the anti-inflammatory function into consideration when integrating the neurogenesis into the nerve repair materials. To this aim, we prepared mesenchymal stem cell (MSC) spheroids-loaded collagen (Col) hydrogels with combined superior anti-inflammatory efficacy and neurogenic activity. The size of the MSC spheroids showed a strong modulation effect on both functions, and the MSC spheroids-100 sample exhibited the best neuronal and anti-inflammatory potentials. The observed dual functions were likely based on the elevated intrinsic cell-cell contacts and cell-extracellular matrix interactions from the MSC spheroids. MSC self-assembly as spheroids expedited the secretions of endogenous trophic factors and extracellular matrix (ECM), which was beneficial to drive neural stem cell differentiation into the neuronal lineage. In addition, the formation of the MSC spheroids secreted more amounts and types of cytokines as well as immunomodulatory paracrine factors to suppress LPS-induced inflammatory reaction. LC-MS/MS analysis further demonstrated that MSC spheroids contributed to the activation of neuroactive ligand-receptor interaction, thereby triggering downstream PI3K-Akt signal pathway, which was likely due to the acceleration of ECM-receptor interaction, gap junction and tight junction. Importantly, inhibiting Akt pathway significantly suppressed the neuronal differentiation, indicating that PI3K-Akt signal pathway was critically involved in the Col-MSC spheroid hydrogel mediated neuroprotection and neurogenesis. Such findings not only provided a simple approach for improving MSC-based therapies for neuron-related diseases, but also shed insight on understanding the underlying mechanisms of MSC-mediated neuronal differentiation.

Fmn2 Regulates Growth Cone Motility by Mediating a Molecular Clutch to Generate Traction Forces

Growth cone–mediated axonal outgrowth and accurate synaptic targeting are central to brain morphogenesis. Translocation of the growth cone necessitates mechanochemical regulation of cell-extracellular matrix interactions and the generation of propulsive traction forces onto the growth environment. However, the molecular mechanisms subserving force generation by growth cones remain poorly characterized. The formin family member, Fmn2, has been identified earlier as a regulator of growth cone motility. Here, we explore the mechanisms underlying Fmn2 function in the growth cone. Evaluation of multiple components of the adhesion complexes suggests that Fmn2 regulates point contact stability. Analysis of F-actin retrograde flow reveals that Fmn2 functions as a clutch molecule and mediates the coupling of the actin cytoskeleton to the growth substrate, via point contact adhesion complexes. Using traction force microscopy, we show that the Fmn2-mediated clutch function is necessary for the generation of traction stresses by neurons. Our findings suggest that Fmn2, a protein associated with neurodevelopmental and neurodegenerative disorders, is a key regulator of a molecular clutch activity and consequently motility of neuronal growth cones.

Bi-functional nanoparticle-stabilized hydrogel colloidosomes as both extracellular matrix and bioactive factor delivery vehicle.

Maintaining both cell-cell and cell-extracellular matrix (ECM) interactions is often a critical component of three-dimensional (3D) tissue regeneration. In high-density cell condensation systems, lack of appropriate cell-ECM interactions can result in limited and/or slow cell differentiation and tissue formation. To address these problems, a colloidosome microsphere system that is composed of a gelatin hydrogel core and a porous nanoparticle shell is developed. The colloidosome microsphere functions as an ECM and morphogen carrier for the induction of cartilage formation of high-density human mesenchymal stem cell (hMSC) in 3D cultures. With the protection of the nanoparticle shell, the colloidosome microspheres can be readily suspended in aqueous solution without clumping, thus incorporated homogeneously within high-density cell condensations. The gelatin-based colloidosome microspheres stimulate chondrogenesis of hMSCs and degrade rapidly to facilitate ECM remodeling for new tissue formation. When loaded with human transforming growth factor-β1, a potent chondrogenic morphogen, the colloidosomes serve as a bioactive factor delivery vehicle as well. The dual functionality of the colloidosomes as an ECM and a growth factor carrier effectively supports the chondrogenic differentiation of high-density hMSC condensations. These capabilities render the colloidosomes a promising platform system amenable to large-scale production of high-density 3D tissue culture constructs.

91P Transcriptomic mapping of integrins and immune activation in Basal-like and HER2+ breast cancer

Integrins, transmembrane receptors that mediate cell-extracellular matrix and cell-cell interaction, have been linked to cancer features. A less explored function of integrins in cancer is their role in leukocyte homing and activation. Understanding their role and relationship with immune infiltrates and immune checkpoints is an area of interest in cancer research.

Spheroids as a Type of Three-Dimensional Cell Cultures-Examples of Methods of Preparation and the Most Important Application.

Cell cultures are very important for testing materials and drugs, and in the examination of cell biology and special cell mechanisms. The most popular models of cell culture are two-dimensional (2D) as monolayers, but this does not mimic the natural cell environment. Cells are mostly deprived of cell–cell and cell–extracellular matrix interactions. A much better in vitro model is three-dimensional (3D) culture. Because many cell lines have the ability to self-assemble, one 3D culturing method is to produce spheroids. There are several systems for culturing cells in spheroids, e.g., hanging drop, scaffolds and hydrogels, and these cultures have their applications in drug and nanoparticles testing, and disease modeling. In this paper we would like to present methods of preparation of spheroids in general and emphasize the most important applications.

Ready to go 3D? A semi-automated protocol for microwell spheroid arrays to increase scalability and throughput of 3D cell culture testing

3-dimensional (3D) cell cultures are being increasingly recognized as physiologically more relevant in vitro models than traditional monolayer cultures, because they better mimic in vivo-like microenvironment, cell-cell and cell-extracellular matrix interactions. Nevertheless, the broader use of 3D models might be limited by requirements for special consumables, equipment, or skills for 3D cell cultures, and by their limited throughput and scalability. In this study, we optimized and adapted a commercially available agarose-micromolding technique to produce scaffold-free spheroid cultures. Brightfield microscopy was used for routine nondestructive and noninvasive evaluation of spheroid formation and growth. The workflow is compatible with manual, as well as high speed automated microscopic image acquisition, and it is supplemented with an in-house developed macro ‘Spheroid_Finder’ for open source software Fiji to facilitate rapid automated image analysis. This protocol was used to characterize and quantify spheroid formation and growth of two different hepatic cell lines, hTERT immortalized, but non-cancerous, adult human liver stem cell line HL1-hT1, and human hepatocellular carcinoma cell line HepG2, as well as their responses to a model antiproliferative and cytotoxic agent, 5-fluorouracil. The complete protocol provides a simple and ready-to-use solution to initiate scaffold-free spheroid cultures in any laboratory with standard equipment for mammalian in vitro cell culture work. Thus, it allows to increase throughput and scale of spheroid culture experiments, which can be greatly utilized in different areas of biomedical, pharmaceutical and toxicological research.

Abstract 326: Differences of drug sensitivities between 2-dimensional and 3-dimensional culture systems in triple negative breast cancer cell lines

Abstract [BACKGROUND] In vitro chemosensitivity tests are not always consistent to clinical results (Hutchinston L et al., Nat Rev Clin Oncol. 2011), partly because of lacking extracellular matrix-cell interactions in 2-dimensional (2D) cell culture system (Antoni D et al., Int J Mol Sci. 2015). Three-dimensional cell culture (3D) system maintains cells with a higher degree of structural complexity and homeostasis, analogous to tissues and organs compared with 2D cell culture system. Thus, 3D cell culture system will be expected for alteration of the animal experiment in chemosensitivity test (Bokhari M et al., J Anat. 2007). Cytotoxic agents are crucial for treating triple negative breast cancer (TNBC).[AIM] To clarify differences of drug sensitivities between 2D and 3D culture in TNBC cell lines, we compared IC50s for Cisplatin (CDDP), Docetaxel (DTX), Epirubicin (EPI) and Bromodomain inhibitor (JQ1). [METHODS] Thirteen TNBC cell lines were examined; HCC1599, HCC1937, HCC38, MDA-MB-468, HCC1806, HCC70, MDA-MB-436, MDA-MB-231, Hs578T, BT549, MDA-MB-453, BT20, and HCC1395. For 2D culture, cells were plated on general 96 well cell culture plate for 24 hours. For 3D culture, cells were plated on the PrimeSurface® 96 well plate (SUMITOMO BAKELITE) for 72 hours. Then, those were incubated in media including CDDP, DTX, EPI or JQ1 for 72 hours. The cell viability was quantified using CellTiter-Glo 2.0 and CellTiter-Glo 3D cell viability Assay (Promega), respectively. [RESULTS] For CDDP, IC50s were highly correlated between 2D and 3D culture (r=0.928) . The mean of IC50 value showed higher in 3D culture (27.67±8.40μM) than 2D culture (12.86 ± 17.23μM). For EPI, the correlation was r=0.593. The mean value showed higher in 3D culture (1.41±1.30μM) than 2D culture (0.62±0.72μM). For DTX and JQ1, the mean values were 1.78 ± 2.05nM and 4.31 ±4.72μM in 2D culture, respectively. However, IC50 failed to be obtained in several TNBC cell lines (HCC1937, HCC38, HCC1806, Hs578T and HCC1395 for JQ1 and MDA-MB-436, MDA-MB-453 and BT20 for DTX) in 3D culture. [CONCLUSION] IC50 value was higher in 3D culture than in 2D culture for CDDP and EPI. The correlation of IC50s between 2D and 3D depended on drugs with the highest in CDDP. We are under investigation on reasons why sensitivity tests for DTX and JQ1 failed in 3D culture system. Citation Format: Masako Muguruma, Saeko Teraoka, Kana Miyahara, Ai Ueda, Takahiko Kawate, Takashi Ishikawa. Differences of drug sensitivities between 2-dimensional and 3-dimensional culture systems in triple negative breast cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 326.

Functional Characterization of Three-Dimensional Cortical Cultures for InVitro Modeling of Brain Networks.

SummaryThree-dimensional (3D) in vitro cultures recapitulate key features of the brain including morphology, cell-cell and cell-extracellular matrix interactions, gradients of factors, and mechanical properties. However, there remains a need for experimental and computational tools to investigate network functions in these 3D models. To address this need, we present an experimental system based on 3D scaffold-based cortical neuron cultures in which we expressed the genetically encoded calcium indicator GCaMP6f to record neuronal activity at the millimeter-scale. Functional neural network descriptors were computed with graph-theory-based network analysis methods, showing the formation of functional networks at 3 weeks of culture. Changes to the functional network properties upon perturbations to glutamatergic neurotransmission or GABAergic neurotransmission were quantitatively characterized. The results illustrate the applicability of our 3D experimental system for the study of brain network development, function, and disruption in a biomimetic microenvironment.

Mechanical stimulation of cell microenvironment for cardiac muscle tissue regeneration: a 3D in-silico model

The processes in which cardiac cells are reorganized for tissue regeneration is still unclear. It is a complicated process that is orchestrated by many factors such as mechanical, chemical, thermal, and/or electrical cues. Studying and optimizing these conditions in-vitro is complicated and time costly. In such cases, in-silico numerical simulations can offer a reliable solution to predict and optimize the considered conditions for the cell culture process. For this aim, a 3D novel and enhanced numerical model has been developed to study the effect of the mechanical properties of the extracellular matrix (ECM) as well as the applied external forces in the process of the cell differentiation and proliferation for cardiac muscle tissue regeneration. The model has into account the essential cellular processes such as migration, cell–cell interaction, cell–ECM interaction, differentiation, proliferation and/or apoptosis. It has employed to study the initial stages of cardiac muscle tissue formation within a wide range of ECM stiffness (8–50 kPa). The results show that, after cell culture within a free surface ECM, cells tend to form elongated aggregations in the ECM center. The formation rate, as well as the aggregation morphology, have been found to be a function of the ECM stiffness and the applied external force. Besides, it has been found that the optimum ECM stiffness for cardiovascular tissue regeneration is in the range of 29–39 kPa, combined with the application of a mechanical stimulus equivalent to deformations of 20–25%.