2D Culture Plates Research Articles

Abstract P2160: High-throughput Imaging Of Cardiomyocytes Using Biomimetic Tools Help Identify Therapeutic Targets

Cardiovascular disease is a leading cause of death and continues to require novel forms of therapies. Mutations in sarcomeres, the basic contractile units of cardiac muscle, causing disarray and loss of function are causal in many genetic cardiomyopathies. Here, we model sarcomeric disarray in vitro by culturing human induced pluripotent stem cell-derived cardiomyocytes (human iPSC-CMs) on tissue culture plates with biomimetic patterned grooves designed to simulate extracellular matrix. This culture system models the natural structure and phenotypic development of human physiology. Next, we created an Adeno-associated virus open reading frame (AAV-ORF) library of ~100 heart failure associated genes of interest (GOI) identified using publicly available human genetic data. We developed a high throughput screening (HTS) process to test the effects of overexpression of these GOIs on sarcomere morphology. Images of human iPSC-CMs treated with overexpression libraries were run through a custom MATLAB HTS image processing script named ‘Tamarack’, developed for quantifying human iPSC-CM sarcomere morphology. Tamarack enables quantification of sarcomere count, length, and orientation. Analysis of sarcomeres from the Tamarack code in the biomimetic plates allows for enhanced quantification of sarcomere structures, in particular, alignment, a metric missing from standard 2D culture plates. We found that treating cells with siRBM20 induced a sarcomere misalignment phenotype which we looked to rescue with overexpression of our GOI. Genes that increased alignment in an RBM20 background were considered top hits and moved to further analysis.

Morus alba extract suppresses IL-17-induced abnormal proliferation in 3D-reconstructed epidermis

IntroductionPsoriasis is a chronic disease that is characterized by the abnormal proliferation of keratinocytes. The pathogenesis of psoriasis is initiated by the infiltration of Th17 cells, which secrete proinflammatory cytokines, including interleukin (IL)-17. IL-17 subsequently affects keratinocytes, causing abnormal proliferation and the release of cytokines and chemokines, which results in a vicious cycle of increased IL-17 production by immune cells. In this study, we aimed to search for a new remedy for treating psoriasis from natural sources. MethodsWe screened plant extracts that suppress IL-17-induced chemokine, chemokine (C-C motif) ligand 20 (CCL20) by qRT–PCR from the in-house library. The hit extract was evaluated by a 2D-wound healing assay and a 3D-reconstruction epidermal model. ResultMorus alba extract (MAE) was discovered as a candidate for a suppressor of IL-17-induced expression of CCL20. MAE also suppressed the migration of keratinocytes on wound-healing assay on a 2D culture plate. Additionally, MAE normalized the abnormal proliferation of the 3D-reconstruction model of keratinocytes induced by IL-17. ConclusionMAE may offer promise as a natural treatment for psoriasis.

Probing the interaction between metastatic breast cancer cells and osteoblasts in a thread-based breast-bone co-culture device.

Breast cancer metastasis to bone is a leading killer in breast cancer patients. A type I collagen-modified thread (Col-I@thread) was prepared for 3-dimensional cell culture and breast cancer bone metastasis co-culture device assembly. First, the coating of Col-I on nylon threads for promoting cell adhesion and growth was studied. Through SEM, XPS, and protein concentration measurements, it was found that the lyophilization method remarkably preserved the Col-I activity and the internal structure of the thread, thereby promoting cell attachment and proliferation. RNA-sequencing (RNA-Seq) and quantitative PCR analysis showed that osteoblast cells (MC3T3-E1) grown on Col-I@thread had elevated RUNX2, ALP, OPN, and Col-I gene expression to promote osteoblast differentiation. Single-cell analysis found that osteoblast MC3T3-E1 cells growing on Col-I@thread had higher Ca2+ secretion activity and mineralized nodules, suggesting robust cell activity and bone matrix formation than cells growing on 2D culture plates. Col-I@threads were knotted in an interdigital cross-finger frame to assemble the breast cancer-bone co-culture model. Confocal microscopy and flow cytometry tests quantified the invasive breast cancer cells. Moreover, the thread-based co-culture devices allowed us to isolate the invasive and non-invasive breast cancer cells to compare their molecular characteristics. qPCR results showed that expression of CX43, CXCR5, and CSPG4 genes was significantly increased in breast cancer cells with bone metastasis. Meanwhile, the expression of RUNX2 and OPG genes in osteoblasts was inhibited. The co-culture model based on the Col-I@thread mimics the bone tissue microenvironment to reveal the cross-talk between cancer cells and bone tissue. Moreover, the thread-based co-culture device is easy to fabricate and operate, providing a platform for exploring the cellular and molecular mechanisms of breast cancer bone metastasis, and holds potential for high-throughput screening of anti-breast cancer bone metastasis drugs.

Coating-Free Culture Medium for Establishing and Maintaining Human Induced Pluripotent Stem Cells

Cell expansion of human pluripotent stem cells (hPSCs) commonly depends on Matrigel as a coating matrix on two-dimensional (2D) culture plates and 3D microcarriers. However, the xenogenic Matrigel requires sophisticated quality-assurance processes to meet clinical requirements. In this study, we develop an innovative coating-free medium for expanding hPSCs. The xenofree medium supports the weekend-free culture and competitive growth of hPSCs on several cell culture plastics without an additional pre-coating process. The pluripotent stemness of the expanded cells is stably sustained for more than 10 passages, featured with high pluripotent marker expressions, normal karyotyping, and differentiating capacity for three germ layers. The expression levels of some integrins are reduced, compared with those of the hPSCs on Matrigel. This medium also successfully supports the clonal expansion and induced pluripotent stem cell establishment from mitochondrial-defective MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) patient’s peripheral blood mononuclear cells. This innovative hPSC medium provides a straightforward scale-up process for producing clinical-orientated hPSCs by excluding the conventional coating procedure.

Sericin nano-gel agglomerates mimicking the pericellular matrix induce the condensation of mesenchymal stem cells and trigger cartilage micro-tissue formation without exogenous stimulation of growth factors in vitro.

Mesenchymal stem cells (MSCs) are excellent seed cells for cartilage tissue engineering and regenerative medicine. Though the condensation of MSCs is the first step of their differentiation into chondrocytes in skeletal development, the process is a challenge in cartilage repairing by MSCs. The pericellular matrix (PCM), a distinct region surrounding the chondrocytes, acts as an extracellular linker among cells and forms the microenvironment of chondrocytes. Inspired by this, sericin nano-gel soft-agglomerates were prepared and used as linkers to induce MSCs to assemble into micro-spheres and differentiate into cartilage-like micro-tissues without exogenous stimulation of growth factors. These sericin nano-gel soft-agglomerates are composed of sericin nano-gels prepared by the chelation of metal ions and sericin protein. The MSCs cultured on 2D culture plates self-assembled into cell-microspheres centered by sericin nano-gel agglomerates. The self-assembly progress of MSCs is superior to the traditional centrifugation to achieve MSC condensation due to its facility, friendliness to MSCs and avoidance of the side-effects of growth factors. The analysis of transcriptomic results suggested that sericin nano-gel agglomerates offered a soft mechanical stimulation to MSCs similar to that of the PCM to chondrocytes and triggered some signaling pathways as associated with MSC chondrogenesis. The strategy of utilizing biomaterials to mimic the PCM as a linker and as a mechanical micro-environment and to induce cell aggregation and trigger the differentiation of MSCs can be employed to drive 3D cellular organization and micro-tissue fabrication in vitro. These cartilage micro-masses reported in this study can be potential candidates for cartilage repairing, cellular building blocks for 3D bio-printing and a model for cartilage development and drug screening.

A Functional in Vitro 3D Model of Human Hematopoietic Niches

Niches in the bone marrow (BM) regulate hematopoietic stem, progenitor and precursor cell fate and behaviour through cell-cell interactions and soluble factor secretion. Niche biology and niche-hematopoietic crosstalk are very complex processes not completely elucidated yet, due essentially to human material accessibility difficulties and limits of existing in vitro culture models. Here, we develop a 3D fully human bone marrow model recapitulating all hematopoietic and non-hematopoietic niche elements to aid further investigation of normal and pathological haematopoiesis and to move away from expensive, time-consuming and imperfect animal models. At first, we have developed a protocol to in vitro production and culture of all human main cellular non-hematopoietic components of bone marrow microenvironment. This comprehensive stroma is generated from human primary cells from femoral head, without any external cell lines contribution, and it reflects accurately the characteristics of the donor. In a 2-step culture process, mesenchymal stromal cells and endothelial cells are first selected and amplified simultaneously in an amplification medium, then they are cultured in the amplification medium as control or in a specific differentiation medium patented by the authors in conventional 2D culture plates or in 3D cultures: in spheroids, by self-organization in ultra-low-adherence plates or on a biomaterial (β-TriCalcium Phosphate), in the U-CUP perfused bioreactor. The resulting cellular stroma composition is analysed by quantifying the gene expression of markers of the osteoblastic (RUNX2, OSX, BSP), adipocytic (LPL, PPARγ, ADIPOQ) and endothelial (CD31) lineages . Moreover, specific markers of hematopoietic niche as ANG1, SCF, CXCL12, LEPR, VCAM, OPN are investigated by RT-qPCR. The spatial organization and the proportion of the different cellular stroma components are analysed by immunofluorescence (Osteoblastic cells: OSX marker, adipocytic cells: Bodipy probe, endothelial cells: CD31). The resulting tissues exhibited all compositional and structural features of human physiological BM: all stroma cells are presents and they express the main niche soluble factors. So, we explore if medullary stroma differentiated using our protocol is able to support the maintenance of HSPCs, to do this we performed coculture experiments in 2D with CD34+ cells sorted from umbilical cord blood. After 14 and 21 days of coculture, hematopoietic cells are recovered, counted and analysed by flow cytometry. Results show that our differentiated stroma is capable of maintaining, expanding and functionally regulated healthy human cord blood-derived HSPCs. An immature compartment (CD34+CD38-) is still present at day 21. Importantly, our primary in vitro niche model supports HSPCs with no cytokine addition. The same results are obtained with healthy human peripheral blood-derived HSPCs cocultured in 3D with the stroma organised in spheroids. Moreover, we demonstrated that our BM model is capable not only to maintain HSPC compartment but also to drive hematopoietic differentiation. In 2D coculture we detected mature blood cells and in 3D cultures, peripheral blood-derived HSPCs cocultured with spheroids are able to engage in erythroid lineage and recapitulate the early phases of erythropoiesis. These results demonstrate the fully complexity and huge versatility of our functional in vitro 3D model of human hematopoietic niches. Our exhaustive fully human hematopoietic niche model prepared from the primary cells of single donor, enables the design of advanced, tuneable in vitro BM proxies for the study of human physiological and pathological haematopoiesis and for screening molecules for predictive purposes in personalized medicine.

Transdifferentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Dopaminergic Neurons in a Three-Dimensional Culture.

The induction of human umbilical cord-derived mesenchymal stem cells (HUC-MSCs) toward dopaminergic neurons is a major challenge in tissue engineering and experimental and clinical treatments of various neurodegenerative diseases, including Parkinson disease. This study aims to differentiate HUC-MSCs into dopaminergic neuron-like cells. Following the isolation and characterization of HUC-MSCs, they were transferred to Matrigel-coated plates and incubated with a cocktail of dopaminergic neuronal differentiation factors. The capacity of differentiation into dopaminergic neuron-like cells in 2-dimensional culture and on Matrigel was assessed by real-time polymerase chain reaction, immunocytochemistry, and high-performance liquid chromatography. Our results showed that dopaminergic neuronal markers' transcript and protein levels were significantly increased on the Matrigel differentiated cells compared to 2D culture plates. Overall, the results of this study suggest that HUC-MSCs can successfully differentiate toward dopaminergic neuron-like cells on Matrigel, having great potential for the treatment of dopaminergic neuron-related diseases.

Human Cholangiocytes Form a Polarized and Functional Bile Duct on Hollow Fiber Membranes.

Liver diseases affect hundreds of millions of people worldwide; most often the hepatocytes or cholangiocytes are damaged. Diseases of the biliary tract cause severe patient burden, and cholangiocytes, the cells lining the biliary tract, are sensitive to numerous drugs. Therefore, investigations into proper cholangiocyte functions are of utmost importance, which is restricted, in vitro, by the lack of primary human cholangiocytes allowing such screening. To investigate biliary function, including transepithelial transport, cholangiocytes must be cultured as three-dimensional (3D) ductular structures. We previously established murine intrahepatic cholangiocyte organoid-derived cholangiocyte-like cells (CLCs) and cultured them onto polyethersulfone hollow fiber membranes (HFMs) to generate 3D duct structures that resemble native bile ducts at the structural and functional level. Here, we established an efficient, stepwise method for directed differentiation of human intrahepatic cholangiocyte organoids (ICOs) into CLCs. Human ICO-derived CLCs showed key characteristics of cholangiocytes, such as the expression of structural and functional markers, formation of primary cilia, and P-glycoprotein-mediated transport in a polarized fashion. The organoid cultures exhibit farnesoid X receptor (FXR)-dependent functions that are vital to liver bile acid homeostasis in vivo. Furthermore, human ICO-derived CLCs cultured on HFMs in a differentiation medium form tubular architecture with some tight, confluent, and polarized monolayers that better mimic native bile duct characteristics than differentiated cultures in standard 2D or Matrigel-based 3D culture plates. Together, our optimized differentiation protocol to obtain CLC organoids, when applied on HFMs to form bioengineered bile ducts, will facilitate studying cholangiopathies and allow developing therapeutic strategies.

Ready-to-use human neuron plates for pharmacological assays～ 384 well 2D culture plates～

Ready-to-use human neuron plates for pharmacological assays～ 384 well 2D culture plates～

An injectable, self-assembled multicellular microsphere with the incorporation of fibroblast-derived extracellular matrix for therapeutic angiogenesis.

Decellularized human lung fibroblast-derived matrix (hFDM) has demonstrated its excellent proangiogenic capability. In this study, we propose a self-assembled, injectable multicellular microspheres containing human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cell (MSCs), collagen hydrogel (Col), and hFDM toward therapeutic angiogenesis. Those multicellular microspheres are spontaneously formed by the mixtures of cell and hydrogel after being dropped on the parafilm for several hours. The size of microspheres can be manipulated via adjusting the initial volume of droplets and the culture period. The cells in the microspheres are highly viable. Multicellular microspheres show good capability of cell migration on 2D culture plate and also exhibit active cell sprouting in 3D environment (Col) forming capillary-like structures. We also find that multiple angiogenic-related factors are significantly upregulated with the multicellular microspheres prepared via Col and hFDM (Col/hFDM) than those prepared using Col alone or single cells (harvested from cocultured HUVECs/MSCs in monolayer). For therapeutic efficacy evaluation, three different groups of single cells, Col and Col/hFDM microspheres are injected to a hindlimb ischemic model, respectively, along with PBS injection as a control group. It is notable that Col/hFDM microspheres significantly improve the blood reperfusion and greatly attenuate the fibrosis level of the ischemic regions. In addition, Col/hFDM microspheres show higher cell engraftment level than that of the other groups. The incorporation of transplanted cells with host vasculature is detectable only with the treatment of Col/hFDM. Current results suggest that hFDM plays an important role in the multicellular microspheres for angiogenic cellular functions in vitro as well as in vivo. Taken together, our injectable multicellular microspheres (Col/hFDM) offer a very promising platform for cell delivery and tissue regenerative applications.

Abstract 174: Functional characterization of ZNF750 in esophageal squamous cell carcinoma

Abstract Background: Esophageal squamous cell carcinoma (ESCC) is a lethal and aggressive malignancy that is a threat worldwide. It is of significant prevalence in regions of Asia including Iran, Korea, Japan and especially China. Epidemiological studies of ESCC reveal genetic variations as a risk-factor to ESCC incidence. Genomic profiling of ESCC primary tumors have already identified candidate genes involved in different cellular processes such as cell cycle and apoptosis. Data from our whole exome sequencing study of primary tumors and lymph node metastases also reveal mutations involved in these pathways; however, we also uncovered an interesting candidate involved in differentiation namely, ZNF750. ZNF750 has been reported to play important roles in other squamous cell carcinomas, and as a candidate tumor suppressor gene in ESCC. Methods and Results: We exogenously upregulated ZNF750 expression by lentiviral system and used this model to investigate ZNF750 function in ESCC. By MTT 2D cell proliferation assay, we showed that in vitro cell proliferation in the ZNF750 group was hindered compared to controls. In addition, we set up 2D and 3D colony formation assays and found that the ZNF750 groups produced fewer colonies in 2D culture plates and produced significantly smaller colonies, when embedded in Matrigel. All of these findings are consistent with previous studies. We also studied the potential functional role of ZNF750 in 3D invasion/migration. Transwell permeable inserts were used to support a thick Matrigel culture with a nutrient gradient. Cells were embedded in Matrigel and moved along the gradient within the gel. After prolonged incubation (>2 weeks), location and size of the colonies were examined by confocal microscopy scanning. Cells with upregulated ZNF750 were found to exhibit greater mobility compared to the control cells. We also subjected ZNF750 and control groups to cisplatin to test for chemoresistance in ESCC. We found that ZNF750 expression increases chemoresistance, as opposed to the control cells. Conclusion: From these results, ZNF750 expression inhibits cell proliferation, while enhancing cell mobility and drug resistance, all of which are characteristics of cells undergoing epithelial-mesenchymal transition (EMT). We, therefore, hypothesize that ZNF750 functions in ESSC beyond that of a tumor suppressor, but as a putative EMT regulator. Acknowledgements: Research Grants Council Collaborative Research Fund grant number 106150246 and Asian Cancer Research Fund to MLL. Citation Format: Sheyne SA Choi, Valen Zhuoyou Yu, Josephine Mun-Yee Ko, Wei Dai, Maria L. Lung. Functional characterization of ZNF750 in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 174.

Cell shape affects nanoparticle uptake and toxicity: An overlooked factor at the nanobio interfaces

HypothesisIt is now being increasingly accepted that cells in their native tissue show different morphologies than those grown on a culture plate. Culturing cells on the conventional two-dimensional (2D) culture plates does not closely resemble the in vivo three-dimensional (3D) structure of cells which in turn seems to affect cellular function. This is one of the reasons, among many others, that nanoparticles uptake and toxicology data from 2D culture plates and in vivo environments are not correlated with one another. In this study, we offer a novel platform technology for producing more in vivo-like models of in vitro cell culture. ExperimentsThe normal fibroblast cells (HU02) were cultured on “pseudo-3D” substrates, made from cell imprinting approach. The respond of the cells to a model nanoparticle (gold nanorod) were compared in 2D and “pseudo-3D” cultures modes, by cytotoxicological assays. FindingsIt is illustrated here that the cells’ respond to the exact same type of nanoparticles is majorly dependant in their shape. The use of “pseudo-3D” substrates which could partially mimic the shape of cells in vivo is strongly proposed as a means of better predicting the efficacy of the 2D cell culture plates.

Flat Cell Culturing Surface May Cause Misinterpretation of Cellular Uptake of Nanoparticles

AbstractIn vitro cellular uptake of nanoparticles (NPs) is typically evaluated using a monolayer of cells seeded on a 2D culture plate, with the assumption of reliable and reproducible outcomes. However, recent developments reveal that 2D culture may produce errors in the measurement of cellular uptake of NPs due to issues including sedimentation and diffusion of NPs in cell‐culture media. To shed more light on the effect of culture methods on the uptake of NPs, the same number of prostate cancer cells is cultured in 2D and 3D substrates and their uptake of quantum dots (QDs, as a model NP) and entrance mechanisms are assessed. Significantly fewer QDs are taken up, but they are more evenly distributed among the cells, in the 3D compared to the 2D culture method; in addition, QDs enter the cells via different mechanisms of endocytosis in 2D than they do in 3D approaches. Findings regarding cell cycle phase distribution also vary between 3D and 2D samples, which results in a significantly lower percentage of QDs being taken up in 3D compared to 2D culture. These findings indicate that the culture environment drastically influences NP–cell readouts, which may lead to misinterpretation of in vitro outcomes.

Probing characteristics of cancer cells cultured on engineered platforms simulating different microenvironments

In this study, we demonstrate cell culture platforms that can provide a microenvironment similar to in vivo conditions so that in vivo-compatible drug testing results can be obtained from the in vitro experiments. To realize such in vivo microenvironment-mimetic platforms, different culture platforms such as a three-dimensional (3D) cell aggregate film, fluidic environment within a microfluidic system or extracellular matrix (ECM) coating were established. The tumor cell growth rate and sensitivity to doxorubicin (DOX) were studied using the glioblastoma cell line T98G. When 3 D spheroids were cultured, they grew significantly slower than under other culture conditions. When the cells were treated with DOX, the anticancer drug could not efficiently penetrate the 3 D spheroids to inhibit cell growth. When cultured on the Matrigel-coated culture vessel, T98G cells grew even in the presence of DOX, demonstrating chemoresistance. Nonetheless, in the 2D culture plate and in the microfluidic chip, cell growth decreased with DOX treatment and the binding ability was lost. These results indicate that the cells reacted differently to the same anticancer drug depending on the culture microenvironment. We believe that the development of a more physiologically relevant tumor cell culture platform will lead to more reliable antitumor drug responses.

Hepatocyte Aggregate Formation on Chitin-Based Anisotropic Microstructures of Butterfly Wings.

Scaffold nanotopography plays the most significant role in the mimicry of the in vivo microenvironment of the hepatocytes. Several attempts have been made to develop methods and substrates suited to growing hepatocytes into aggregates. Functional biomaterials, particularly biodegradable polymers, have been used in several studies aimed to develop improved scaffolds with ordered geometry and nanofibrous architecture for tissue engineering. However, there are still some limitation in their fabrication: it is not cost-efficient, is time-consuming, and exhibits some technological complications. The synthetic scaffolds are usually non-biodegradable and can be non-biocompatible compared to the naturally derived biomaterials. Here, we utilized a simple, cost-effective, and green method with two-step chemical treatment to get more selected hydrophilic butterfly wings from Morpho menelaus, Papilio ulysses telegonus, and Ornithoptera croesus lydius as a chitin-based natural scaffolds to growing hepatocyte aggregates. We established a three-dimensional (3D) in vitro model for culture of HepG2 cells and aggregate formation that maintained the hepatocytes function on these natural anisotropic microstructures. Cells cultured on these substrates show higher viability than those cultured on a two-dimensional (2D) culture plate. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay results revealed excellent viability of HepG2 cells on P. u. telegonus wings (fibrous area). The results also demonstrated appropriate cell activity, cell retention, and stable and functional expression in terms of albumin secretion and urea synthesis activity compared to the 2D monolayer culture of hepatocytes on the culture dish surface. With a slightly different degree, the other substrates also shown similar results. We anticipate that these natural anisotropic, biodegradable, and biocompatible substrates can maintain long-term hepatic culture as an in vitro 3D model for potential therapeutic applications and regenerative tissue applications. The model presented here provides a feasible alternative to the synthetic scaffolds and is expected to be more reliable for 3D organotypic liver culture models based on such scaffolds.

Porcine spermatogonial stem cells self-renew effectively in a three dimensional culture microenvironment.

Generally, self-renewal of spermatogonial stem cells (SSCs) is maintained in vivo in a three-dimensional (3D) microenvironment consisting of the seminiferous tubule basement membrane, indicating the importance of the 3D microenvironment for in vitro culture of SSCs. Here, we report a 3D culture microenvironment that effectively maintains porcine SSC self-renewal during culture. Porcine SSCs were cultured in an agarose-based 3D hydrogel and in 2D culture plates either with or without feeder cells. Subsequently, the effects of 3D culture on the maintenance of undifferentiated SSCs were identified by analyzing cell colony formation and morphology, AP activity, and transcriptional and translational regulation of self-renewal-related genes and the effects on proliferation by analyzing cell viability and single cell-derived colony number. The 3D culture microenvironment constructed using a 0.2% (w/v) agarose-based 3D hydrogel showed the strongest maintenance of porcine SSC self-renewal and induced significant improvements in proliferation compared with 2D culture microenvironments. These results demonstrate that self-renewal of porcine SSCs can be maintained more effectively in a 3D than in a 2D culture microenvironment. Moreover, this will play a significant role in developing novel culture systems for SSCs derived from diverse species in the future, which will contribute to SSC-related research.

Abstract 3165: 3D culture may better represent trastuzumab resistance associated with PIK3CA mutation than 2D culture

Abstract Background: It is becoming clear that presence of PIK3CA mutations is associated with lower pathological complete response rate in patients with HER2-overexpressing breast cancer when treated with trastuzumab-based chemotherapy in neo-adjuvant settings. On the other hand, in in vitro studies using traditional 2-dimentional (2D) cell culture, differential cellular or biochemical response to trastuzumab between PIK3CA-mutant (mt) and -wild-type (wt) cells has not been clearly demonstrated. Further, while tumor shrinkage is occasionally observed in breast cancer patients who are treated with trastuzumab as a single agent, cyto-toxic effect of trastuzumab is not simulated in 2D culture models. Recently, many studies reported 3-dimensional (3D) cell culture mimics in vivo environment better than 2D culture. Therefore, we hypothesized that 3D culture better represents clinically-observed trastuzumab resistance associated with PIK3CA mutation than 2D culture, and decided to comparatively investigate cellular and biochemical response to trastuzumab in HER2-amlified PIK3CA-mt and -wt cell lines cultured in 2D and 3D environments. Method: HER2-amplified breast cancer cell lines, BT474 (PIK3CA-wt), and UACC893 and MDA-MB361 (PIK3CA-mt) were seeded (day 0) and allowed to grow in 2D and 3D (NanoCluture Plate®, ORGANOGENIX, Kanagawa, Japan) cell culture plates. On day 3, trastuzamab (10 µg/ml) and/or BKM120 (1 and 5 µM), a PI3K inhibitor, were added. The effect of the drugs on cell growth was evaluated with WST-8 assay on days 3 through 7. Apoptosis and cell signaling were evaluated using Western blot on day 6 and days 3 through 5, respectively. Result: In PIK3CA-wt BT474, treatment with trastuzumab led to decrease in cell number, indicating cyto-toxic effect, only in 3D culture but not in 2D culture. In PIK3CA-mt UACC893 and MDA-MB-361 cell lines, treatment with trastuzumab resulted in no cellular reduction either in 2D or 3D cultures. Consistently, increase in cleaved PARP, indicative for apoptosis, was observed only in 3D-cultured BT474 but not in 2D-cultured BT474 or two PIK3CA-mt cell lines. Furthermore, in BT474, greater decrease in phosphorylation of AKT (p-AKT) was observed in 3D culture than in 2D culture. In PIK3CA-mutant cell lines, trastuzumab did not change level of p-AKT regardless of cell culture conditions. In PIK3CA-mutant UACC893, combined treatment with trastuzumab and BKM120 resulted in greater increase in expression of cleaved PARP than either drug alone. Conclusion: Trastuzumab-induced inhibition of PI3K/AKT pathway and resultant apoptosis in HER2-overxpressing PIK3CA-wt cells may be observed in 3D culture, which may be simulating cyto-toxic effect of trastuzumab by itself observed in clinic. Furthermore, 3D cell culture represents the resistance to trastuzumab associated with PIK3CA mutation better than 2D cell culture. Citation Format: Takashi Tatara, Toru Mukohara, Rina Tanaka, Yohei Shimono, Masanori Toyoda, Naomi Kiyota, Midori Hirai, Yoshihiro Kakeji, Hironobu Minami. 3D culture may better represent trastuzumab resistance associated with PIK3CA mutation than 2D culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3165. doi:10.1158/1538-7445.AM2017-3165

What qualifies depression to be an aetiological risk factor for coronary artery disease?

It is now being increasingly accepted that cells in their native tissue show different morphologies than those grown on a culture plate. Culturing cells on the conventional two-dimensional (2D) culture plates does not closely resemble the in vivo three-dimensional (3D) structure of cells which in turn seems to affect cellular function. This is one of the reasons, among many others, that nanoparticles uptake and toxicology data from 2D culture plates and in vivo environments are not correlated with one another. In this study, we offer a novel platform technology for producing more in vivo-like models of in vitro cell culture.The normal fibroblast cells (HU02) were cultured on “pseudo-3D” substrates, made from cell imprinting approach. The respond of the cells to a model nanoparticle (gold nanorod) were compared in 2D and “pseudo-3D” cultures modes, by cytotoxicological assays.It is illustrated here that the cells’ respond to the exact same type of nanoparticles is majorly dependant in their shape. The use of “pseudo-3D” substrates which could partially mimic the shape of cells in vivo is strongly proposed as a means of better predicting the efficacy of the 2D cell culture plates.

Engineered 3D Cardiac Fibrotic Tissue to Study Fibrotic Remodeling.

Activation of cardiac fibroblasts into myofibroblasts is considered to play an essential role in cardiac remodeling and fibrosis. A limiting factor in studying this process is the spontaneous activation of cardiac fibroblasts when cultured on two-dimensional (2D) culture plates. In this study, a simplified three-dimensional (3D) hydrogel platform of contractile cardiac tissue, stimulated by transforming growth factor-β1 (TGF-β1), is presented to recapitulate a fibrogenic microenvironment. It is hypothesized that the quiescent state of cardiac fibroblasts can be maintained by mimicking the mechanical stiffness of native heart tissue. To test this hypothesis, a 3D cell culture model consisting of cardiomyocytes and cardiac fibroblasts encapsulated within a mechanically engineered gelatin methacryloyl hydrogel, is developed. The study shows that cardiac fibroblasts maintain their quiescent phenotype in mechanically tuned hydrogels. Additionally, treatment with a beta-adrenergic agonist increases beating frequency, demonstrating physiologic-like behavior of the heart constructs. Subsequently, quiescent cardiac fibroblasts within the constructs are activated by the exogenous addition of TGF-β1. The expression of fibrotic protein markers (and the functional changes in mechanical stiffness) in the fibrotic-like tissues are analyzed to validate the model. Overall, this 3D engineered culture model of contractile cardiac tissue enables controlled activation of cardiac fibroblasts, demonstrating the usability of this platform to study fibrotic remodeling.

Cytotoxic activity of fumonisin B1 in Vero cells: comparison between 2D and 3D structural microplates

The present study aimed to estimate cytotoxic activity of fumonisin B1 (FB1) in Vero cells using crystal violet staining assay (CVA) and lactate dehydrogenase assay (LDH) performed in flattened (2D) and three dimensional (3D) bottom (V, U, and M) microplates. Vero cells were exposed to different concentrations of FB1 ranging from 0 to 10 μg/ml for 24 and 48 h. Tween 20 was used as a positive control and acetonitrile 50% in water was used as a negative control. Results revealed that CVA could be performed in the F type microplate for 24 and 48 h but it was useless in the 3D microplates. LDH assay showed that FB1 induced significant cytotoxicity on Vero cells after 24 h exposure in all microplates. Using 3D bottom microplates allowed formation of spheroid cell aggregates and reduced cell adhesion. LDH release in the U type microplate showed a significant dose-dependent increase, while V type and M type microplates showed a significant dose-independent increase in all exposed cells. Forty-eight hours exposure to FB1 in 2D microplate induced time-related cytotoxicity in all exposed cells pronounced as increased percentage of dead cells and significant increase in LDH release at 0.01, 0.1, 1, and 10 μg/ml. It was concluded that 2D culture plate is suitable for both LDH and CVA while 3D microplates are suitable only for the LDH assay. LDH assay could detect the cytotoxic effect of FB1 at lower concentrations than crystal violet staining assay.