Two-dimensional Monolayer Culture Research Articles

Abstract 54: Characterization of patient-derived organoids cultured on a gas-rich, liquid-liquid interface

Abstract Modeling tissues in vitro has long consisted of two-dimensional (2D) monolayer cultures grown in a dish, flask, or multi-well plate. While these methods remain adequate for some applications, the advent of personalized cancer therapies has necessitated the development of more complex models that more accurately represent the in vivo environment. Attempting to characterize tumor cell biology and response to candidate pharmaceuticals in 2D cultures has led to results that fail to translate to clinical therapies. A key source of this failure is the loss of cell diversity due to selective pressure resulting in cell populations that favor adhering to a rigid substrate. Three-dimensional (3D) cell culture methods have produced models that capture many of the dynamics of the in vivo tissue environment. Patient-derived organoids (PDOs) are quickly becoming the most attractive solution for creating predictive, patient-specific tumor models in vitro. Typically grown as spheroids, these models still present limitations in terms of cell survival and recapitulation of tumor development. Lena Biosciences has previously developed complex, vascularized tissue models using patented 3D scaffolds and a perfusion system. Hepatocytes grown in this system demonstrated increased cell viability and recovery of cytochrome P450 enzyme activity compared to 2D controls. Integral to the perfusion system is a pure, synthetic liquid that is twice as dense as and immiscible with cell culture medium and reagents. It is permissive to atmospheric gases, providing a gas-rich, liquid-liquid interface below 3D cultures maintained in the system. We hypothesized that PDOs could be cultured without a 3D scaffold, directly at the interface between the cell culture medium and this synthetic liquid. A specific preparation of this interface resulted in lung-derived PDOs that spontaneously formed dense, 3D, tissue-like structures that showed increased viability (as measured by a 50% reduction in LDH release) and cellular respiration (three-fold increase as measured by an alamarBlue assay). Ongoing studies are focused on characterizing PDOs from other patients and other tissues as they develop in long-term in vitro studies. Should this PDO model prove to be a faithful representation of in vivo tumor development, this system would be ideal for applications such as testing drug efficacy and studying mechanisms of tumor metastasis. Citation Format: James T. Shoemaker, Katherine R. Richardson, Jamie Arnst, Adam Marcus, Jelena Vukasinovic. Characterization of patient-derived organoids cultured on a gas-rich, liquid-liquid interface [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 54.

Abstract 51: Propagation of human colon, mammary, and lung cancer organoids in growth medium utilizing tissue-specific reagent kits and ready-to-use Wnt-3a and R-Spondin1 conditioned media

Abstract Three-dimensional “organoid” growth of tumors may represent a more physiologically relevant in vitro model system than traditional two-dimensional monolayer cultures of cancer cell lines. With the increased availability of cryopreserved human cancer organoids generated by academic laboratories, large-scale biobanking initiatives, and commercial sources, there is an unmet need for simplified, standardized, and cost-effective methods for preparation of the complex growth media required by these models. Human organoid culture media contains a variety of recombinant proteins, small molecules, and other growth factors that are costly to purchase in small-scale, time consuming to reconstitute and aliquot, and have varying stability and shelf life once prepared. Organoid culture media often also utilizes undefined conditioned media (CM) from one or more engineered cell lines that must be cultured separately, requiring additional time and resources to maintain. These lines secrete critical growth factors and the CM generated must be carefully prepared, collected, and stored. CM is subject to variability in activity levels due to batch-to-batch and protocol-to-protocol differences that can affect subsequent organoid culture performance. To address these challenges and to facilitate the wide-spread adoption of human organoid culture we are developing reagent kits containing essential growth medium components in an individually lyophilized format for long-term storage and easy single-use preparation of organoid growth media for a variety of tissue types including human colon, mammary, and lung. We compared growth medium formulated with our components with small-scale “homebrew” preparations and found equivalent or better culture performance (as calculated by doubling times) and similar morphology. Both approaches were able to generate media capable of supporting growth for at least three passages (~30 days) in 15 different organoid models. Additionally, we compared protocols and cell lines for CM generation to support organoid culture and found that an optimized, scaled-up production method could generate Wnt-3a and R-spondin1 CM with higher activity levels (up to 52%) and reduced lot-lot variability in comparison with commonly used small-scale protocols. We found that the optimized protocols produced CM that was stable for at least 2 months at 4°C and at least 6 months at -20°C, and was resilient against freezing and thawing. Previously frozen CM was also able to support organoid growth in culture. These results show that our tissue-specific kits and ready-to-use CM can provide a simplified, cost effective method to support the long-term propagation of human cancer organoids from multiple tissues and cancer types. Citation Format: James Clinton, Allison Ruchinskas, Himanshi Desai, Nanda Mahashetty, Dezhong Yin. Propagation of human colon, mammary, and lung cancer organoids in growth medium utilizing tissue-specific reagent kits and ready-to-use Wnt-3a and R-Spondin1 conditioned media [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 51.

Cytotoxicity of New Diterpene Alkaloids, Ceylonamides G-I, Isolated From Indonesian Marine Sponge of Spongia sp.

In the course of the search for cancer cell growth inhibitors, 3 new diterpene alkaloids, designated ceylonamides G-I (1-3), together with ceylonamide F (4) were isolated from an Indonesian marine sponge of Spongia sp. The chemical structures of compounds 1-3 were determined using spectroscopic analysis and compared with those of compound 4. Among the isolated compounds, 1 and 4 inhibited the growth of human prostate cancer DU145 cells in a two-dimensional monolayer culture, with an IC50 of 6.9 and 18.8 µM, respectively. Furthermore, these compounds are also effective on spheroid of three-dimensional cell culture model, which was prepared from DU145 cells. Based on the morphological changes in the spheroids, the minimum effective concentrations of compounds 1 and 4 were 10 and 25 µM, respectively.

Penetration Efficiency of Antitumor Agents in Ovarian Cancer Spheroids: The Case of Recombinant Targeted Toxin DARPin-LoPE and the Chemotherapy Drug, Doxorubicin.

The efficiency of delivering a therapeutic agent into a tumor is among the crucial factors determining the prospects for its clinical use. This problem is particularly acute in the case of targeted antitumor agents since many of them are high-molecular-weight compounds. In this work, the penetration of therapeutic agents of two distinct molecular weights into the spheroids of ovarian adenocarcinoma overexpressing human epidermal growth factor receptor 2 (HER2) was studied. It was shown that the low-molecular-weight chemotherapy drug, doxorubicin (~0.5 kDa), effectively penetrates through almost the entire depth of a 300 to 400 μm spheroid, while the penetration depth of the HER2-specific recombinant targeted toxin, DARPin-LoPE (~42 kDa), is only a few surface layers of cells and does not exceed 70 μm. The low penetration of the targeted toxin into spheroid was shown along with a significant decrease in its efficiency against the three-dimensional tumor spheroid as compared with the two-dimensional monolayer culture. The approaches to increasing the accumulation of agents in the tumor are presented and prospects of their use in order to improve the effectiveness of therapy are discussed.

Spatial control of oxygen delivery to three-dimensional cultures alters cancer cell growth and gene expression.

Commonly used monolayer cancer cell cultures fail to provide a physiologically relevant environment in terms of oxygen delivery. Here, we describe a three-dimensional (3D) bioreactor system where cancer cells are grown in Matrigel in modified six-well plates. Oxygen is delivered to the cultures through a polydimethylsiloxane (PDMS) membrane at the bottom of the wells, with microfabricated PDMS pillars to control oxygen delivery. The plates receive 3% oxygen from below and 0% oxygen at the top surface of the media, providing a gradient of 3-0% oxygen. We compared growth and transcriptional profiles for cancer cells grown in Matrigel in the bioreactor, 3D cultures grown in 21% oxygen, and cells grown in a standard hypoxia chamber at 3% oxygen. Additionally, we compared gene expression of conventional two-dimensional monolayer culture and 3D Matrigel culture in 21% oxygen. We conclude that controlled oxygen delivery may provide a more physiologically relevant 3D system.

152 Suppression of p38α promotes fibroblast-led human squamous cell carcinoma (SCC) invasion through p38δ-dependent mechanism

Cancer cell invasion is a hallmark of malignancy. Therefore, interfering with key signaling pathways that drive invasion would be expected to provide significant clinical benefits. To evaluate this approach, we are targeting stress-activated p38 kinases p38α and p38δ to modulate cancer cell motility and invasiveness. We recently found that in human squamous carcinoma cell lines SCC9 and SCC12 the rates of scratch wound closure were significantly reduced by pharmacologic or genetic inhibition of p38α alone, or by p38α/p38δ co-inhibition. These data implicated both p38α and p38δ in control of cancer cell motility in two-dimensional (2D) monolayer culture of SCC cells. Here we used pharmacologic inhibitors and RNA interference to investigate the contributions of p38α and p38δ signaling to fibroblast-led SCC invasion in 3D organotypic invasion model in which tumor cells move through collagen matrix remodeled by stromal fibroblasts. In both cell lines inhibition of p38α resulted in a highly invasive SCC tumor cell phenotype that was reversed by a co-inhibition of p38δ. Furthermore, p38α inhibition or depletion led to striking tissue fragility and a marked disruption of tissue architecture, phenotypes which were also largely ameliorated by co-inhibition of p38δ. Weakened E-cadherin-dependent intercellular adhesion likely underlies this fragility as well as increased ability of SCC cells to invade a collagen matrix observed upon p38α inhibition in organotypic, but not in monolayer cultures. Together, our data suggest that while p38α functions to restrict SCC cell invasiveness in 3D organotypic invasion model, p38δ is required for collective invasion of SCC cells. Therefore, blocking p38δ could be a potential strategy to reduce SCC metastasis.

A microparticle approach for non-viral gene delivery within 3D human mesenchymal stromal cell aggregates

Three-dimensional (3D) multicellular aggregates, in comparison to two-dimensional monolayer culture, can provide tissue culture models that better recapitulate the abundant cell-cell and cell-matrix interactions found in vivo. In addition, aggregates are potentially useful building blocks for tissue engineering. However, control over the interior aggregate microenvironment is challenging due to inherent barriers for diffusion of biological mediators (e.g. growth factors) throughout the multicellular aggregates. Previous studies have shown that incorporation of biomaterials into multicellular aggregates can support cell survival and control differentiation of stem cell aggregates by delivering morphogens from within the 3D construct. In this study, we developed a highly efficient microparticle-based gene delivery approach to uniformly transfect human mesenchymal stromal cells (hMSC) within multicellular aggregates and cell sheets. We hypothesized that release of plasmid DNA (pDNA) lipoplexes from mineral-coated microparticles (MCMs) within 3D hMSC constructs would improve transfection in comparison to standard free pDNA lipoplex delivery in the media. Our approach increased transfection efficiency 5-fold over delivery of free pDNA lipoplexes in the media and resulted in homogenous distribution of transfected cells throughout the 3D constructs. Additionally, we found that MCMs improved hMSC transfection by specifically increasing macropinocytosis-mediated uptake of pDNA. Finally, we showed up to a three-fold increase of bone morphogenetic protein-2 (BMP-2) expression and enhanced calcium deposition within 3D hMSC constructs following MCM-mediated delivery of a BMP-2 encoding plasmid and culture in osteogenic medium. The technology described here provides a valuable tool for achieving efficient and homogenous transfection of 3D cell constructs and is therefore of particular value in tissue engineering and regenerative medicine applications. Statement of SignificanceThis original research describes a materials-based approach, whereby use of mineral-coated microparticles improves the efficiency of non-viral gene delivery in three-dimensional human mesenchymal stromal cell constructs. Specifically, it demonstrates the use of mineral-coated microparticles to enable highly efficient transfection of human mesenchymal stromal cells in large, 3D culture formats. The manuscript also identifies specific endocytosis pathways that interact with the mineral coating to afford the improved transfection efficiency, as well as demonstrates the utility of this approach toward improving differentiation of large cell constructs. We feel that this manuscript will impact the current understanding and near-term development of materials for non-viral gene delivery in broad tissue engineering and biofabrication applications, and therefore be of interest to a diverse biomaterials audience.

Studying Human Neurological Disorders Using Induced Pluripotent Stem Cells: From 2D Monolayer to 3D Organoid and Blood Brain Barrier Models.

Neurological disorders have emerged as a predominant healthcare concern in recent years due to their severe consequences on quality of life and prevalence throughout the world. Understanding the underlying mechanisms of these diseases and the interactions between different brain cell types is essential for the development of new therapeutics. Induced pluripotent stem cells (iPSCs) are invaluable tools for neurological disease modeling, as they have unlimited self-renewal and differentiation capacity. Mounting evidence shows: (i) various brain cells can be generated from iPSCs in two-dimensional (2D) monolayer cultures; and (ii) further advances in 3D culture systems have led to the differentiation of iPSCs into organoids with multiple brain cell types and specific brain regions. These 3D organoids have gained widespread attention as invitro tools to recapitulate complex features of the brain, and (iii) complex interactions between iPSC-derived brain cell types can recapitulate physiological and pathological conditions of blood-brain barrier (BBB). As iPSCs can be generated from diverse patient populations, researchers have effectively applied 2D, 3D, and BBB models to recapitulate genetically complex neurological disorders and reveal novel insights into molecular and genetic mechanisms of neurological disorders. In this review, we describe recent progress in the generation of 2D, 3D, and BBB models from iPSCs and further discuss their limitations, advantages, and future ventures. This review also covers the current status of applications of 2D, 3D, and BBB models in drug screening, precision medicine, and modeling a wide range of neurological diseases (e.g., neurodegenerative diseases, neurodevelopmental disorders, brain injury, and neuropsychiatric disorders). © 2019 American Physiological Society. Compr Physiol 9:565-611, 2019.

Role and mechanisms of a three-dimensional bioprinted microtissue model in promoting proliferation and invasion of growth-hormone-secreting pituitary adenoma cells

Growth-hormone-secreting pituitary adenoma (GHSPA) is a benign tumour with a high incidence and large economic burden, which greatly affects quality of life. The aetiological factors are yet to be clarified for GHSPA. Conventional two-dimensional (2D) monolayer culture of tumour cells cannot ideally reflect the growth status of tumours in the physiological environment, and insufficiencies of in vitro models have severely restricted the progress of cancer research. Three-dimensional (3D) bioprinting technology is being increasingly used in various fields of biology and medicine, which allows recapitulation of the in vivo growth environment of tumour cells. In this study, a GHSPA microtissue model was established using 3D bioprinting. Tumour cells in the 3D environment exhibited more active cell cycle progression, secretion, proliferation, invasion, and tumourigenesis compared with those in the 2D environment. Furthermore, the molecular mechanisms of the 3D-printed microtissue model were explored. We demonstrated that the 3D-printed microtissue provides an excellent in vitro model at the tissue level for oncological research and may facilitate in-depth studies on the aetiology, treatment, drug resistance, and long-term prognosis of GHSPA .

Spheroid-3D and Monolayer-2D Intestinal Electrochemical Biosensor for Toxicity/Viability Testing: Applications in Drug Screening, Food Safety, and Environmental Pollutant Analysis

The rise of three-dimensional cell culture systems that provide in vivo-like environments for pharmaco-toxicological models has prompted the need for simple and robust viability assays suitable for complex cell architectural structures. This study addresses that challenge with the development of an in vitro enzyme based electrochemical sensor for viability/cytotoxicity assessment of two-dimensional (2D) monolayer and three-dimensional (3D) spheroid culture formats. The biosensor measures the cell viability/toxicity via electrochemical monitoring of the enzymatic activity of nonspecific esterases of viable cells, through the hydrolysis of 1-naphthyl acetate to 1-naphthol. The proposed sensor demonstrated strong correlation ( r = 0.979) with viable cell numbers. Furthermore, the model intestinal toxicants diclofenac (DFC, pharmaceutical), okadaic acid (OA, food-safety), and mancozeb (MZB, environmental) were used for the functional evaluation of the proposed sensor using 2D and 3D culture formats. Sensor performance showed high consistency with conventional cell viability/cytotoxicity assays (MTT/CFDA-AM) for all toxicants, with the sensor IC50 values matching the relevant viability LC50 values at the 95% confidence interval range for 2D (DCF: 1.19-1.26 mM, MZB: 10.28-14.18 μM, OA: 40.91-77.13 nM) and 3D culture formats (DCF: 1.02-4.78 mM, MZB: 11.26-15.16 μM, OA: 162.09-179.67 nM). The presented results demonstrate the feasibility of the proposed sensor as a robust endpoint screening tool for both 2D and 3D cytotoxicity assessment.

2D- and 3D-Based Intestinal Stem Cell Cultures for Personalized Medicine.

Colorectal cancer (CRC) is one of the most common cancers that have high occurrence and death in both males and females. As various factors have been found to contribute to CRC development, personalized therapies are critical for efficient treatment. To achieve this purpose, the establishment of patient-derived tumor models is critical for diagnosis and drug test. The establishment of three-dimensional (3D) organoid cultures and two-dimensional (2D) monolayer cultures of patient-derived epithelial tissues is a breakthrough for expanding living materials for later use. This review provides an overview of the different types of 2D- and 3D-based intestinal stem cell cultures, their potential benefits, and the drawbacks in personalized medicine in treatment of the intestinal disorders.

Beyond 2D: 3D bioprinting for skin regeneration.

Essential cellular functions that are present in tissues are missed by two-dimensional (2D) cell monolayer culture. It certainly limits their potential to predict the cellular responses of real organisms. Engineering approaches offer solutions to overcome current limitations. For example, establishing a three-dimensional (3D)-based matrix is motivated by the need to mimic the functions of living tissues, which will have a strong impact on regenerative medicine. However, as a novel approach, it requires the development of new standard protocols to increase the efficiency of clinical translation. In this review, we summarised the various aspects of requirements related to well-suited 3D bioprinting techniques for skin regeneration and discussed how to overcome current bottlenecks and propel these therapies into the clinic.

Aggregation of human dental pulp cells into 3D spheroids enhances their migration ability after reseeding.

Multicellular three-dimensional (3D) spheroids allow intimate cell-cell communication and cell-extracellular matrix interaction. Thus, 3D cell spheroids better mimic microenvironment in vivo than two-dimensional (2D) monolayer cultures. The purpose of this study was to evaluate the behaviors of human dental pulp cells (DPCs) cultured on chitosan and polyvinyl alcohol (PVA) membranes. The protein expression of hypoxia-inducible factor 1-α (HIF-1α) and vascular endothelial growth factor (VEGF), and the migration ability of the DPCs from 2D versus 3D environments were investigated. The results showed that both chitosan and PVA membranes support DPCs aggregation to form multicellular spheroids. In comparison to 2D cultures on tissue culture polystyrene, DPC spheroids exhibited higher protein expression of HIF-1α and VEGF. The treatment with YC-1 (inhibitor to HIF-1α) blocked the upregulation of VEGF, indicating a downstream event to HIF-1α expression. When DPC spheroids were collected and subjected to the transwell assay, the cells growing outward from 3D spheroids showed greater migration ability than those from 2D cultures. Moreover, DPCs aggregation and spheroid formation on chitosan membrane were abolished by Y-27632 (inhibitor to Rho-associated kinases), whereas the inhibitory effect did not exist on PVA membrane. This suggests that the mechanism regulating DPCs aggregation and spheroid formation on chitosan membrane is involved with the Rho-associated kinase signaling pathway. In summary, the multicellular spheroid structure was beneficial to the protein expression of HIF-1α and VEGF in DPCs and enhanced the migration ability of the cells climbing from spheroids. This study showed a new perspective in exploring novel strategies for DPC-based research and application.

In Vitro Models for Studying Invasive Transitions of Ductal Carcinoma In Situ.

About one fourth of all newly identified cases of breast carcinoma are diagnoses of breast ductal carcinoma in situ (DCIS). Since we cannot yet distinguish DCIS cases that would remain indolent from those that may progress to life-threatening invasive ductal carcinoma (IDC), almost all women undergo aggressive treatment. In order to allow for more rational individualized treatment, we and others are developing in vitro models to identify and validate druggable pathways that mediate the transition of DCIS to IDC. These models range from conventional two-dimensional (2D) monolayer cultures on plastic to 3D cultures in natural or synthetic matrices. Some models consist solely of DCIS cells, either cell lines or primary cells. Others are co-cultures that include additional cell types present in the normal or cancerous human breast. The 3D co-culture models more accurately mimic structural and functional changes in breast architecture that accompany the transition of DCIS to IDC. Mechanistic studies of the dynamic and temporal changes associated with this transition are facilitated by adapting the in vitro models to engineered microfluidic platforms. Ultimately, the goal is to create in vitro models that can serve as a reproducible preclinical screen for testing therapeutic strategies that will reduce progression of DCIS to IDC. This review will discuss the in vitro models that are currently available, as well as the progress that has been made using them to understand DCIS pathobiology.

Abstract 5012: Feasibility of manufacturing-scale bioproduction of novel next-generation 3D organoid cancer models in support of the Human Cancer Models Initiative

Abstract To meet the need for improved approaches to study cancer in vitro, there has been a surge in the development of novel research models utilizing advanced culture methods. These methods permit in vitro growth of cancer types previously not possible, and/or models with enhanced in vivo relevancy compared to traditional continuous cell lines. However, availability of these early-stage research models is currently limited and there is a lack of data on the ability to scale up production of these models to support the needs of the global research community. We sought to investigate the protocols, expansion capacity, cryopreservation ability, genetic stability, and feasibility of larger-scale bioproduction of a subset of the models generated by the Human Cancer Models Initiative (HCMI), an international collaborative effort between the National Cancer Institute, the foundation Hubrecht Organoid Technology, Cancer Research UK, and the Wellcome Trust Sanger Institute. The HCMI's initial goal is the development of 1,000 novel human cancer models, paired with bioinformatics and patient clinical data, particularly from rare or underrepresented cancer types. One advanced culture method being utilized, three-dimensional organoid “microtissue” culture, potentially poses challenges for traditional large-scale bioproduction processes. It requires growth embedded within an undefined extracellular matrix and complex media formulations containing multiple small molecules and recombinant proteins with unknown stability and shelf-life. Additionally, organoid growth media typically includes multiple sources of undefined conditioned media containing critical growth factors. We cultured organoid models derived from human colon, pancreas, esophagus, and mammary tissues developed by laboratories contributing to the HCMI. Multiple unique donors were available for all tissues and both cancer and non-cancer models were available for two tissue types. Most models were maintained in culture continuously for at least 60 days (7-27 population doublings, > 10 passages). Tissue and donor variability was evident in model characteristics, including morphology (assessed by microscopy and immunocytochemistry), growth rate, and genetic stability (measured by short tandem repeats analysis). All models were amenable to scale up beyond multiwell plates, and all models could recover from cryopreservation. While organoid culture represents a significant divergence from typical two-dimensional monolayer culture of continuous cell lines, our results show that these next-generation in vitro models are suitable for larger-scale bioproduction. This is vital to ensure the widespread availability of these models within the research community to facilitate applications like pre-clinical drug discovery and basic cancer research. Citation Format: James Clinton, Penney McWilliams-Koeppen, Siddhartha Paul, Allison Ruchinskas, Dezhong Yin, Robert Newman. Feasibility of manufacturing-scale bioproduction of novel next-generation 3D organoid cancer models in support of the Human Cancer Models Initiative [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5012.

Three-dimensional contractile muscle tissue consisting of human skeletal myocyte cell line

This paper describes a method to construct three-dimensional (3D) contractile human skeletal muscle tissues from a cell line. The 3D tissue was fabricated as a fiber-based structure and cultured for two weeks under tension by anchoring its both ends. While myotubes from the immortalized human skeletal myocytes used in this study never contracted in the conventional two-dimensional (2D) monolayer culture, myotubes in the 3D tissue showed spontaneous contraction at a high frequency and also reacted to the electrical stimulation. Immunofluorescence revealed that the myotubes in the 3D tissues had sarcomeres and expressed ryanodine receptor (RyR) and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). In addition, intracellular calcium oscillations in the myotubes in the 3D tissue were observed. These results indicated that the 3D culture enabled the myocyte cell line to reach a more highly matured state compared to 2D culture. Since contraction is the most significant feature of skeletal muscle, we believe that our 3D human muscle tissue with the contractile ability would be a useful tool for both basic biology research and drug discovery as one of the muscle-on-chips.

A Novel Three-Dimensional Immune Oncology Model for High-Throughput Testing of Tumoricidal Activity.

The latest advancements in oncology research are focused on autologous immune cell therapy. However, the effectiveness of this type of immunotherapy for cancer remediation is not equivalent for all patients or cancer types. This suggests the need for better preclinical screening models that more closely recapitulate in vivo tumor biology. The established method for investigating tumoricidal activity of immunotherapies has been study of two-dimensional (2D) monolayer cultures of immortalized cancer cell lines or primary tumor cells in standard tissue culture vessels. Indeed, a proven means to examine immune cell migration and invasion are 2D chemotaxis assays in permeabilized supports or Boyden chambers. Nevertheless, the more in vivo-like three-dimensional (3D) multicellular tumor spheroids are quickly becoming the favored model to examine immune cell invasion and tumor cell cytotoxicity. Accordingly, we have developed a 3D immune oncology model by combining 96-well permeable support systems and 96-well low-attachment microplates. The use of the permeable support system enables assessment of immune cell migration, which was tested in this study as chemotactic response of natural killer NK-92MI cells to human stromal-cell derived factor-1 (SDF-1α). Immune invasion was assessed by measuring NK-92MI infiltration into lung carcinoma A549 cell spheroids that were formed in low-attachment microplates. The novel pairing of the permeable support system with low-attachment microplates permitted simultaneous investigation of immune cell homing, immune invasion of tumor spheroids, and spheroid cytotoxicity. In effect, the system represents a more comprehensive and in vivo-like immune oncology model that can be utilized for high-throughput study of tumoricidal activity.

A novel approach to producing uniform 3-D tumor spheroid constructs using ultrasound treatment

Producing three-dimensional (3-D) multicellular tumor spheroids (TSs) is valuable for characterizing anticancer drugs since they provide a more representative model of the 3-D in vivo tumor than conventional two-dimensional (2-D) monolayer culture. The interaction of tumor cells with the extracellular matrix (ECM) in a 3-D culture environment is more similar to a tumor in vivo than in a 2-D environment; cell-cell and cell-ECM interaction can influence cell behaviour, such as in response to drug treatment. In vitro tumor spheroid models have been developed using microfluidic systems to generate 3-D hydrogel beads containing components of alginate and ECM protein, such as collagen, with high uniformity and throughput. Cell-laden hydrogel droplets are formed using a flow focusing process wherein the hydrogel precursors should be a homogeneous mixture. During gelation of the droplets into beads, the alginate acts as a fast gelling component helping to maintain the spherical shape of beads and preventing coalescence as the temperature-sensitive collagen I component gels more slowly. To produce uniform hydrogel droplets using the microfluidic flow focusing system, the mixtures must be homogeneous. However, collagen's sensitivity to temperature can lead to formation of chunks of collagen gel inside of the mixture, causing the mixture to become non-uniform and risking chip clogging. In order to overcome this limitation, previous approaches have used a cooling system during bead encapsulation while tumor cells were also present in the mixture, but this procedure can contribute to a delay in cell proliferation. Here a novel yet simple method is developed to prepare homogeneous pre-bead-encapsulation-mixtures containing collagen type I through ultrasonication. This method allows the cultivation of homogenous TS cultures with high uniformity and compact structure, and not only maintains cell viability but also the proliferation of cells in alginate/collagen hydrogel bead cultures. Depending on the sonication parameters, time and temperature, collagen can form small sized fibrils to thick fibers. Here, the mixtures containing collagen are assessed for morphology of collagen fibers/fibrils, cell viability, and proliferation. Human source Michigan Cancer Foundation-7 (MCF-7) breast cancer cells are successfully incorporated into alginate/collagen mixtures, followed by sonication, and then bead production. After bead gelation, the encapsulated MCF-7 cells remained viable and proliferated to form uniform TSs when the beads contained alginate and collagen. Results indicate that ultrasound treatment (UST) provides a powerful technique to change the structure of collagen from fiber to fibril, and to disperse collagen fibers in the mixture homogeneously for an application to generate uniform hydrogel beads and spheroids while not inhibiting cell proliferation.

Comparative proteome analysis of monolayer and spheroid culture of canine osteosarcoma cells

Osteosarcoma is an aggressive bone tumor with high metastasis rate in the lungs and affects both humans and dogs in a similar way. Three-dimensional tumor cell cultures mimic the in vivo situation of micro-tumors and metastases and are therefore better experimental in vitro models than the often applied two-dimensional monolayer cultures. The aim of the present study was to perform comparative proteomics of standard monolayer cultures of canine osteosarcoma cells (D17) and three-dimensional spheroid cultures, to better characterize the 3D model before starting with experiments like migration assays. Using DIGE in combination with MALDI-TOF/TOF we found 27 unique canine proteins differently represented between these two culture systems, most of them being part of a functional network including mainly chaperones, structural proteins, stress-related proteins, proteins of the glycolysis/gluconeogenesis pathway and oxidoreductases. In monolayer cells, a noticeable shift to more acidic pI values was noticed for several proteins of medium to high abundance; two proteins (protein disulfide isomerase A3, stress-induced-phosphoprotein 1) showed an increase of phosphorylated protein species. Protein distribution within the cells, as detected by immunohistochemistry, displayed a switch of stress-induced-phosphoprotein 1 from the cytoplasm (in monolayer cultures) to the nucleus (in spheroid cultures). Additionally, Western blot testing revealed upregulated concentrations of metastasin (S100A4), triosephosphate isomerase 1 and septin 2 in spheroid cultures, in contrast to decreased concentrations of CCT2, a subunit of the T-complex. Results indicate regulation of stress proteins in the process of three-dimensional organization characterized by a hypoxic and nutrient-deficient environment comparable to tumor micro-metastases. SignificanceOsteosarcoma is an aggressive bone tumor that early spreads to the lungs. Three-dimensional tumor cell cultures represent the avascular stage of micro-tumors and metastases, and should therefore represent a better experimental in vitro model compared to two-dimensional monolayer cultures. Significant differences have been reported in response to drug and radiation treatment between these two culture systems. A gel-based proteomic investigation was performed to compare protein patterns of a canine osteosarcoma cell line cultivated under those two conditions, to learn more about altered cell composition and its impact on cell behaviour. Due to the fact that the canine osteosarcoma is an accepted model for the human disease, results will be relevant for the human species as well.

Doxorubicin resistance in breast cancer cells is mediated by extracellular matrix proteins

BackgroundCancer cell resistance to therapeutics can result from acquired or de novo-mediated factors. Here, we have utilised advanced breast cancer cell culture models to elucidate de novo doxorubicin resistance mechanisms.MethodsThe response of breast cancer cell lines (MCF-7 and MDA-MB-231) to doxorubicin was examined in an in vitro three-dimensional (3D) cell culture model. Cells were cultured with Matrigel™ enabling cellular arrangements into a 3D architecture in conjunction with cell-to-extracellular matrix (ECM) contact.ResultsBreast cancer cells cultured in a 3D ECM-based model demonstrated altered sensitivity to doxorubicin, when compared to those grown in corresponding two-dimensional (2D) monolayer culture conditions. Investigations into the factors triggering the observed doxorubicin resistance revealed that cell-to-ECM interactions played a pivotal role. This finding correlated with the up-regulation of pro-survival proteins in 3D ECM-containing cell culture conditions following exposure to doxorubicin. Inhibition of integrin signalling in combination with doxorubicin significantly reduced breast cancer cell viability. Furthermore, breast cancer cells grown in a 3D ECM-based model demonstrated a significantly reduced proliferation rate in comparison to cells cultured in 2D conditions.ConclusionCollectively, these novel findings reveal resistance mechanisms which may contribute to reduced doxorubicin sensitivity.