Sofia P. Rebelo

Three-dimensional co-culture of human hepatocytes and mesenchymal stem cells: improved functionality in long-term bioreactor cultures

The development of human cell models that can efficiently restore hepatic functionality and cope with the reproducibility and scalability required for preclinical development poses a significant effort in tissue engineering and biotechnology. Primary cultures of human hepatocytes (HHs), the preferred model for in vitro toxicity testing, dedifferentiate and have short‐term viability in two‐dimensional (2D) cultures. In this study, hepatocytes isolated from human liver tissue were co‐cultured with human bone marrow mesenchymal stem cells (BM‐MSCs) as spheroids in automated, computer‐controlled, stirred‐tank bioreactors with perfusion operation mode. A dual‐step inoculation strategy was used, resulting in an inner core of parenchymal liver tissue with an outer layer of stromal cells. Hepatocyte polarization and morphology as well as the mesenchymal phenotype of BM‐MSCs were maintained throughout the culture period and the crosstalk between the two cell types was depicted. The viability, compact morphology and phenotypic stability of hepatocytes were enhanced in co‐cultures in comparison to monocultures. Gene expression of phase I and II enzymes was higher and CYP3A4 and CYP1A2 activity was inducible until week 2 of culture, being applicable for repeated‐dose toxicity testing. Moreover, the excretory activity was maintained in co‐cultures and the biosynthetic hepatocellular functions (albumin and urea secretion) were not affected by the presence of BM‐MSCs. This strategy might be extended to other hepatic cell sources and the characterization performed brings knowledge on the interplay between the two cell types, which may be relevant for therapeutic applications. Copyright

Adaptable stirred-tank culture strategies for large scale production of multicellular spheroid-based tumor cell models.

Currently there is an effort toward the development of in vitro cancer models more predictive of clinical efficacy. The onset of advanced analytical tools and imaging technologies has increased the utilization of spheroids in the implementation of high throughput approaches in drug discovery. Agitation-based culture systems are commonly proposed as an alternative method for the production of tumor spheroids, despite the scarce experimental evidence found in the literature. In this study, we demonstrate the robustness and reliability of stirred-tank cultures for the scalable generation of 3D cancer models. We developed standardized protocols to a panel of tumor cell lines from different pathologies and attained efficient tumor cell aggregation by tuning hydrodynamic parameters. Large numbers of spheroids were obtained (typically 1000-1500 spheroids/mL) presenting features of native tumors, namely morphology, proliferation and hypoxia gradients, in a cell line-dependent mode. Heterotypic 3D cancer models, based on co-cultures of tumor cells and fibroblasts, were also established in the absence or presence of additional physical support from an alginate matrix, with maintenance of high cell viability. Altogether, we demonstrate that 3D tumor cell model production in stirred-tank culture systems is a robust and versatile approach, providing reproducible tools for drug screening and target verification in pre-clinical oncology research.

Drug screening in 3D in vitro tumor models: overcoming current pitfalls of efficacy read-outs

There is cumulating evidence that in vitro 3D tumor models with increased physiological relevance can improve the predictive value of pre‐clinical research and ultimately contribute to achieve decisions earlier during the development of cancer‐targeted therapies. Due to the role of tumor microenvironment in the response of tumor cells to therapeutics, the incorporation of different elements of the tumor niche on cell model design is expected to contribute to the establishment of more predictive in vitro tumor models. This review is focused on the several challenges and adjustments that the field of oncology research is facing to translate these advanced tumor cells models to drug discovery, taking advantage of the progress on culture technologies, imaging platforms, high throughput and automated systems. The choice of 3D cell model, the experimental design, choice of read‐outs and interpretation of data obtained from 3D cell models are critical aspects when considering their implementation in drug discovery. In this review, we foresee some of these aspects and depict the potential directions of pre‐clinical oncology drug discovery towards improved prediction of drug efficacy.

3D-3-culture: A tool to unveil macrophage plasticity in the tumour microenvironment

The tumour microenvironment (TME) shapes disease progression and influences therapeutic response. Most aggressive solid tumours have high levels of myeloid cell infiltration, namely tumour associated macrophages (TAM). Recapitulation of the interaction between the different cellular players of the TME, along with the extracellular matrix (ECM), is critical for understanding the mechanisms underlying disease progression. This particularly holds true for prediction of therapeutic response(s) to standard therapies and interrogation of efficacy of TME-targeting agents. In this work, we explored a culture platform based on alginate microencapsulation and stirred culture systems to develop the 3D-3-culture, which entails the co-culture of tumour cell spheroids of non-small cell lung carcinoma (NSCLC), cancer associated fibroblasts (CAF) and monocytes. We demonstrate that the 3D-3-culture recreates an invasive and immunosuppressive TME, with accumulation of cytokines/chemokines (IL4, IL10, IL13, CCL22, CCL24, CXCL1), ECM elements (collagen type I, IV and fibronectin) and matrix metalloproteinases (MMP1/9), supporting cell migration and promoting cell-cell interactions within the alginate microcapsules. Importantly, we show that both the monocytic cell line THP-1 and peripheral blood-derived monocytes infiltrate the tumour tissue and transpolarize into an M2-like macrophage phenotype expressing CD68, CD163 and CD206, resembling the TAM phenotype in NSCLC. The 3D-3-culture was challenged with chemo- and immunotherapeutic agents and the response to therapy was assessed in each cellular component. Specifically, the macrophage phenotype was modulated upon treatment with the CSF1R inhibitor BLZ945, resulting in a decrease of the M2-like macrophages. In conclusion, the crosstalk between the ECM and tumour, stromal and immune cells in microencapsulated 3D-3-culture promotes the activation of monocytes into TAM, mimicking aggressive tumour stages. The 3D-3-culture constitutes a novel tool to study tumour-immune interaction and macrophage plasticity in response to external stimuli, such as chemotherapeutic and immunomodulatory drugs.

Establishing Liver Bioreactors for In Vitro Research.

In vitro systems that can effectively model liver function for long periods of time are fundamental tools for preclinical research. Nevertheless, the adoption of in vitro research tools at the earliest stages of drug development has been hampered by the lack of culture systems that offer the robustness, scalability, and flexibility necessary to meet industrys demands. Bioreactor-based technologies, such as stirred tank bioreactors, constitute a feasible approach to aggregate hepatic cells and maintain long-term three-dimensional cultures. These three-dimensional cultures sustain the polarity, differentiated phenotype, and metabolic performance of human hepatocytes. Culture in computer-controlled stirred tank bioreactors allows the maintenance of physiological conditions, such as pH, dissolved oxygen, and temperature, with minimal fluctuations. Moreover, by operating in perfusion mode, gradients of soluble factors and metabolic by-products can be established, aiming at resembling the in vivo microenvironment. This chapter provides a protocol for the aggregation and culture of hepatocyte spheroids in stirred tank bioreactors by applying perfusion mode for the long-term culture of human hepatocytes. This in vitro culture system is compatible with feeding high-throughput screening platforms for the assessment of drug elimination pathways, being a useful tool for toxicology research and drug development in the preclinical phase.

Abstract A21: 3D tumor models in bioreactors recapitulate microenvironment and disease progression

The high attrition rates observed in cancer drug discovery (up to 95% of failure of drugs tested in phase I trials) have raised the awareness of the scientific and industrial communities towards the need for more predictive pre-clinical models. These models should be more representative of the disease and consequently help to eliminate at pre-clinical stages drug candidates that lack efficacy or safety. Tumor microenvironment is composed by a network of fibroblasts, endothelial cells, immune-competent cells within the extracellular matrix (ECM). Interactions between these components are critical for tumor initiation, proliferation, migration and metastasis. The design of in vitro cancer cell models that recapitulate the tumor microenvironment and 3D architecture provides higher physiological relevance as they more closely resemble the in vivo cellular context. We have established methodologies for scalable generation of 3D cancer cell models in stirred-tank culture systems, and applied these to a large panel of tumor cell lines from different pathologies, including breast, colon, hepatic and lung tumor cell lines. Large numbers of spheroids were obtained per culture (typically 1000-1500 spheroids/mL) with representative characteristics of native tumors, such as morphology, proliferation and hypoxia gradients, in a cell-line dependent mode. With the aim of increasing the relevance of spheroids as tumor cell models, several aspects of tumor microenvironment were incorporated, such as the presence of stromal cells (fibroblasts and monocytes) and specific physical parameters, namely by embedding cells in a polymeric matrix. Heterotypic 3D breast and Non-Small Cell Lung Carcinoma (NSCLC) cancer models, based on co-cultures of tumor cells with stromal cells were established by using an alginate matrix to provide physical support to cells. Tumor spheroids were microencapsulated alone or with fibroblasts and monocytes, thus allowing the establishment of an epithelial tumor compartment and a stromal compartment of increasing complexity. Cultures were performed in stirred-tank vessels for 15 days with continuous monitoring. In both breast and lung tumor models, the presence of fibroblasts was associated with secretion of pro-inflammatory cytokines and accumulation of collagen in the microcapsules. Long-term culture (up to 15 days) resulted in phenotypic alterations in co-cultured breast tumor spheroids, such as loss of cell polarity, reduced cell-cell adhesions, collective cell migration and increased angiogenic potential. In contrast, the effects of fibroblasts were not as significant in NSCLC co-cultures using H1650, H1437 and H157 cell lines suggesting that the effect of tumor-stroma cross-talk is cell line dependent. Moreover, these models have also been shown as feasible tools for drug screening by assessing the effect of chemotherapeutic and specific inhibitors compounds on mono- and co-cultures. In conclusion, we have developed scalable, robust and versatile methodologies for the generation and culture of 3D cancer models, enabling long-term in vitro recapitulation of tumor-stroma crosstalk, via reconstruction of key aspects of the tumor microenvironment, allowing continuous monitoring of disease progression events in vitro. In addition, it is easily transferable to industry for feeding high-throughput systems or miniaturized bioreactors used in drug development, target validation and target identification. Acknowledgments: We acknowledge support from the Innovative Medicines Initiative Joint Undertaking (IMI grant agreement n° 115188), resources composed of financial contribution from EU - FP7 and EFPIA companies in kind contribution. iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), which is cofunded by Fundacao para a Ciencia e Tecnologia / Ministerio da Ciencia e do Ensino Superior, through national funds, and by FEDER under the PT2020 Partnership Agreement, is acknowledged. This research has also received support from Fundacao para a Ciencia e Tecnologia, Portugal—PTDC/BBB-BIO/1240/2012. MFE, SA and CP are the recipients of PhD fellowships from FCT (SFRH/BD/52208/2013, PD/BD/105768/2014 and SFRH/BD/52202/2013, respectively). Citation Format: Vitor E Santo, Marta F Estrada, Sofia P Rebelo, Sofia Abreu, Catarina Pinto, Elizabeth Anderson, Wolfgang Sommergruber, Paula Alves, Erwin Boghaert, Catarina Brito. 3D tumor models in bioreactors recapitulate microenvironment and disease progression. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A21.

Abstract 321: In vitro recapitulation of 3D tumor microenvironment with defined oxygen and pH levels through a novel scalable bioreactor-based strategy

Drug discovery for cancer therapy has faced tremendous inefficacy at advanced stages of clinical trials. The scientific community has been trying to tackle this issue by improving preclinical models for target validation, in which the complexity and heterogeneity found in human tumours can be recapitulated. In particular, the tumour microenvironment is known to influence tumour progression and drug resistance through tumour-stroma crosstalk. Three dimensional (3D) cell models allow maintenance of cell-cell and cell-extracellular matrix (ECM) interactions, mimicking in vivo tissue organization. However, current 3D cell models are typically generated in non-scalable culture systems, with poor robustness and use undefined, biologically active matrices. In this work, we present a novel strategy for in vitro recapitulation of solid tumour microenvironments. H1650 (human NSCLC lung adenocarcinoma) and MCF7 (human ER+ breast cancer) cells were selected for the establishment of lung and breast cancer models respectively. An alginate microencapsulation strategy for co-culture of either lung or breast tumour cells with fibroblasts was developed. The constructs were then cultured in stirred-tank bioreactors, with precise definition and control of oxygen and pH levels for several weeks, and characterized by analysis of cell viability, proliferation and apoptosis along culture time, as well as metabolic profiling, phenotypic characterization and analysis of ECM deposition The strategy developed allowed the formation of stable hydrogel microcapsules, with tumour aggregates presenting an epithelial phenotype with partial polarization, surrounded by fibroblasts, recapitulating tumour-stroma organization in vivo. Co-cultures of lung or breast cancer cell aggregates with fibroblasts led to the deposition and accumulation of collagen I type inside the capsules, along with altered tumour cell phenotype, with loss of epithelial character. Perturbation studies with chemotherapeutic agents revealed differences between models established with mono-cultures of tumour cells and co-cultures of tumour cells and fibroblasts. In conclusion, we have developed a scalable, robust and versatile strategy for establishment of in vitro long-term recapitulation of tumour-stroma crosstalk and hypoxic microenvironment, providing tools for characterization of disease progression mechanisms, target validation and drug response in different cancer types. Acknowledgements: The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking (grant agreement n° 115188), resources composed of financial contribution from EU- FP7and EFPIA companies in kind contribution. M.Estrada acknowledges support from FCT, Portugal (SFRH/BD/52208/2013).The authors are also thankful to Cathrin Brisken, Heiko van der Kuip, Moshe Oren and Erwin Boghaert. Citation Format: Vitor E. Santo, Marta Estrada, Sofia Rebelo, Elizabeth Anderson, Paula M. Alves, Catarina Brito. In vitro recapitulation of 3D tumor microenvironment with defined oxygen and pH levels through a novel scalable bioreactor-based strategy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 321. doi:10.1158/1538-7445.AM2015-321

Evaluation of the impact of matrix stiffness on encapsulated HepaRG spheroids

Background The drug development process is widely hampered by the lack of human models that recapitulate liver functionality and efficiently predict toxicity of new chemical compounds. Moreover, liver failure is a global medical problem, with transplantation being the only effective treatment currently available. The bipotent liver progenitor cell line HepaRG can be differentiated into cholangiocyte and hepatocyte-like cells that express major functions of mature hepatocytes, representing a valuable tool to model hepatic function [1]. Current two-dimensional (2D) protocols for the differentiation into mature hepatocyte-like cells fail to recapitulate the complex cell-cell interactions, which are crucial for maintaining polarity and inherent mature hepatic functionality. Herein, we present a three-dimensional (3D) strategy for the culture of HepaRG cells based on the encapsulation of aggregates. The effect of matrix stiffness on expansion and differentiation was evaluated through encapsulation with different concentrations of alginate (1.1% and 2%). Further characterization of the hepatic features will reveal the extent of the hepatic functionality of the generated spheroids. Materials and methods HepaRG cells were routinely propagated in static conditions as previously described [2]. Briefly, culture medium Williams E was supplemented with 1% (v/v) Glutamax, 1% (v/v) pen/strep, 5 μ g/ml insulin and 50 μ M hydrocortisone hemissuccinate and 10% (v/v) FBS and cultures were maintained at 37 ° C, 5% CO2 .S pinner vessels with ball impeller (Wheaton) were inoculated with inoculums ranging from 5 to 8 × 10 5 cell/mL and an agitation ranging from 35 to 45 rpm to attain the desired aggregation conditions. Aggregate size was determined by measuring Ferret’s diameter using the Image J software (NIH). After 3 days of aggregation, spheroids were encapsulated in 1.1% and 2% (w/v) of Ultra Pure MVG alginate (UP MVG NovaMatrix, Pronova Biomedical) in NaCl 0.9% (w/v) solution. Encapsulation was performed in an electrostatically driven microencapsulation unit VarV1 (Nisco) and cultures were maintained for 14 days in stirred culture conditions. Viability was determined by the double stain viability test - alginate beads were collected from stirred cultures, incubated with fluorescein diacetate (10 μg/ mL) and TO-PRO3 ® (1 μM) and observed on a fluores