Publications

The involvement of the extracellular matrix (ECM) in tumor progression has motivated the development of biomaterials mimicking the tumor ECM to develop more predictive cancer models. Particularly, polypeptides based on elastin could be an interesting approach to mimic the ECM due to their tunable properties. Here, we demonstrated that elastin-like recombinamer (ELR) hydrogels can be suitable biomaterials to develop breast cancer models. This hydrogel was formed by two ELR polypeptides, one containing sequences biodegradable by matrix metalloproteinase and cyclooctyne and the other carrying arginylglycylaspartic acid and azide groups to allow cell adhesion, biodegradability, and suitable stiffness through "click-chemistry" cross-linking. Our findings show that breast cancer or nontumorigenic breast cells showed high viability and cell proliferation for up to 7 days. MCF7 and MCF10A formed spheroids whereas MDA-MB-231 formed cell networks, with the expression of ECM and high drug resistance in all cases, evidencing that ELR hydrogels are a promising biomaterial for breast cancer modeling.

JTD Keywords: clinical-trials, collagen i, discovery, mcf-7 cells, phenotype, progression, spheroids, translation, tumor microenvironment, Extracellular-matrix

Technologies to cryogenically preserve (a.k.a. cryopreserve) living tissue, cell lines and primary cells have matured greatly for both clinicians and researchers since their first demonstration in the 1950s and are widely used in storage and transport applications. Currently, however, there remains an absence of viable cryopreservation and thawing methods for bioengineered, three-dimensional (3D) cell models, including patients' samples. As a first step towards addressing this gap, we demonstrate a viable protocol for spheroid cryopreservation and survival based on a 3D carboxymethyl cellulose scaffold and precise conditions for freezing and thawing. The protocol is tested using hepatocytes, for which the scaffold provides both the 3D structure for cells to self-arrange into spheroids and to support cells during freezing for optimal post-thaw viability. Cell viability after thawing is improved compared to conventional pellet models where cells settle under gravity to form a pseudo-tissue before freezing. The technique may advance cryobiology and other applications that demand high-integrity transport of pre-assembled 3D models (from cell lines and in future cells from patients) between facilities, for example between medical practice, research and testing facilities.

JTD Keywords: 3d cell culture, biofabrication, biomaterials, carboxymethyl cellulose, cryopreservation, hepatocytes, 3d cell culture, Biofabrication, Biomaterials, Carboxymethyl cellulose, Cell culture techniques, three dimensional, Cell survival, Cryopreservation, Freezing, Hepatocytes, Humans, Prevention, Scaffolds, Spheroids

JTD Keywords: Breast cancer, Hydrogels, Spheroids

BACKGROUND: The embryo implantation process is crucial for the correct establishment and progress of pregnancy. During implantation, the blastocyst trophectoderm cells attach to the epithelium of the endometrium, triggering intense cell-to-cell crosstalk that leads to trophoblast outgrowth, invasion of the endometrial tissue, and formation of the placenta. However, this process, which is vital for embryo and foetal development in utero, is still elusive to experimentation because of its inaccessibility. Experimental implantation is cumbersome and impractical in adult animal models and is inconceivable in humans. OBJECTIVE AND RATIONALE: A number of custom experimental solutions have been proposed to recreate different stages of the implantation process in vitro, by combining a human embryo (or a human embryo surrogate) and endometrial cells (or a surrogate for the endometrial tissue). In vitro models allow rapid high-throughput interrogation of embryos and cells, and efficient screening of molecules, such as cytokines, drugs, or transcription factors, that control embryo implantation and the receptivity of the endometrium. However, the broad selection of available in vitro systems makes it complicated to decide which system best fits the needs of a specific experiment or scientific question. To orient the reader, this review will explore the experimental options proposed in the literature, and classify them into amenable categories based on the embryo/cell pairs employed. The goal is to give an overview of the tools available to study the complex process of human embryo implantation, and explain the differences between them, including the advantages and disadvantages of each system. SEARCH METHODS: We performed a comprehensive review of the literature to come up with different categories that mimic the different stages of embryo implantation in vitro, ranging from initial blastocyst apposition to later stages of trophoblast invasion or gastrulation. We will also review recent breakthrough advances on stem cells and organoids, assembling embryo-like structures and endometrial tissues. OUTCOMES: We highlight the most relevant systems and describe the most significant experiments. We focus on in vitro systems that have contributed to the study of human reproduction by discovering molecules that control implantation, including hormones, signalling molecules, transcription factors and cytokines. WIDER IMPLICATIONS: The momentum of this field is growing thanks to the use of stem cells to build embryo-like structures and endometrial tissues, and the use of bioengineering to extend the life of embryos in culture. We propose to merge bioengineering methods derived from the fields of stem cells and reproduction to develop new systems covering a wider window of the implantation process.

JTD Keywords: in vitro models, blastocyst, blastocyst-like structures, early-pregnancy, endometrial cells, epidermal-growth-factor, gene-expression, implantation, in vitro models, in-vitro model, indian hedgehog, organoids, receptivity, self-organization, spheroids, trophoblast, trophoblast invasion, uterine receptivity, Blastocyst, Blastocyst-like structures, Early-pregnancy, Endometrial cells, Endometrial stromal cells, Epidermal-growth-factor, Gene-expression, Implantation, In vitro models, In-vitro model, Indian hedgehog, Organoids, Receptivity, Self-organization, Spheroids, Trophoblast, Trophoblast invasion, Uterine receptivity

© 2020, Springer Science+Business Media, LLC, part of Springer Nature. In many solid tumors, collective cell invasion prevails over single-cell dissemination strategies. Collective modes of invasion often display specific front/rear cellular organization, where invasive leader cells arise from cancer cell populations or the tumor stroma. Collective invasion involves coordinated cellular movements which require tight mechanical crosstalk through specific combinations of cell–cell interactions and cell–matrix adhesions. Cancer Associated Fibroblasts (CAFs) have been recently reported to drive the dissemination of epithelial cancer cells through ECM remodeling and direct intercellular contact. However, the cooperation between tumor and stromal cells remains poorly understood. Here we present a simple spheroid invasion assay to assess the role of CAFs in the collective migration of epithelial tumor cells. This method enables the characterization of 3D spheroid invasion patterns through live cell fluorescent labeling combined with spinning disc microscopy. When embedded in extracellular matrix, the invasive strands of spheroids can be tracked and leader/follower organization of CAFs and cancer cells can be quantified.

JTD Keywords: 3d spheroid invasion, cancer associated fibroblasts, collective migration, dissemination, epithelial cancer cells, leader/follower cells, 3d spheroid invasion, Cancer associated fibroblasts, Cancer-associated fibroblasts, Cell culture techniques, Cell line, tumor, Cell movement, Cell tracking, Collective invasion, Collective migration, Epithelial cancer cells, Extracellular matrix, Humans, Imaging, three-dimensional, Leader/follower cells, Microscopy, fluorescence, Spheroids, cellular, Tumor cells, cultured