3/30/2019 — 4/3/2019

Exosomes are subcellular nanoparticles (50–200 nm in size) that are released from cells through a fusion of multicellular bodies with plasma membrane. Exosomes are currently being evaluated as potential diagnostic tools in a number of diseases including cancer. Exosomes are stable carriers of cell-free cargo in the form of DNA, RNA, and protein, thereby making them an attractive candidate for early detection of cancer via liquid biopsy. Tumor exosomes have also been linked to stimulation of tumor cell growth, angiogenesis, metastasis, and suppression of the immune system. However, isolating a consistent population of exosomes can be challenging and the need exists for highly characterized exosomes for use as reference standards for research and diagnostic applications. We have developed a novel method employing tangential flow filtration for  isolation of  large quantities of pure and sterile exosomes from cell culture media.

Among women, breast cancer continues to be the most common cancer, with metastasis being the leading cause of mortality in patients around the world. Epithelial to mesenchymal transition (EMT)—the process by which epithelial cells shift to the mesenchymal state—has been implicated in many aspects of breast cancer tumorigenesis, metastasis, and drug resistance. However, despite the extensive accumulation of data on the association of EMT with cancer over the years, EMT has not been an active target for therapeutic development. This is due in part to the lack of appropriate in vitro models. Here, we have exploited some of the basic biology of EMT to create an advanced in vitro metastatic breast cancer reporter cell line model for use in basic research and the discovery of new EMT inhibitors.

3/30/2019 — 4/3/2019

World-wide, metastasis continues to be the leading cause of death in cancer patients. Although epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) have been implicated in the incidence of cancer metastasis and drug resistance, their impact in cancer progression and patient survival is not fully understood. This is partly due to the lack of suitable in vitro models. Thus, to facilitate the utility of the EMT concept in therapeutic development, we have utilized some of the basic biology of EMT/MET to create a novel advanced in vitro model for use in both basic research and discovery of new anti-EMT drugs.

Epithelial-to-mesenchymal transition (EMT) describes a dynamic and reversible process where cells lose their epithelial characteristics and acquire mesenchymal properties. Accumulating evidence indicates that EMT displays an array of intermediate states, a phenotype referred to as “partial EMT”.1 EMT is executed in response to signaling pathway molecules and microRNAs (miRNAs) that induce the expression of specific EMT-associated transcription factors (EMT-TFs), including Zeb1/2, Snail1/2, and Twist. There is clinical evidence and an ever-growing body of research indicating that EMT plays an important role in cancer cell dissemination and distal metastasis.2 Therefore, targeting EMT is considered a novel opportunity in anti-cancer treatment and drug development.

Metastasis is responsible for most cancer-related deaths. One mechanism of metastasis involves epithelial-to-mesenchymal transition (EMT), a process characterized by the decrease in cell adhesion and increase in cell motility. Cells undergoing EMT often display downregulation of epithelial markers (such as E-cadherin; ECAD) and upregulation of mesenchymal markers (such as vimentin; VIM). Besides metastasis, EMT has also been reported to be associated with other pathological conditions, such as acquired therapeutic drug resistance. Given the roles that EMT plays in the pathological processes, it is of increasing interest as a target for anti-cancer treatment and drug discovery. In vitro reporter models have proven to be a valuable tool for dissecting the signaling pathways that regulate the EMT process and for screening compounds targeting EMT. In previously developed EMT reporter cell lines, the reporter gene was driven by a truncated EMT marker gene promoter. Therefore, the establishment of a more physiologically relevant reporter cell model is critical for advancing our knowledge of EMT.

Propagation of Human Organoids

Utilizing Tissue-specific Reagent Kits and Ready-to-use Wnt-3a and R-Spondin1 Conditioned Media

3/30/2019 — 4/3/2019

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 demonstrate 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.

There is an increasing demand for in vitro models to replace animal models in toxicity testing. Drivers for this change include decreased overall cost for cell-based models and the ability to do more high-throughput screening. The development of cell-based in vitro models for toxicity testing is a challenging task. Primary cells can best represent the in vivo situation; however, donor variability and replicative senescence restrict the potential usefulness of this cell model in the study of toxicity. Conversely, continuous cell lines often have altered genomes and do not fully represent the parental cells as a result of their altered genomic state. Human telomerase reverse transcriptase (hTERT)-immortalized primary cells provide a better solution—these cells can be continuously cultured while retaining the physiological characteristics of the parental primary cell.

3/10/2019 — 3/13/2019

Human primary cells are useful pre-clinical models as they more closely mimic the physiology of cells in vivo than continuous cell lines. Here, we explored some of the applications of human primary cells for cytotoxicity assays and drug screening. Cancer is a leading cause of female mortality worldwide and gynecologic cancers have a low survival rate. Further, cytotoxicity is a common side effect of all anti-cancer drugs. We investigated the cytotoxic effects of three anti-cancer drugs (alone or in combination) on three types of normal reproductive cells in vitro. Primary human uterine fibroblasts, cervical epithelial cells, and vaginal epithelial cells along with cervical (SiHa) and vaginal (VK2/E6E7) epithelial cancer cell lines were treated for two days with topotecan, paclitaxel, cisplatin, or a combination of topotecan and cisplatin at concentrations of 0 µM, 0.1 µM, 1 µM, 10 µM, or 100 µM. Cytotoxicity of these chemotherapeutic drugs was assessed by using Reliablue™ Cell Viability Reagent.

Skin pigmentation is a complex process; melanocytes produce melanin and package it into melanosomes that are in turn exocytosed into the surrounding extracellular matrix and adjacent cells. Numerous genes play a role in controlling pigmentation at various levels of melanin production. Mutations in these genes are characteristic of multiple skin disorders, including hyperpigmentation, hypopigmentation, and mixed hyper/hypopigmentation. Additionally, extrinsic factors secreted by the surrounding resident cell types also regulate the melanin expression in adult melanocytes. Human primary cells can be a useful model for elucidating melanocyte biology. However, primary cells have their limitations such as donor variability, a limited lifespan and loss of melanin. Therefore, there is a need for a more robust human cell model system for studying skin pigmentation.

The scientific community is currently experiencing an outpouring of research surrounding extracellular vesicles (EVs) and, more specifically, exosomes. This is due not only to their critical role in intercellular communication, but also to their potential to be used as diagnostic tools and/or therapeutic agents in a wide range of pathological conditions1. The rising interest in exosomes, coupled with the immense volume of research, underlies significant needs for both the isolation of high quality exosomes from large-scale batches and the development of industry standards for the characterization and quality control testing of exosomes. While traditional methods, which include the use of ultra-centrifugation and density gradients, are suitable for small-scale studies, the development of scalable and robust processes for the isolation of functional exosomes is essential to meet the growing needs of the scientific community. Here, we report the use of tangential flow filtration (TFF) for the isolation and concentration of functional exosomes from large batches of conditioned culture medium to include lung carcinoma cells, human mesenchymal stem cells (MSCs), and human induced pluripotent stem cells (iPSCs).