MR spectroscopy of cultured cells allows non-invasive analyses of the metabolism of cells with specific phenotypes under defined conditions. This technique can be used to investigate the intracellular metabolism of cells or extended to critically evaluate phenomena observed by in vivo MRS. In this paper, a cell maintenance system is described which allows MR analyses with unparalleled spectral resolution, S/N and stability. This system consists of a 25 mm diameter hollow fiber bioreactor and a supporting circuit. The hollow fiber reactor was chosen because it yields a large filling factor which can be perfused through defined volumes. The fibers were 300 microns diameter microporous (0.2 micron) cellulose acetate/cellulose nitrate membranes with high porosity, which allow bulk convective flow throughout the extracapillary space. This flow (Starling flow) is necessary to disrupt steady-state gradients in substrates and waste products. In many respects, the design of the supporting circuit is more important than the bioreactor itself, since it provides the reactor with the proper chemical and physical environment. Hence, this circuit can be applied to a variety of bioreactor configurations. The circuit consists of a hollow fiber oxygenator and a bleed-and-feed system housed in a temperature-controlled cabinet. Culture of mammalian cells in this reactor yields 31P spectra which have excellent spectral and temporal resolution. At confluence, endogenous 31P line widths were typically < 10 Hz (at 162 MHz) and well resolved spectra were obtained in < 30 s.
Reliable detection and measurement of cell proliferation are essential in the preclinical assessment of carcinogenic risk of therapeutics. In this context, the assessment of mitogenic potential on mammary glands is crucial in the preclinical safety evaluation of novel insulins. The existing manual counting is time-consuming and subject to operator bias. To standardize the processes, make it faster, and resistant to errors, we developed a semiautomated image analysis system (CEPA software, which is open-source) for counting of proliferating cells in photomicrographs of mammary gland sections of rats labeled with Ki-67. We validated the software and met the predefined targets for specificity, accuracy, and reproducibility. In comparison to manual counting, the respective mean differences in absolute labeling indices (LIs) for CEPA software were 3.12% for user 1 and 3.05% for user 2. The respective regression analysis revealed a good correlation between the CEPA software user and manual counting. Moreover, the CEPA software showed enhanced reproducibility between independent users. The interuser variability is centered around 0 and the absolute difference was about 0.53% LI. Based on validation data, our software has superiority to the manual counting and is a valid and reliable tool for the routine analysis of cell proliferation in mammary glands from rats exposed to insulin analogs.
Applications of nuclear magnetic resonance (NMR) spectroscopy to isolated or cultured mammalian cells have been limited because of technical difficulties in maintaining cultures at the extremely high densities required by NMR. Among the well-engineered systems available for such analyses, hollow fiber bioreactors (HFBRs) can maintain the greatest cell density. This attribute of HFBRs makes them ideal for application to NMR-based studies. These systems are currently being applied in biotechnology, where they are used for the production of mammalian cell-derived products, such as monoclonal antibodies. In this paper, the application of a HFBR system designed especially for NMR-based investigations is described. Performance of this system is monitored by NMR and the resulting stability and density of hybridoma cultures are reported. The resulting signal-to-noise per unit time is the highest seen to date for a mammalian cell system.
The pattern recognition receptor RIG-I plays an important role in the recognition of nonself RNA and antiviral immunity. RIG-I's natural ligand, triphosphate RNA (ppp-RNA), is proposed to be a valuable addition to the growing arsenal of cancer immunotherapy treatment options. In this study, we present comprehensive data validating the concept and utility of treatment with synthetic RIG-I agonist ppp-RNA for the therapy of human cancer, with melanoma as potential entry indication amenable to intratumoral treatment. Using mRNA expression data of human tumors, we demonstrate that RIG-I expression is closely correlated to cellular and cytokine immune activation in a wide variety of tumor types. Furthermore, we confirm susceptibility of cancer cells to ppp-RNA treatment in different cellular models of human melanoma, revealing unexpected heterogeneity between cell lines in their susceptibility to RNA agonist features, including sequence, secondary structures, and presence of triphosphate. Cellular responses to RNA treatment (induction of type I IFN, FasR, MHC-I, and cytotoxicity) were demonstrated to be RIG-I dependent using KO cells. Following ppp-RNA treatment of a mouse melanoma model, we observed significant local and systemic antitumor effects and survival benefits. These were associated with type I IFN response, tumor cell apoptosis, and innate and adaptive immune cell activation. For the first time, we demonstrate systemic presence of tumor antigen-specific CTLs following treatment with RIG-I agonists. Despite potential challenges in the generation and formulation of potent RIG-I agonists, ppp-RNA or analogues thereof have the potential to play an important role for cancer treatment in the next wave of immunotherapy.
Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease that can lead to irreversible liver cirrhosis and cancer. Early diagnosis of NASH is vital to detect disease before it becomes life-threatening, yet noninvasively differentiating NASH from simple steatosis is challenging. Herein, bifunctional probes have been developed that target the hepatocyte-specific asialoglycoprotein receptor (ASGPR), the expression of which decreases during NASH progression. The results show that the probes allow longitudinal, noninvasive monitoring of ASGPR levels by positron emission tomography in the newly developed rat model of NASH. The probes open new possibilities for research into early diagnosis of NASH and development of drugs to slow or reverse its progression.
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