Equipment and Methods for Concurrently Housing Germfree and Gnotobiotic Mice in the Same Room.

Here, we combined the use of 2 technologies that have not previously been used together-a positively pressurized isolator IVC (IsoIVC-P) and a modular isolator with integrated vaporized hydrogen peroxide (VHP) technology???to develop highly tractable and scalable methods to support long-term maintenance of germfree mouse colonies and the concurrent use of germfree and gnotobiotic mice in the same room. This space-efficient system increases the practicality of microbiome studies. Specifically, the exterior surfaces of microbially similar IsoIVC-P were sterilized by using VHP prior to opening the cages and handling the mice therein. This space-efficient system increases the feasibility of microbiome studies. After over 74 wk of experimentation and handling equivalent to more than 1,379,693 germfree mouse-days, we determined that the method and practices we developed have a weekly performance metric of 0.0001 sterility breaks per husbandry unit; this rate is comparable to the isolator 'gold standard.' These data were achieved without adverse incidents while maintaining an Altered Schaedler Flora colony and multiple gnotobiotic studies involving fecal microbial transplants in the same room. Our novel IsoIVC-P???VHP workstation housing system thus improves microbiome research efficiency, eliminates hazards, and reduces risks associated with traditional methods.

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease.

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the "Murine Ommaya." As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Ovarian monocyte progenitor cells: phenotypic and functional characterization.

Leukocytes of the macrophage lineage are abundant in the ovarian tissues and have an important function in both follicular development and regression of postovulatory follicles. In this study, we tested the hypothesis that continuous production of macrophages in the ovarian stroma is maintained by a resident population of progenitors. We established a long-term culture of ovarian follicular stromal cells from BALB/c and green fluorescent protein-transgenic (GFP-TG) C57BL/6 mice. Nonadherent cells were collected and tested for hematopoietic function in vitro and in vivo. Histological and ultrastructural analyses revealed a homogenous population of monocyte-like rounded cells. Nonadherent cells continued to proliferate in culture for several months without senescence. When plated at very low density in methylcellulose, these cells formed colonies consisting of monocyte-like cells. Ovarian monocyte-like cells reacted with CD45, CD11b, CD11c, and Ly6-Gr-1 cell surface markers. A distinct CD45low population within these cells reacted with CD117 (C-kit) surface marker, suggestive of a primitive hematopoietic progenitor. Fifty thousand nonadherent cells failed to provide radioprotection to lethally irradiated mice and thus were not considered to be equivalent to pluripotent hematopoietic stem cells. Ovarian nonadherent stromal cells were positive for alkaline phosphatase but lacked embryonic cell antigens stage-specific embryonic antigen (SSEA-1) and Oct-4. We conclude that in the ovaries, a higher requirement for macrophages is provided by a resident stromal population of progenitors whose progeny is restricted to the production of cells of the monocyte-macrophage lineage.