3D printed mold leachates in PDMS microfluidic devices

The introduction of poly(dimethylsiloxane) (PDMS) and soft lithography in the 90’s has revolutionized the field of microfluidics by almost eliminating the need for a clean-room environment for device fabrication. More recently, 3D printing has been introduced to fabricate molds for soft lithography, the only step for which a clean-room environment is still often necessary, to further support the rapid prototyping of PDMS microfluidic devices. However, toxicity of most of the commercial 3D printing resins has been established, and little is known regarding the potential for 3D printed molds to leak components into the PDMS that would, in turn, hamper cells and/or tissues cultured in the devices. In the present study, we investigated if 3D printed molds produced by stereolithography can leach components into PDMS, and compared 3D printed molds to their more conventional SU-8 counterparts. Different leachates were detected in aqueous solutions incubated in the resulting PDMS devices prepared from widely used PDMS pre-polymer:curing agent ratios (10:1, 15:1 and 20:1), and these leachates were identified as originating from resins and catalyst substances. Next, we explored the possibility to culture cells and tissues in these PDMS devices produced from 3D printed molds and after proper device washing and conditioning. Importantly, we demonstrated that the resulting PDMS devices supported physiological cultures of HeLa cells and ovarian tissues in vitro, with superior outcomes than static conventional cultures.</p

A dog oviduct-on-a-chip model of serous tubal intraepithelial carcinoma

Ovarian cancer is the fifth cause of cancer-related mortality in women, with an expected 5-year survival rate of only 47%. High-grade serous carcinoma (HGSC), an epithelial cancer phenotype, is the most common malignant ovarian cancer. It is known that the precursors of HGSC originate from secretory epithelial cells within the Fallopian tube, which first develops as serous tubal intraepithelial carcinoma (STIC). Here, we used gene editing by CRISPR-Cas9 to knock out the oncogene p53 in dog oviductal epithelia cultured in a dynamic microfluidic chip to create an in vitro model that recapitulated human STIC. Similar to human STIC, the gene-edited oviduct-on-a-chip, exhibited loss of cell polarization and had reduced ciliation, increased cell atypia and proliferation, with multilayered epithelium, increased Ki67, PAX8 and Myc and decreased PTEN and RB1 mRNA expression. This study provides a biomimetic in vitro model to study STIC progression and to identify potential biomarkers for early detection of HGSC.</p

CHROMIC AND IRON OXIDES AS FECAL MARKERS TO IDENTIFY INDIVIDUAL WHOOPING CRANES

The whooping crane (Grus americana) is listed as endangered under the IUCN Red List, the United States Endangered Species Act, and the Canadian Species at Risk Act (BirdLife International 2012, CWS and USFWS 2007). A major focus of recovery efforts for this endangered species is reintroduction to establish new populations (CWS and USFWS 2007). Captive populations are critical as a source of individuals for reintroduction efforts and also serve as insurance populations. Currently, there are a total of 157 whooping cranes held in captive breeding centers across North America, with the largest at the USGS Patuxent Wildlife Research Center (PWRC) in Laurel, Maryland. Birds produced in this facility are currently being released as part of efforts to establish the Eastern Migratory Population (EMP, Urbanek et al. 2005) and in an effort to establish a non-migratory population in Louisiana. In the past decade, PWRC has produced and released annually an average of 18 birds into the wild; however, reproductive performance of birds at this facility is lower than desired. PWRC had a 60% fertility rate for eggs laid from 2000 through 2010 (J. N. Chandler, personal communication, 2011). Furthermore, reproductive onset in this captive population appears to be delayed compared to wild populations. In wild populations, reproductive onset (production of sperm and eggs) normally occurs ~5 years of age in both males and females, ~2 years after initial pair formation occurs (Ellis et al., 1996), while some females in the EMP have laid eggs earlier than 5 years of age (Converse et al. 2011). However, PWRC females in some cases do not start to lay eggs until 7 years of age (Mirande et al. 1996). Currently, the PWRC population consists of a total of 74 whooping cranes, including 22 pairs. Six of these pairs (27%) are consistently infertile (i.e., no production of fertile eggs) and 3 other pairs (14%) have low fertility (30- 45% fertility in eggs laid), which is variable from year to year. Six pairs (27%) are recently formed and have not produced eggs, and so have unknown fertility. This leaves only 7 pairs (33%) which contribute maximally to PWRC’s chick production (J. N. Chandler, personal communication, 2011). Because of the challenges occurring within this captive colony, PWRC and Smithsonian National Zoo have initiated a joint research project to identify potential underlying causes of poor reproduction in captive whooping cranes

Conserving, Distributing and Managing Genetically Modified Mouse Lines by Sperm Cryopreservation

Sperm from C57BL/6 mice are difficult to cryopreserve and recover. Yet, the majority of genetically modified (GM) lines are maintained on this genetic background.Reported here is the development of an easily implemented method that consistently yields fertilization rates of 70+/-5% with this strain. This six-fold increase is achieved by collecting sperm from the vas deferens and epididymis into a cryoprotective medium of 18% raffinose (w/v), 3% skim milk (w/v) and 477 microM monothioglycerol. The sperm suspension is loaded into 0.25 mL French straws and cooled at 37+/-1 degrees C/min before being plunged and then stored in LN(2). Subsequent to storage, the sperm are warmed at 2,232+/-162 degrees C/min and incubated in in vitro fertilization media for an hour prior to the addition of oocyte cumulus masses from superovulated females. Sperm from 735 GM mouse lines on 12 common genetic backgrounds including C57BL/6J, BALB/cJ, 129S1/SvImJ, FVB/NJ and NOD/ShiLtJ were cryopreserved and recovered. C57BL/6J and BALB/cByJ fertilization rates, using frozen sperm, were slightly reduced compared to rates involving fresh sperm; fertilization rates using fresh or frozen sperm were equivalent in all other lines. Developmental capacity of embryos produced using cryopreserved sperm was equivalent, or superior to, cryopreserved IVF-derived embryos.Combined, these results demonstrate the broad applicability of our approach as an economical and efficient option for archiving and distributing mice

Effects of body size on estimation of mammalian area requirements.

Accurately quantifying species' area requirements is a prerequisite for effective area-based conservation. This typically involves collecting tracking data on species of interest and then conducting home range analyses. Problematically, autocorrelation in tracking data can result in space needs being severely underestimated. Based on the previous work, we hypothesized the magnitude of underestimation varies with body mass, a relationship that could have serious conservation implications. To evaluate this hypothesis for terrestrial mammals, we estimated home-range areas with global positioning system (GPS) locations from 757 individuals across 61 globally distributed mammalian species with body masses ranging from 0.4 to 4000 kg. We then applied blockcross validation to quantify bias in empirical home range estimates. Area requirements of mammals 1, meaning the scaling of the relationship changedsubstantially at the upper end of the mass spectrum

Effects of body size on estimation of mammalian area requirements

Accurately quantifying species’ area requirements is a prerequisite for effective area‐based conservation. This typically involves collecting tracking data on species of interest and then conducting home‐range analyses. Problematically, autocorrelation in tracking data can result in space needs being severely underestimated. Based on previous work, we hypothesized the magnitude of underestimation varies with body mass, a relationship that could have serious conservation implications. To evaluate this hypothesis for terrestrial mammals, we estimated home‐range areas with GPS locations from 757 individuals across 61 globally distributed mammalian species with body masses ranging from 0.4 to 4,000 kg. We then applied block cross‐validation to quantify bias in empirical home‐range estimates. Area requirements of mammals 1, meaning the scaling of the relationship changed substantially at the upper end of the mass spectrum