Calcium Alginate

The coaxial electrospray system and a cross-sectional view of the coaxial needle are illustrated in Fig. 1A and B, respectively. The system consists of two syringe pumps for pushing the core fluid with living cells and shell fluid of alginate through the concentric inner (28 G) and outer (21 G) lumens in the coaxial needle, respectively. Under an open electric field from a voltage generator, concentric drops of the two coaxial fluids at the needle tip were broken up into microdrops and sprayed into the gelling bath containing 100 mM calcium chloride solution to instantly gel alginate in the shell fluid before the two fluids got mixed. The core fluid contained ES cells at 5 × 10⁶ per ml, 0.25 M aqueous mannitol (instead of medium or physiologic saline according to our previous study^{38 (link)}), and either 2% sodium alginate (w/v) for making microcapsules with an alginate hydrogel core or 1% (w/v) sodium carboxymethyl cellulose for making microcapsules with a liquid core. The shell fluid consisted of 2% purified alginate (w/v) in 0.25 M aqueous mannitol solution. After encapsulation, the microcapsules with ES cells in the gelling solution were washed with 0.5 M mannitol solution for 7 min and suspended in ES cell medium after removing mannitol for further culture.^{38 (link)}

All mouse work was approved by the Office of Lab Animal Care at San Diego State University and conducted under accepted veterinary standards. Female CD1 and BALB/c mice (8–16 weeks old) were obtained from Charles River Laboratories and used for colonization assays adapted from previous work (Sheen et al., 2011 (link)). Breeding pairs of CXCr2 (CXCL2 receptor) knock out (KO) mice (formerly IL8r KO mice), were originally purchased (C.129S2(B6)-Cxcr2^tm1Mwm/J, Jackson Laboratories). The mutation was crossed onto a BALB/c background prior to being deposited at Jackson Laboratories. We established a homozygous×homozygous breeding colony at the UCSD VA Hospital using mice that were maintained on water containing co-trimoxazole (200 µg/mL sulfamethoxazole and 40 µg/mL trimethoprim). For the 17 week old females used in this study, antimicrobial treatment was terminated 48 hours prior to inoculation with GBS. To synchronize estrus and promote bacterial colonization (Furr et al., 1989 (link), Cheng et al., 2005 (link)), mice were injected intraperitoneally with 0.5mg 17β-estradiol suspended in sesame oil (Sigma) 24 hours prior to inoculation. Mice were inoculated with ~1×10⁷ cfu (in 10 µL PBS) GBS in the vaginal lumen. Immediately prior to inoculation, vaginal lavage was performed by pipetting the lumen with 20 µL of PBS several times to collect cells and cytokines as described elsewhere (Sonoda et al., 1998 (link), Caligioni, 2009 ). On successive days, the vaginal lumen of each mouse was first lavaged for cytokine analysis and then swabbed with ultrafine calcium alginate-tipped swabs. Bacterial load was determined by serial dilution plating of swab samples. WT or mutant GBS strains were identified as mauve or light pink-pigmented colonies on CHROMagar Strep B agar (DRG International Inc.) (Poisson et al., 2011 (link)). For tissue collection, mice were sacrificed using CO₂ asphyxiation and reproductive tracts excised from mid-uterine horn to just proximal of the vulva. Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. ELISA assays were performed on vaginal lavages for KC (R&D Systems), MIP-2 (R&D Systems) and IL-1β (eBiosciences) as described by manufacturer.