0.2 μm membrane

Exosomes were isolated by adapting the protocol of Peinado et al. (13 (link)) from melanoma cell culture supernatant. B16F1 supernatants were harvested, supplemented by complete protease inhibitor cocktail (Roche, Mannheim, Germany) and centrifuged at 780 g for 5 min at 4°C to remove intact cells. Then, the supernatants were centrifuged at 3,900 g for 15 min at 4°C and filtered through a 0.2 μm membrane (Millipore, Billerica, MA) to remove larger cell fragments and microvesicles. Exosomes were pelleted by ultracentrifugation at 150,000 g (T-1270 rotor at 40,500 rpm) for 1 h at 4°C. The pellet was washed twice and resuspended in DPBS and stored at −80°C.
The concentration of exosomal proteins was determined using a Pierce BCA Protein assay kit (Thermo Fisher Scientific, Waltham, MA) and a Benchmark Microplate Reader (Bio-Rad, Hercules, CA) according to the manufacturer's instructions.

MAP strain ATCC 19698 was used in all the MAP PPD production processes. PPD lots produced at the NVSL in 1998 (9801), and ten years later in 2008 (0802, 0803A) were produced by traditional production methods involving autoclaving of production flasks containing floating cultures with subsequent protein precipitation using trichloroacetic acid (TCA) as described previously [15 (link), 16 ]. Lot 9801 currently serves as a reference lot and has been used in the field for cattle skin testing purposes since its production in 1998. Lot 0902 was produced by an alternative production method involving sterile filtration through a 0.2 μm membrane (Millipore) in place of autoclaving followed by protein precipitation using TCA. Lots 0902A and 0902B, which comprised the larger PPD 0902 bulk preparation, were harvested on different days from the same culture inoculum. In production of large PPD batches (lots), multiple concentrated harvest bulks are commonly combined to reduce the number of potency tests conducted and allow for larger final product release volumes. Due to the alternative production process used for PPD 0902 these two smaller bulks were held as reserve on each harvest date to evaluate possible differences in this revised procedure. Purity testing was conducted in accordance with the Code of Federal Regulations, 9CFR part 113.26.

The oral formulations of insulin-conjugated ORLN (ORLN-PHI) were prepared through a two-step method (Fig. 1). First, small empty unilamellar liposomes were obtained by probe sonication as follows: 0.01–0.05 g lipoid S100 was dissolved in 15 mL chloroform, the solvent was evaporated by a rotary evaporator, and the dispersion was obtained by adding 10 mL pure water and shaking vigorously. The dispersion was sonicated using a probe-type sonicator at 200 W for 20 min and filtered through a 0.2-μm membrane (Millipore, Billerica, MA, USA). The PHI solution was prepared by dissolving 1.0 mg PHI in 10 mL acidic water. Then, a 2-mL mixture of the two solutions described above was added to a 7-mL vial at a ratio of 1:1 (v/v) and lyophilized in a freeze drier for 48 h (FD-1; Beijing SiHuan Technology Company, Beijing, China) to remove all water. Lyophilization was performed under the following conditions: freezing at − 50 °C for 4 h, primary at − 45 °C to − 10 °C for 20 h, secondary at − 10 °C to 20 °C for 15 h, and maintaining at 20 °C for 9 h. Finally, oral formulations of ORLN-PHI with different PC contents were obtained by dissolving the lyophilized dry cake in 0.5 mL MCT. The resulting formulation contained PHI at a concentration of 0.2 mg/mL.Fig. 1

Preparation and structure of “oil-soluble” reversed lipid nanoparticle (ORLN)-insulin system

To determine the effect of stress hormones on the in vitro growth of A. hydrophila, the isolated A. hydrophila strain was cultured in TSB at 37 °C for 18 h, and the turbidity was adjusted to match that of 0.5 McFarland standards (1.5 × 10⁸/mL) for inoculation. The hormones norepinephrine and epinephrine (Sigma-Aldrich, St. Louis, MO, USA) were prepared at 0.1 M in PBS, and each solution was filtered through a 0.2-μm membrane (Millipore). These solutions were further diluted 10-fold in PBS to prepare solutions ranging from 10⁻² to 10⁻⁵ M [12 (link)], which were added to the broth. The growth of A. hydrophila was estimated using McFarland standards after incubation for 0, 24, 48, and 72 h at room temperature.