Streamlined Methods for Processing and Cryopreservation of Cell Therapy Products Using Automated Systems.

Streamlined procedures for processing and cryopreservation of cell therapies using good laboratory practices are integral to biomanufacturing process development and clinical applications. The protocol herein begins with the preparation of human cell types cultured as adherent (i.e., mesenchymal stromal cells, MSCs) or suspension cells (i.e., peripheral blood mononuclear cells, PBMCs) to comprehensively demonstrate procedures that are applicable to commonly used primary cell cultures. Cell processing steps consist of preparing high yields of cells for cryopreservation using instruments routinely used in cell manufacturing, including the Finia® Fill and Finish System and a controlled-rate freezer. The final steps comprise the storage of cells at subzero temperatures in liquid nitrogen vapor phase followed by the analysis of cell phenotypes before and after processing and cryopreservation, along with cell quality metrics for validation. Additionally, the protocol includes important considerations for the implementation of quality control measures for equipment operation and cell handling, as well as Good Laboratory Practices for cell manufacturing, which are essential for the translational use of cell therapies. Key features • The protocol applies to small- or large-scale manufacturing of cell therapy products. • It includes streamlined procedures for processing and cryopreservation of cells cultured as adherent cells (MSCs) and suspension cells (PBMCs). • Provides temperature control and rapid partitioning of sample in cryopreservation solution to maintain high viability of a range of cell types throughout the procedures. • This protocol employs the Finia® Fill and Finish System and a controlled-rate freezer. Graphical overview.

Refractive states of eyes and associations between ametropia and age, breed, and axial globe length in domestic cats.

OBJECTIVE: To determine the refractive states of eyes in domestic cats and to evaluate correlations between refractive error and age, breed, and axial globe measurements. ANIMALS: 98 healthy ophthalmologically normal domestic cats. PROCEDURES: The refractive state of 196 eyes (2 eyes/cat) was determined by use of streak retinoscopy. Cats were considered ametropic when the mean refractive state was ≥ ± 0.5 diopter (D). Amplitude-mode ultrasonography was used to determine axial globe length, anterior chamber length, and vitreous chamber depth. RESULTS: Mean ± SD refractive state of all eyes was -0.78 ± 1.37 D. Mean refractive error of cats changed significantly as a function of age. Mean refractive state of kittens (≤ 4 months old) was -2.45 ± 1.57 D, and mean refractive state of adult cats (> 1 year old) was -0.39 ± 0.85 D. Mean axial globe length, anterior chamber length, and vitreous chamber depth were 19.75 ± 1.59 mm, 4.66 ± 0.86 mm, and 7.92 ± 0.86 mm, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Correlations were detected between age and breed and between age and refractive states of feline eyes. Mean refractive error changed significantly as a function of age, and kittens had greater negative refractive error than did adult cats. Domestic shorthair cats were significantly more likely to be myopic than were domestic mediumhair or domestic longhair cats. Domestic cats should be included in the animals in which myopia can be detected at a young age, with a likelihood of progression to emmetropia as cats mature.

Effects of the topically applied calcium-channel blocker flunarizine on intraocular pressure in clinically normal dogs.

OBJECTIVE: To determine effects of the topically applied calcium-channel blocker flunarizine on intraocular pressure (IOP) in clinically normal dogs. ANIMALS: 20 dogs. PROCEDURES: Baseline diurnal IOPs were determined by use of a rebound tonometer on 2 consecutive days. Subsequently, 1 randomly chosen eye of each dog was treated topically twice daily for 5 days with 0.5% flunarizine. During this treatment period, diurnal IOPs were measured. In addition, pupillary diameter and mean arterial blood pressure (MAP) were evaluated. Serum flunarizine concentrations were measured on treatment day 5. Intraday fluctuation of IOP was analyzed by use of an ANOVA for repeated measures and a trend test. Changes in IOP from baseline values were assessed and compared with IOPs for the days of treatment. Values were also compared between treated and untreated eyes. RESULTS: A significant intraday fluctuation in baseline IOP was detected, which was highest in the morning (mean +/- SE, 15.8 +/- 0.63 mm Hg) and lowest at night (12.9 +/- 0.61 mm Hg). After 2 days of treatment, there was a significant decrease in IOP from baseline values in treated (0.93 +/- 0.35 mm Hg) and untreated (0.95 +/- 0.34 mm Hg) eyes. There was no significant treatment effect on pupillary diameter or MAP. Flunarizine was detected in serum samples of all dogs (mean +/- SD, 3.89 +/- 6.36 microg/L). CONCLUSIONS AND CLINICAL RELEVANCE: Topically applied flunarizine decreased IOP in dogs after 2 days of twice-daily application. This calcium-channel blocker could be effective in the treatment of dogs with glaucoma.