Decellularization of precision-cut kidney slices-application of physical and chemical methods.

The extracellular matrix (ECM) obtained by decellularization provides scaffolds with the natural complex architecture and biochemical composition of the target organ. Whole kidney decellularization by perfusion uses the vasculature to remove cells leaving a scaffold that can be recellularized with patient-specific cells. However, decellularization and recellularization are highly complex processes that require intensive optimization of various parameters. In pursuit of this, a huge number of animals must be sacrificed. Therefore, we used precision-cut kidney slices (PCKS) as a source of natural scaffolds, which were decellularized by immersion in chemical reagents allowing the examination of more parameters with less animals. However, chemical reagents have a damaging effect on the structure and components of the ECM. Therefore, this study aimed at investigating the effects of physical treatment methods on the effectiveness of PCKS decellularization by immersion in chemical reagents (CHEM). PCKS were treated physically before or during immersion in chemicals (CHEM) with high hydrostatic pressure (HHP), freezing-thawing cycles (FTC) or in an ultrasonic bath system (UBS). Biochemical and DNA quantification as well as structural evaluation with conventional histology and scanning electron microscopy (SEM) were performed. Compared to decellularization by CHEM alone, FTC treatment prior to CHEM was the most effective in reducing DNA while also preserving glycosaminoglycan (GAG) content. Moreover, while UBS resulted in a comparable reduction of DNA, it was the least effective in retaining GAGs. In contrast, despite the pretreatment with HHP with pressures up to 200 MPa, it was the least effective in DNA removal. Histological scoring showed that HHP scaffolds received the best score followed by UBS, FTC and CHEM scaffolds. However further analysis with SEM demonstrated a higher deterioration of the ultrastructure in UBS scaffolds. Altogether, pretreatment with FTC prior to CHEM resulted in a better balance between DNA removal and structural preservation.

MyoBio: An Automated Bioreactor System Technology for Standardized Perfusion-Decellularization of Whole Skeletal Muscle.

OBJECTIVE: Decellularizing solid organs is a promising top-down process to produce acellular bio-scaffolds for 'de novo' regrowth or application as tissue 'patches' that compensate, e.g., large volumetric muscle loss in reconstructive surgery. Therefore, generating standardized acellular muscle scaffolds marks a pressing area of need. Although animal muscle decellularization protocols were established, those are mostly manually performed and lack defined bioreactor environments and metrologies to assess decellularization quality in real-time. To close this gap, we engineered an automated bioreactor system to provide chemical decellularization solutions to immersed whole rat gastrocnemius medialis muscle through perfusion of the main feeding arteries. RESULTS: Perfusion control is adjustable according to decellularization quality feedback. This was assessed both from (i) ex situ assessment of sarcomeres/nuclei through multiphoton fluorescence and label-free Second Harmonic Generation microscopy and DNA quantification, along with (ii) in situ within the bioreactor environment assessment of the sample's passive mechanical elasticity. CONCLUSION: We find DNA and sarcomere-free constructs after 72 h of 0.1% SDS perfusion-decellularization. Furthermore, passive elasticity can be implemented as additional online decellularization quality measure, noting a threefold elasticity decrease in acellular constructs. SIGNIFICANCE: Our MyoBio represents a novel and useful automated bioreactor environment for standardized and controlled generation of acellular whole muscle scaffolds as a valuable source for regenerative medicine.

Comparison of Loading Doses of Ziv-Aflibercept and Aflibercept in Neovascular Age-Related Macular Degeneration.

PURPOSE: The aim of this study was to compare outcomes of 3 loading doses of ziv-aflibercept and aflibercept in treatment-naïve neovascular age-related macular degeneration (nAMD). DESIGN: Retrospective, nonrandomized, comparative study. METHODS: This was a retrospective chart review which included cases with treatment-naïve nAMD. The patients were divided into 2 groups (group 1, ziv-aflibercept; group 2, aflibercept). Groups 1 and 2 received 1.25 mg/0.05 mL of intravitreal ziv-aflibercept and 2 mg/0.05 mL aflibercept, respectively every month for 3 months. Best-corrected visual acuity (BCVA) in Snellen and logarithm of minimum angle of resolution (logMAR), central subfoveal thickness (CSFT), subretinal hyperreflective material height, neurosensory detachment height, and pigment epithelial detachment height were recorded at baseline and 3 monthly follow-up. RESULTS: Twenty-three eyes of 23 patients were included (males 14, females 9). Twelve and 11 eyes were included in group 1 and group 2, respectively. Group 1 showed statistically significant improvement in BCVA (P < 0.001) and CSFT (P=0.007) through 3 months compared with baseline. There was significant change in BCVA from baseline at 1st month (P = 0.007), 2nd month (P = 0.002) and 3rd month (P = 0.008). In group 2, there was no significant improvement in BCVA, CSFT, subretinal hyperreflective material height, neurosensory detachment, and pigment epithelial detachment height from baseline through 3 months. CONCLUSIONS: After 3 loading doses, ziv-aflibercept showed efficacy in terms of improved BCVA and reduction of CSFT from baseline whereas aflibercept did not show such improvement. Considering the cost- effectiveness and the proven safety of ziv-aflibercept, it is a viable option for the crucial, initial 3 doses in the treatment of nAMD.