Page 178 - ESHRE2019

The Rabbit Model: Materials and Methods
Partial digestion: solubilizing ECM using pepsin in acid
environment.
• 48h pepsin digestion at RT in
0.01 M HCl.
• Put on ice and normalizing pH.
Hydrogel characterization:
• Scanning electron microscopy. • Gelation kinetic.
• Proteomics.
• Sterility.
Biological characteristics of coatings and hydrogels:
• Embryo culture.
Foundation
(Campo et al., 2019) 29
48h
• Aliquots flash frozen and stored at ‐80°C.
Innovation
The Rabbit Model: Materials and Methods
Embryo culture
Bioengineered substrata:
• Coating (C): 4°C overnight, wash with PBS and add 1ml culture medium.
Hydrogel (H): 1h incubation at 37 °C , leaving in culture medium overnight.
Control Conditions:
Positive control (C+FBS): Culture medium with fetal bovine serum.
Negative control (C‐FBS).
Culture medium without FBS, except for C+FBS condition.
ECM coating or hydrogel with biochemical cues from 3 conditions (M,NS,S) and uncoated controls.
Matrigel: M • Non‐synchronous: NS Synchronous: S
Day 3 embryo at 38.5°C for 48h.
• •
Compare and analyze:
• Hatching efficiency rates. • Morphometry analysis.
• Pluripotency markers.
Foundation
(Campo et al., 2019) 30
Innovation
8 culture conditions in total: MH, MSC , NSH, NSC, SH, SC, C‐FBS, C+FBS.
176
173