Page 171 - ESHRE2019

The Pig Model: Materials and Methods
Experimental Design
D1
Assessment scaffold: finding a balance between DC and ECM preservation.
Protocol 1: Fresh uterus (F)
DC protocol
D2
&
D4 D3
Reagent Concentration Duration
Day 1
Day 2
PBS 1X 1 hour
SDS 0,10% 18 hours
H2Od ‐ 30 min
Triton‐X100 1% 30 min
PBS 1X 5 hours
SDS 0,10% 18 hours
H2Od ‐ 30 min
Triton X‐100 1% 30 min
Foundation
PBS 1X 5 hours
Unperfused
Disks: D1, D2, D3, D4
Successful DC
Impact on ECM
(Campo et al., 2017) 15
Protocol 2: Freeze/thawed uterus (F/T)
Innovation
The Pig Model: Materials and Methods
Experimental Design Successful decellularization
• Qualitatively: • HE.
Impact on ECM
• Protein quantification.
• Qualitatively: • Stainings.
• Trichrome (collagen).
• Alcian blue (GAGs). • Electron microscopy
(ultrastructure).
• Scanning (SEM).
• Transmission (TEM).
• Immunofluorescence. • Collagen I & IV.
• Elastin.
• Fibronectin.
• Laminin.
• Vascular integrity:
• Vascular corrosion cast.
Testing biocompatibility
Assessing bio‐inductive capacities of decellularized endometrium.
• DAPI.
• DNA quantification & size
recellularization of acellular scaffold disks.
(Campo et al., 2017)
fragment determination.
Innovation
Foundation
16
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PRECONGRESS COURSE 11 I VIENNA, AUSTRIA – 23 JUNE 2019 169
Cycle 2 Cycle 1