Fig 1.
Increasing salinity of fibrinogen solvent with NaCl impacts fibrin opacity.
pH (A) and CaCl2 concentration (B) of gel precursor solutions do not impact gel transparency or morphology. Increasing fibrinogen concentration decreases transparency (C). However, increasing NaCl concentration in fibrinogen solution prior to gelation increases final gel transparency (D, E). For reference, PBS absorbance of 352 nm light is 0.06.
Fig 2.
Changing salinity of fibrinogen solution alters fibrin gel properties.
SEM images of fiber morphology (A, B) reveal that increasing salt concentration decreases porosity and fiber size (C). Young’s moduli of individual fibers measured by AFM (D), Young’s and Storage Modulus of the bulk materials measured by parallel plate rheometry (E,F), Swelling Ratio (G), and growth factor sequestering (F) are also significantly affected by salinity during gelation.
Fig 3.
Increased salinity does not affect viability of encapsulated cells or ability to form endothelial networks.
LIVE/DEAD® analysis stains Live cells green and Dead cells red. No significant difference in viability detected between amniotic fluid cells seeded in HS and PS gels (A-F), as quantified in (I). Both HS and PS gels support capillary-like network formation when seeded with GFP-HUVEC (green) and HDF (stained red with anti-α-smooth muscle actin) (G, H). Scale bars 50um.
Fig 4.
Transparent high salt (HS) fibrin degrades slowly in vitro.
Increasing salt concentration yields fibrin with decreasing degradation kinetics when treated with Papain (no cells, panel A) and when seeded with AFC (B). 1 mg/mL 6-aminocaproic acid (ACA) prevents cell-mediated fibrinolysis, but HS gels are stable without ACA for at least 14d. Images of AFC-seeded gels degrading over 14d shown in panels C-I. By day 14, PS gels are completely degraded while HS gels are stable and continue to support 3D AFC culture (G-I). Scale bars of 200x images = 50um.
Fig 5.
HS gels are stable in vivo after 7d, but PS gels degraded completely.
HS and PS precursor solutions were mixed with GFP-HUVEC (green) and HDF (stained with Vimentin, red) and injected subcutaneously into athymic mice (N). After 7d, HS gels remained intact (H&E, A and B) and delivered cells remained viable (IF, C-G). Remaining gel indicated with arrows. PS gels degraded completely; no gel or delivered cells were detectable after 7d (H-M). Scale bars 500um (A, H), 200um (B-M), 50um (G).
Fig 6.
PS and HS gels support iPSC expansion and pluripotency.
Both gel types maintain expression of pluripotency genes POU5F1 (E) and NANOG (F) compared to standard 2D Matrigel® culture. HS gels appear to drive formation of spheroid-like iPSC colonies (A,B) while PS gels appear to maintain singularized iPSC (C,D). Scale bars 50um.