Compositions and methods for induction and modulation of angiogenesis and methods and assays for identifying angiogenesis modulators
View Patent ↗The present disclosure provides methods and compositions, including a placental extract, for inducing and/or modulating angiogenesis; methods of identifying modulators of angiogenesis, and assays for identifying modulators of angiogenesis. The present disclosure also provides methods of making a composition, including a placental extract that can induce and/or modulate angiogenesis.
1. A method of making a human placental extract, the method comprising:
obtaining a sample from a human placenta;
removing blood from the placental sample to produce a crude placental extract;
mixing the crude placental extract with a protein solubilization agent to solubilize and linearize proteins in the crude extract;
separating and removing solid materials from the solubilized protein-placental extract;
performing dialysis on the solubilized protein-placental extract to remove the protein solubilization agent; and
after dialysis, removing remaining solids from the extract to produce the human placental extract, whereby the resulting human placental extract contains less than 2% normalized protein abundance of collagen IV or laminin.
2. The method of claim 1 , wherein removing blood from the placental sample comprises homogenizing the human placenta sample with a buffer, centrifuging the homogenized sample, and discarding the supernatant containing blood.
3. The method of claim 1 , wherein the protein solubilization agent is urea.
4. The method of claim 1 , wherein separating solid materials from the solubilized protein-placental extract comprises centrifuging the solubilized protein-placental extract and removing solids.
5. The method of claim 1 , wherein the method is conducted at a temperature between about −86° C. and about 5° C.
6. The method of claim 1 , wherein removing remaining solids from the dialyzed protein-placental extract comprises centrifuging the dialyzed protein-placental extract and removing remaining solids.
7. The method of claim 1 , wherein the resulting human placental extract contains less than 2% normalized protein abundance of laminin.
8. A method of making a human placental extract, the method comprising:
obtaining a sample from a human placenta;
removing blood from the placental sample to produce a crude placental extract;
mixing the crude placental extract with a protein solubilization agent to solubilize and linearize proteins in the crude extract;
separating and removing solid materials from the solubilized protein-placental extract;
performing dialysis on the solubilized protein-placental extract to remove the protein solubilization agent; and
after dialysis, removing remaining solids from the extract to produce the human placental extract, whereby the resulting human placental extract contains less than 2% normalized protein abundance of collagen IV.
9. A method of making a human placental extract, the method comprising:
obtaining a sample from a human placenta;
removing blood from the placental sample to produce a crude placental extract;
mixing the crude placental extract with a protein solubilization agent to solubilize and linearize proteins in the crude extract;
separating and removing solid materials from the solubilized protein-placental extract;
performing dialysis on the solubilized protein-placental extract to remove the protein solubilization agent; and
after dialysis, removing remaining solids from the extract to produce the human placental extract, whereby the resulting human placental extract contains less than 2% normalized protein abundance of collagen IV and less than 2% normalized protein abundance of laminin.