The document discusses methods for decellularizing and isolating extracellular matrices (ECM) from tissues, focusing on both mammalian organs and plant tissues, particularly spinach leaves. It highlights various physical, chemical, and enzymatic treatments for decellularization and demonstrates successful recellularization of human cells in decellularized plant structures, revealing similarities between plant and human vascular systems. The findings suggest potential applications in regenerative medicine using decellularized plant scaffolds.

1. Impact Factor: 8.40 (2016)
5 year IF : 8.94 (2016)
Date : 10 Feb 2017
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2. • Process to isolate ECM from inhabiting cells
• Pioneer- Stephen F. Badylak,
McGowan Institute for Regenerative Medicine,
University of Pittsburgh.
• Produces natural biomaterial – scaffold for cell growth, differentiation and tissue
regeneration
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3. Figure : Combinations of agents used for the decellularization of (A) thin laminate tissues, (B) thicker
laminate tissues, (C) adipose tissues, (D) whole simple organs, (E) essential and complex organs .
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• Physical Treatments : a. Freeze-thaw, b. Pressure, and c. Electric disruption [Non
thermal irreversible electroporation (NTIRE)].
•Chemical Treatment : a. Ionic detergents (SDS), b. Alkaline & acid treatments, and
c. Non-ionic detergents (Triton X-100, EDTA)
•Enzymatic Treatments : Lipase, Thermolysine, Galactosidase, Nuclease, Trypsin, Dipase,
and Collagenase
Perfusion and Immersion Decellularization Techniques

4. Figure 1
Ref : Trends in Molecular Medicine , Volume 17, Issue 8, Pages 424-432 (August 2011) ,
DOI: 10.1016/j.molmed.2011.03.005
Perfusion-decellularized whole organ scaffolds. The native ECMs of cadaveric organs can be isolated by the perfusion of the native vascular system with detergent solutions. The resulting
scaffolds are acellular, but maintain the structure of the native organ. (a) Rat heart scaffold generated from cadaveric heart by perfusion decellularization (i). The ascending aorta was cannulated
for perfusion. Cadaveric human heart before and after perfusion decellularization (ii). (b) Rat lung scaffold generated from cadaveric lung by perfusion decellularization (i). Perfusion was
performed via the pulmonary artery. Cadaveric sheep lung before and after decellularization (ii). (c) Rat kidney scaffold generated from cadaveric kidney by perfusion decellularization (i). The
abdominal aorta was cannulated for perfusion. Porcine kidney before and after perfusion decellularization (ii). (d) Rat pancreas scaffold generated from cadaveric pancreas by perfusion
decellularization (i). The abdominal aorta was cannulated for perfusion. Human pancreas before and after perfusion decellularization (ii).
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5. • Why Crossing Kingdoms???
• Why Plant tissues???
• Why not mammalian tissues???
Fig : Comparison of animal and plant vascular network pattern branching and structures. A decellularized rat heart perfused with a
Ponceau Red stain to visualize the vasculature. A Buddleja davidii leaf perfused with fluorescein-labeled PEGDA to visualize the leaf
vasculature. 5

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10% SDS
for 5 days
&
Triton X-
100 in sod.
chlorite
bleach,
48 hrs

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Figure : Depiction of the cannulation of a spinach leaf. (A) The petiole of the
spinach leaf is identified. (B, C) A cannula is inserted into the petiole of the stem of
the spinach leaf and is held in place with suture.

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Fig : Time lapse of spinach leaf decellularization. (A) Leaf is dark green and opaque Day 0.
(B) Day 1, loss of dark color (C) Day 5, completely translucent while a light green hue. (D)
After being treated and sterilized with sodium chlorite, the leaf loses the remainder of its
coloring and becomes completely decellularized on Day 7.

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Fig : Characterization of plant scaffolds before and after decellularization. Spinach leaf
(A) before and (B) after decellularization; (C) Native and (D) decellularized leaf stained
with Safranin and Fast Green. (E) native and (F) decellularized spinach leaves. (G) DNA
content (H) Total protein before and after leaf decellularization

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Figure : Loss of plant total protein during the decellularization process

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Fig : Comparison of the mechanical properties of native and decellularized spinach
leaves. (A) dogbone shape to ensure uniformity, (B) The maximum tangent
modulus (MTM), (C) ultimate tensile strength (UTS), and (D) strain at failure

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Fig. 4. Spinach leaf vascular scaffolds retain patency and perfusion capabilities after
decellularization. Decellularized leaf (A) before and (B) after perfusion of Ponceau Red.
Fluorescent microspheres of various diameters (1, 10, 50, and 100 mm)

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Fig. (A) Dil-Ac-LDL-labeled human umbilical vein endothelial cells (HUVECs)
Scale bar: 250 mm. (B) Human mesenchymal stem cells (hMSCs) labeled with
quantum dots Scale bars: 250 mm for main image, 50 mm for insert.

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Fig : HUVECs remain viable after recellularization in decellularized spinach stems.
Unlabeled HUVECs incubated in medium containing Dil-Ac-LDL. (A, B) prior to
incubation in LDL; (C, D) 24 and (E, F) 48 hours incubation
Cell viability determination through Low Density
Lipoprotein (Dil-Ac-LDL) uptake assay

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Fig : (A) hPS-CMs adhere and form cell clusters. Scale bar: 50 mm. (B) Contractile strain of
hPS-CMs quantified at 1% strain. (C) tissue culture platiic,Contractile through a heatmap.
(D) Contractile strain 21 days (E) Day 21 lowered contractile strain magnitude. (F) hPS-
CMs modified with a GCaMP3 reporter

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• Decellularization of Spinach leaves was successfully performed
through perfusion.
• Plant vasculature determined similarity with human vasculature.
• Recellularization and cell viability achieved
• Human pluripotent stem cell derived cardiomyocytes (hPS-Ms)
attachment and contractile ability determined.

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