ELECTRONIC SUPPLEMENTARY MATERIAL
I. DETAILED METHODS
Myocardial patch (MP) isolation
Whole hearts were acquired from Charles River Laboratories (CD,CRL, Kingston,
NY). Hearts were isolated from 11-12 week old male skeletally mature adult Sprague
Dawley rats, stored in storage solution containing Dulbecco’s Modified Eagles Medium
(DMEM) with 15% dimethyl sulfoxide (DMSO) (v/v) and shipped to UC Davis on dry ice,
where they were placed at -80oC immediately upon arrival. On Day 1 of MP isolation,
hearts were defrosted and blood removed by antegrade coronary perfusion with 4 oC
heparinized filtered ice-cold PBS (10 IU/ml). MPs were prepared as follows: The left
ventricle (LV) was isolated by removal of the great vessels, atria and right ventricle and
planarized by incision of the LV free wall between the papillary muscles. A 3.5 mm
biopsy punch (Miltex) was utilized to cut adjacent cylindrical pieces of LV tissue from
endocardium to epicardium, herein designated as Myocardial Patches (MP) (Suppl. Fig.
1A). This approach allowed for the generation of 10-12 identical MPs from each LV
(Suppl. Fig.1). The 3.5 mm diameter punch was chosen because we were able to obtain
MPs with a consistent CV of 10%. Additionally, the recording of wet weights was
optimized and timed with precision. MPs were patted twice in sterile gauge for 30 s
each, and then patted for a third time at all sides before being weighed in scales with
closed compartments. This procedure was employed to accomplish identical pieces of
1
myocardial tissue that could be used as anatomical controls for the various optimization
experiments as well as the biochemical normalizations. Following wet weight recording,
MPs were placed in MP isolation buffer (20 mM Tris, 500 mM NaCl, pH 7.5) on ice.
Antigen removal (AR) procedure and Research Design
All AR solutions were made fresh and filtered. All steps of the AR protocol were
performed in 6-well plates, at 125 rpm, in 3 ml working volume at RT with solutions
changed every 12 h unless otherwise stated. MPs from a single heart (and its
correspondent anatomical controls for the residual antigenicity assessments) were
randomly assigned to each experimental group. Therefore, each group included
replicates from different parts of the LV of different rat hearts. This randomized design
ensured that biological and anatomical variability were controlled. Suppl. Table I depicts
the timeline of the procedure.
Histology
Histologic analysis was performed on a total of seven experimental groups, with n=6
replicates per group (Supplemental Table 1). Samples were fixed in 10% formalin,
paraffin embedded, sectioned (4 μm thick), deparaffinized and histological stains
performed according to standard procedures. Hematoxylin and Eosin (H&E) staining
was used for assessment of removal of cellular components under light microscopy.
Verhoeff van Gieson (VVG) staining was used to evaluate the presence and
2
organization of elastin fibers. Masson’s trichrome stain was performed to further
evaluate structure and organization of collagen fibers and cytoplasmic/muscle remnants
since collagen fibers are expected to be blue and cytoplasmic/muscle remnants are
expected to be red with this stain.1 Finally, Picrosirius red and Light Green
counterstaining was also performed to highlight the presence and organization of
collagen fibers and also confirm presence of cellular remnants.2
Immunohistochemistry
Negative technique controls where the primary antibody was omitted and negative
tissue controls, using tissues where the antigen was not present were included in
immunohistochemical stainings. Presence of GFP-labeled hMSCs on the scaffolds after
7 days of recellularization was confirmed by immunostaining against GFP. Briefly, 4 μm
sections were prepared, deparaffinized, rehydrated, blocked against endogenous
peroxidase activity with 3% H2O2 in methanol and antigen retrieval performed with
Target Retrieval Solution pH 9 (DAKO) using steaming. Sections were blocked at RT
with 4% non fat dry milk (Lab Scientific) diluted in deionized water followed by 1 h
incubation at 37oC with a monoclonal primary antibody against GFP (6AT316; Abcam,
Cambridge, MA) 1:4000 diluted in PBS with 0.01% Tween (PBS-T). Sections were
washed in PBS-T 3 x 5 min and GFP positivity was detected incubating for 30 min at RT
with
anti-mouse
IgG
peroxidase-labeled
polymer
(DAKO)
followed
by
3,3'-
Diaminobenzidine substrate (DAB, DAKO) for 4 min. Slides were counterstained in
3
Mayer’s Hematoxylin for 10 min and mounted in Cytoseal 60 medium (Thermo-Fisher
Scientific).
Scanning Electron Microscopy
Samples were fixed in Kornofsky’s fixative at 4oC O/N. Samples were dehydrated in
three steps of ethanol (5min in 25 % ethanol, followed by 5 min in 50% ethanol and then
stored in 70% ethanol at 4oC until needed for imaging). Prior to imaging samples were
further dehydrated with 100% ethanol, critically point dried, mounted on studs, sputter
coated and stored in a desiccator until imaging with a Philips XL30 TMP scanning
electron microscope.
Protein Extraction
Samples (~20mg starting wet weight) stored in storage solution at -80oC were
thawed at RT and rinsed in wash buffer for 3 x 5 min. To minimize protein degradation
all steps of the extraction procedure were in solution on ice. For hydrophilic protein
extraction, samples were placed into 1.5 ml microcentrifuge tubes containing 0.5 ml of
ice-cold standard extraction solution (10mM pH 8.0 Tris-HCl, 1mM DTT, 2mM Mg2Cl2,
10mM KCl, 0.5 mM Pefabloc) with 0.1 % SDS. Samples were homogenized manually
with single-use microtube pestles (7-10 strokes/sample) and shaken at 1000 rpm, at
4oC for 1 h. Samples were centrifuged at 17,000 x g, 4 oC for 25 min, supernatants
collected and designated as the hydrophilic fraction. The pellet was washed twice in 0.5
4
ml of hydrophilic extraction solution by repeating the above extraction procedure, with
the supernatant from each wash discarded. The pellet was resuspended in 0.25 ml
lipophilic extraction solution buffer (1% SDS in standard extraction solution) and shaken
at 1400 rpm, 4oC, followed by centrifugation at 17,000 x g at 4oC for 25 min and the
supernatant designated as the lipophilic protein fraction. Total protein concentration of
hydrophilic and lipophilic extracts was determined using a DC protein assay kit (BioRad). Protein extracts were stored at -80oC.
One-dimensional electrophoresis (SDS-PAGE) and western blotting
Equal volumes of extracts (hydrophilic and lipophilic) from each group were loaded
into each well. After one-dimensional electrophoresis at 200 V for 47 min, gels were
immersed in 20% ethanol for 5-10 min and blotted to nitrocellulose membranes (0.2 μm
pores) according to the manufacturer’s recommendations using the iBlot Dry Blotting
system at 20 V for 8 min.
Following primary antibody incubations with anti-rat LV serum, membranes were
washed in 10 mM TBS with 0.05% Tween-20 (TBS-T) and incubated with horseradish
peroxidase-conjugated goat monoclonal anti-mouse IgG light chain specific antibody
(Jackson ImmunoResearch Laboratories) in 10mM Tris buffered saline (TBS, pH 8.8).
Blots
were
washed
and
protein
bands
visualized
with
Immobilon Western
Chemiluminescent HRP Substrate (Millipore), and imaged using FluorChem Xplor CCD
Bioimaging system and AlphaView acquisition and analysis software (Alpha Innotech
5
Corp). To ensure direct comparison between groups irrespective of the individual gel
and/or transfer discrepancies, each nitrocellulose membrane contained all six
experimental groups and their respective 1-min anatomically adjacent controls.
Densitometry was used to quantify banding intensity. Residual antigenicity of each
sample was defined as the ratio of banding intensity for extracts following AR compared
its anatomically adjacent 1-min AR control.3, 4 Normalized values were then compared
between experimental AR groups and controls (SDS and native control).
For α-sarcomeric actin quantification, gel loading and running conditions were as
described above. Following transfer with the iBlot system, membranes were blocked
and subsequently incubated with 1:30,000 of primary antibody (mouse monoclonal antiα-sarcomeric actin, Sigma-Aldrich) diluted in PFBB-T20 blocking buffer, overnight at
4oC. Blots were the washed and incubated for 1.5 h with peroxidase-conjugated donkey
anti-mouse IgM, μ chain specific (Jackson Immunoresearch) at 1:20,000 in TBS (pH
8.8), followed by imaging and analysis as described above.
Quantitative Biochemistry
Prior to AR each MP half was weighed and its wet weight recorded. Following AR,
samples were frozen at -20oC overnight, lyophilized for 72 h, and final dry weights
recorded. Collagen content was quantified using a modified hydroxyproline colorimetric
assay following the manufacturer’s protocol (Chondrex). Briefly, samples were
hydrolyzed in 5N hydrochloric acid for 26 h at 100 oC with intermittent mild shaking. After
6
cooling to room temperature, samples were centrifuged for 3 min at 10,000 x g and
supernatants collected and cooled to RT. Sample supernatants and hydroxyproline
standards were added into 96-well plates with Chloramine-T solution and plates were
incubated for 20 min at RT. Then, DMAB solution was added into all wells, followed by
manual mixing of plates by gentle tapping. Plates were then incubated at 60 oC for 30
min in a hot bead bath and were read at 540 nm. Hydroxyproline assay standards were
run in triplicates and samples were conducted in duplicates.
For the determination of sulfated GAG content, samples were digested in 0.2 M Na
phosphate buffer (pH 6.4) containing 1200 mg sodium acetate, 600 mg EDTA disodium
salt, 120 mg cysteine HCl and 100 μg/ml papain for 20 h at 65oC. Extracts were cooled
to RT, centrifuged for 10 min at 10,000 x g and supernatants collected and assayed for
GAG content following the manufacturer’s protocol. Elastin content was determined
using the Fastin assay (Biocolor). Briefly, samples were incubated in 0.25 M oxalic acid
at 100oC for 60 min. Samples were cooled to RT, centrifuged for 10 min at 10,000 x g,
supernatants collected and assayed following the manufacturer’s procedure. GAG and
elastin assays were conducted in duplicates.
Uniaxial Tensile Testing
In collaboration with the UC Davis TEAM Design, Prototyping and Fabrication
facilities lab, we designed and fabricated razor punches that could cut full thickness
endo- to epicardial left ventricular tissue sections, in a dogbone shape which conformed
7
to ASTM soft tissue testing standards, that is 4:1 gauge length to dogbone width ratio
(see Suppl. Fig 1C for desired shape and dimensions). Dogbone punches were taken
along the longitudinal axis of the rat heart. Thus, they had the same starting thickness
as a MP, while including all the structural elements of the LV wall (endocardium,
myocardium, epicardium). Briefly, 3D printed blade holders were designed in 3D CAD,
into which razor blades could fit tightly in order to form a dogbone shape. Razor blades
were bent to shape before fitting into the 3D printed holder using a custom designed
metal mold press. Since several of the AR procedures under investigation resulted in
changes in geometry of the MP, tensile testing specimens were cut from native rat LV
prior to AR, providing essentially identical dog bone specimens which then underwent
the various AR and/or decellularization protocols. Fifteen rat hearts were prepared as
described for myocardial patch isolation up to the point of LV planarization and then
dogbone specimens cut (Suppl. Fig.1C). Minimum cross sectional area of the dogbone
region was measured before and after AR and decrease in cross sectional area
computed as follows: [(dogbone width before treatment) x (dogbone thickness before
treatment) - (dogbone width after treatment) x (dogbone thickness after treatment)] /
(dogbone width before treatment) x (dogbone thickness before treatment) x 100 %.
Cross sectional area after treatment was used to calculate material properties.
eGFP-labeled human Mesenchymal Stem Cell (hMSCs) Recellularization
Human bone marrow was acquired from Lonza (Allendale, NJ), plated in T75 flasks
(Corning) and incubated overnight at 37oC in 5% CO2. The following day, phosphate
8
buffered saline (PBS) wash was used to discard non-adherent cells from the culture,
and then medium was added (1% mM L-glutamine, 1% penicillin/streptomycin solution
in Dulbecco’s modified eagle medium (DMEM), all from Hyclone Laboratories) and 20%
fetal bovine serum (FBS) premium select (Atlanta Biologicals). Human Mesenchymal
Stem Cells (hMSCs) isolated via adherence were then cultured in T225 flasks at
37oC/5% CO2. hMSCs were transduced for 3 days with a lentiviral vector (VSV-G,
MNDU3-Luc-PGK-eGFP-WPRE) expressing green fluorescent protein (GFP) and 20
ng/ml protamine sulfate. After 3 days, cells were harvested.
Recellularization experiments with eGFP+hMSCs were conducted on three groups:
Native control, SDS control and RelYDisY, and were performed in 48-well plates
(Corning) at a seeding density of 1000 cells/ mm2. Incubations were performed at
37oC/5% CO2 with hMSC growth medium changed every two days. GFP+ hMSCs were
expanded to P4. All groups were prepared in advance and stored at -80oC in storage
solution until use. They were thawed, rinsed with wash buffer at 4oC for 48 h with 2
changes per day, and left overnight in hMSC medium at 4 oC. hMSCs were passaged,
counted with a hematocytometer and 140 μl were pipetted onto the surface of the
scaffold. After 1.5 h ~600μl media was added to cover the scaffold, and constructs were
incubated for 24h. Media were replaced the following day. After 3d static culture,
recellularized scaffolds were transferred to new well plates to avoid over-confluence and
constructs were cultured statically for a total of 7d, with media changes every other day.
Scaffolds were then fixed in formalin and paraffin embedded.
9
SUPPLEMENTAL REFERENCES
1.
Penney D. P., J. M. Powers, M. Frank, C. Willis and C. Churukian. Analysis and
testing of biological stains--the Biological Stain Commission Procedures. Biotech
Histochem 77: 237-275, 2002.
2.
Sarasquete C. and M. Gutiérrez. New tetrachromic VOF stain (Type III-G.S) for
normal and pathological fish tissues. Eur J Histochem 49: 211-227, 2005.
3.
Wong M. L., J. K. Leach, K. A. Athanasiou and L. G. Griffiths. The role of protein
solubilization in antigen removal from xenogeneic tissue for heart valve tissue
engineering. Biomaterials 32: 8129-8138, 2011.
4.
Wong M. L., J. L. Wong, K. A. Athanasiou and L. G. Griffiths. Stepwise
solubilization-based antigen removal for xenogeneic scaffold generation in tissue
engineering. Acta Biomater 9: 6492-6501, 2013.
Supplemental Table I:
Detailed Antigen Removal Procedure Timeline. Groups examined in Antigen
Removal (AR) experiments. ASB-14, Amidosulfobetaine-14; mSARB, myocardial
Standard Antigen Removal Buffer; SDS, Sodium Dodecyl Sulfate. When No is
indicated, samples were incubated with Wash buffer to account for the hypotonic effects
of time in solution (see 2.2. for details of each step). Note that “No ASB” group did not
pass the primary decellularization assessment (no clearance of cellular and nuclear
components and was thus not evaluated further).
10
Supplemental Table I:
Group
Step 1
Step 2
Step 3
Step 4
Step 5
Relax
Lipophile
(Sarcomeric.
Repeat Lipophile
Hydrophile
Disassembly)
Solubilization
solubilization
h)
(48h)
(48 h)
(24h)
(1hr) solubilization (48
Step 6
Step 7
Nuclease Washout(48h)
(24h)
RelNDisN
No
2% ASB-14
No
2% ASB-14
mSARB
Nuclease
Wash buffer
RelNDisY
No
2% ASB-14
Yes
2% ASB-14
mSARB
Nuclease
Wash buffer
RelYDisN
Yes
2% ASB-14
No
2% ASB-14
mSARB
Nuclease
Wash buffer
RelYDisY
Yes
2% ASB-14
Yes
2% ASB-14
mSARB
Nuclease
Wash buffer
No
No
No
Nuclease
Wash buffer
Wash buffer
Wash buffer
Wash buffer
Wash buffer
Nuclease
Wash buffer
No
Yes
No
mSARB
Nuclease
Wash buffer
SDS
1% SDS 24 h, then
2x15 min in 1% Triton-X100, then Wash buffer
Native
Wash
Control
buffer
No ASB
Yes
11
II. Supplementary Data
Supplemental Table II: Densitometry analysis to determine scaffold residual α-sarcomeric actin. Values are
expressed as percentage of residual α-sarcomeric actin compared to anatomically adjacent 1-min controls (see Methods
for details). Non-repeated measures analysis of variance and Tukey-Kramer HSD post hoc analysis were performed on
sample means for each analysis. All data are presented as mean ± standard deviation. For each analysis, groups not
connected by the same letter in the ANOVA column are statistically significantly different from one another. SD, standard
deviation. (n=6/group).
Group
NatCntr
Mean Residual
SD
ANOVA
Mean Residual
Hydrophilic
Lipophilic
α-sarcomeric actin
α-sarcomeric actin
86.28
10.57
A
93.9
SD
ANOVA
5.18
A
12
RelNDisN
41.17
9.49
B
58.86
7.12
B
RelNDisY
4.64
1.52
C
32.88
12.98
C
RelYDisN
25.69
7.74
B
39.57
24.06
B,C
RelYDisY
3.84
1.31
C
9.38
3.46
D
SDS
4.32
1.4
C
0.8
0.8
D
13
Supplemental Table III: Densitometry analysis to determine Residual Antigenicity. Values are expressed as
percentage of residual antigenicity compared to anatomically adjacent 1-min controls (see Methods for details). All data
are presented as mean ± standard deviation. For each analysis, groups not connected by the same letter in the ANOVA
column are statistically significantly different from one another. SD, standard deviation. (n=6/group).
Group
Mean Residual Standard ANOVA Mean Residual Standard ANOVA
Hydrophilic
Deviation
Lipophilic
Antigenicity
Deviation
Antigenicity
NatCntr
82.37
11.1
A
84.01
12.78
A
RelNDisN
6.59
6.91
B
17.58
12
B
RelNDisY
7.39
8.96
B
18.54
15.27
B
RelYDisN
4.05
4.33
B
13.14
12.05
B
RelYDisY
3.79
3.59
B
12.67
13.23
B
SDS
6.10
5.45
B
19.96
19.91
B
14
Supplemental Table IV: Quantitative Biochemistry. All data are presented as mean ± standard deviation. For each
analysis, groups not connected by the same letter in the ANOVA column are statistically significantly different from one
another. SD, standard deviation. GAG, glycosaminoglycan. (n=6/group).
Group
Mean
SD
ANOVA Collagen
SD
ANOVA
(μg/mg)
% Water
Elastin
SD
ANOVA
(μg/mg)
GAG
SD
ANOVA
(μg/mg)
Content
NatCntr
89.77
0.37
A
3.8
0.62
NS
34.76
10.88
A
1.29
0.16
A
RelNDisN
99.24
0.17
B
3.23
0.88
NS
15.4
3.65
B
0.1
0.07
B
RelNDisY
99.24
0.28
B
2.89
0.23
NS
20.05
5.59
A,B
0.05
0.02
B
RelYDisN
99.4
0.09
B
4.39
1.37
NS
17.57
5.78
B
0.07
0.03
B
RelYDisY
99.44
0.1
B
4.32
1.17
NS
22.55
7.77
A,B
0.05
0.02
B
SDS
99.55
0.2
B
4.26
0.67
NS
14.40
9.77
B
0.06
0.02
B
15
Supplemental Table V: MP Dry Weights: All data are presented mean ± standard deviation. For each analysis, groups
not connected by the same letter in the ANOVA column are statistically significantly different from one another. SD,
standard deviation.
Group
Mean MP
SD
ANOVA
Dry weight
(mg)
NatCntr
1.267
0.210
A
RelNDisN
0.089
0.025
B
RelNDisY
0.105
0.042
B
RelYDisN
0.067
0.013
B
RelYDisY
0.069
0.011
B
SDS
0.071
0.040
B
16
Supplemental Table VI: Tensile properties. All data are presented as mean ± standard deviation. For each analysis,
groups not connected by the same letter in the ANOVA column are statistically significantly different from one another.
SD, standard deviation. (n=6/group). UTS, Ultimate Tensile Strength. (n=8/group).
Group
Mean
SD
ANOVA
Mean
SD
ANOVA
Mean
% Change
Young’s
UTS
cross sectional
Modulus
(kPa)
area
(kPa)
SD
ANOVA
NatCntr
23.53
18.27
B
166.66
69.63
B
72.32
23.54
A,B
RelNDisN
41.46
5.51
A,B
117.24
59.91
B
55.51
12.95
B
RelNDisY
43.07
14.65
A,B
182.13
127.18
B
59.24
36.7
B
RelYDisN
34.45
16.74
B
116.36
59.35
B
48.03
18.07
B
RelYDisY
36.95
21.52
B
119.71
43.71
B
48.19
19.43
B
SDS
62.33
11.57
A
386.46
210.67
A
119.99 57.07
A
17
Supplemental Figure Captions:
Suppl. Fig. 1. System for comparison between Antigen Removal (AR)/decellularization
methods. (A). Workflow for production of anatomically adjacent myocardial patches
(MPs). Blood was removed by antegrade coronary perfusion with heparinized PBS. Left
ventricle (LV) was isolated, planarized and biopsy punches used to cut 10-12
MPs/heart. (B). Gross morphology of MPs after AR/decellularization. All AR groups
were translucent compared to native control. Note that SDS MPs are more opaque than
AR groups. (C). Uniaxial tensile testing procedure. Razor blades were cut, placed into a
custom made press and bent into half dogbone shape. Two blades inserted into custom
designed 3D-printed holders formed the final dogbone-shaped punch for generation of
LV samples conforming to ASTM uniaxial tensile testing standards (4:1 ratio).
Suppl. Fig.2. Sample mass and dimensions post-AR/decellularization treatment. (A).
MP dry weight of all AR/decellularization groups was significantly decreased compared
to native control (p<0.0001). (B). Dogbone region cross sectional area (CSA) was
computed by multiplying dogbone thickness by dogbone width. CSA was recorded
before and after treatment. No statistically significant changes in dogbone CSA were
found following treatment for all AR groups, but SDS-decellularization significantly
reduced CSA compared to native control, which served as control for time in solution
(p<0.05, n=8/group).
Suppl. Fig. 3. Recellularization with eGFP+ human Mesenchymal Stem Cells (hMSCs)
after 7 days of static seeding. Representative GFP immunohistochemical (a-c and a’-c’)
18
and H&E (d-f) images. Native control group (left column) showed minimal
recellularization with eGFP+ hMSCs at the periphery of the construct (a, a’ and d).
Group RelYDisY (middle column) displayed widespread invasion of hMSCs into the
scaffold (b, b’ and e). SDS group (right column) was also infiltrated by eGFP+ hMSCs
(c, c’ and f) but at seemingly lower levels than RelYDisY (n=3/group). Scale bars
represent 100 μm.
19