Testing performed by a 501c3 non-profit on various products being advertised by manufacturers as amniotic stem cell therapy.

1. Do Amniotic Products Have Viable
Stem Cells or Can They Help
Autologous Stem Cells?
[All slides are protected by copyright-Interventional Orthopedics Foundation-2015]

2. Marketing materials and/or communications from
multiple companies vending FDA 361 registered
amniotic tissues claim that these products contain
viable stem cells. Many physicians who purchase these
products for injection have web-sites that refer to these
injections as “stem cell therapy”.

3. Typical communications from amniotic vendors:

4. Provider Web-sites…

5. Overall Results of Viability Testing:
Flowable Amniotic Product
Cells
Present?
Culture
Expandable?
Amniofix Injectable No No
BioD Restore No No
FloGraft Freedom No No
Ovation Cellular Repair Matrix Yes No
PalinGen Sport Flow No No
Are there cells
visible under
inversion
microscopy?
Did any cells grow
under ideal
nutrition and other
conditions after
plating the
product?

6. AmnioFix Injectable
(1:10 Dilution in CCM)
48 Hours (Pre-Wash)
48 Hours (Post-Wash)
Flowable Amniotic Product Cells Present? Culture Expandable?
Amniofix Injectable No No
BioD Restore No No
FloGraft Freedom No No
Ovation Cellular Repair Matrix Yes No
PalinGen Sport Flow No No

7. 48 Hours (Pre-Wash)
48 Hours (Post-Wash)
BioD Restore
(1:10 Dilution in CCM)
Flowable Amniotic Product Cells Present? Culture Expandable?
Amniofix Injectable No No
BioD Restore No No
FloGraft Freedom No No
Ovation Cellular Repair Matrix Yes No
PalinGen Sport Flow No No

8. 48 Hours (Pre-Wash)
48 Hours (Post-Wash)
FloGraft Freedom
(1:10 Dilution in CCM)
Flowable Amniotic Product Cells Present? Culture Expandable?
Amniofix Injectable No No
BioD Restore No No
FloGraft Freedom No No
Ovation Cellular Repair Matrix Yes No
PalinGen Sport Flow No No

9. 48 Hours (Pre-Wash)
48 Hours (Post-Wash)
Ovation Cellular Repair Matrix
(1:10 Dilution in CCM)
Flowable Amniotic Product Cells Present? Culture Expandable?
Amniofix Injectable No No
BioD Restore No No
FloGraft Freedom No No
Ovation Cellular Repair Matrix Yes No
PalinGen Sport Flow No No
Adhered object
exhibits cellular
morphology

10. 48 Hours (Pre-Wash)
48 Hours (Post-Wash)
PalinGen Flow Sport
(1:10 Dilution in CCM)
Flowable Amniotic Product Cells Present? Culture Expandable?
Amniofix Injectable No No
BioD Restore No No
FloGraft Freedom No No
Ovation Cellular Repair Matrix Yes No
PalinGen Sport Flow No No

11. Can Amniotic Products Help Human MSCs?

12. Human bone marrow MSCs from several donors
were seeded with various amniotic products to
observe:
• Morphology
• Proliferation-PicoGreen DNA Assay
• Metabolic activity (Alamar Blue)
• Reactive Oxygen Species (ROS)-A test of cell stress and
overall health

13. Complete Culture
Medium (CCM)
hMSC Morphology (CCM has 16.5% PL)

14. PicoGreen Assay Results (SF vs PL)
S
e
ru
m
-F
re
e
M
e
d
iu
m
A
m
n
io
F
ix
S
p
o
rts
M
e
d
B
io
D
R
e
s
to
re
F
lo
G
ra
ft
F
re
e
d
o
m
O
v
a
tio
n
R
e
p
a
ir
M
a
trix
C
o
m
p
le
te
C
u
ltu
re
M
e
d
iu
m
A
m
n
io
F
ix
S
p
o
rts
M
e
d
B
io
D
R
e
s
to
re
F
lo
G
ra
ft
F
re
e
d
o
m
O
v
a
tio
n
R
e
p
a
ir
M
a
trix
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
d s D N A Q u a n tific a tio n (P ic o G re e n )
FluorescenceIntensity(a.u.)
P la te le t L ysa teS e ru m -F re e
***
***
*** p < 0 .0 0 0 5
** p < 0 .0 0 5
* p < 0 .0 5
If the amniotic tissues were
causing the MSCs to
proliferate more, then total
DNA volume would be
higher than CCM (Complete
Culture Medium). This does
happen when serum free
(low growth factor) media
is used. However, these
effects are largely washed
out when high growth
factor culture media
(platelet lysate) is used.
This could indicate that
while there are active
growth factors in these
products, but that these GF
levels are likely less
biologically active than
those found in platelets.

15. AlamarBlue Assay Results (SF vs PL)
C
o
m
p
le
te
C
u
ltu
re
M
e
d
iu
m
(S
F
)
A
m
n
io
F
ix
S
p
o
rts
M
e
d
B
io
D
R
e
s
to
re
F
lo
G
ra
ft
F
re
e
d
o
m
O
v
a
tio
n
R
e
p
a
ir
M
a
trix
C
o
m
p
le
te
C
u
ltu
re
M
e
d
iu
m
A
m
n
io
F
ix
S
p
o
rts
M
e
d
B
io
D
R
e
s
to
re
F
lo
G
ra
ft
F
re
e
d
o
m
O
v
a
tio
n
R
e
p
a
ir
M
a
trix
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
M e ta b o lic A c tiv ity (A la m a rB lu e )
FluorescenceIntensity(a.u.)
P la te le t L ysa teS e ru m -F re e
**
***
***
* *
*** p < 0 .0 0 0 5
** p < 0 .0 0 5
* p < 0 .0 5
*
If the amniotic
tissues were helping
the MSCs, then
metabolic activity
would be higher
than CCM (Complete
Culture Medium).
Here it is generally
lower.

16. AlamarBlue Assay Results (SF vs PL vs FBS)
C
o
m
p
le
te
C
u
ltu
re
M
e
d
iu
m
(S
F
)
A
m
n
io
F
ix
S
p
o
rts
M
e
d
B
io
D
R
e
s
to
re
F
lo
G
ra
ft
F
re
e
d
o
m
O
v
a
tio
n
R
e
p
a
ir
M
a
trix
C
o
m
p
le
te
C
u
ltu
re
M
e
d
iu
m
A
m
n
io
F
ix
S
p
o
rts
M
e
d
B
io
D
R
e
s
to
re
F
lo
G
ra
ft
F
re
e
d
o
m
O
v
a
tio
n
R
e
p
a
ir
M
a
trix
C
o
m
p
le
te
C
u
ltu
re
M
e
d
iu
m
(F
B
S
)
A
m
n
io
F
ix
S
p
o
rts
M
e
d
B
io
D
R
e
s
to
re
F
lo
G
ra
ft
F
re
e
d
o
m
O
v
a
tio
n
R
e
p
a
ir
M
a
trix
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
A la m a r B lu e
B a s e M e d ia C o m p a ris o n
FluorescenceIntensity(a.u.)
B o v in e S e ru mP la te le t L ysa teS e ru m -F re e
**
***
***
* *
**
**
**
*** p < 0 .0 0 0 5
** p < 0 .0 0 5
* p < 0 .0 5
*
If the amniotic tissues
were helping the MSCs,
then metabolic activity
would be higher than
CCM (Complete Culture
Medium). Here it is
generally lower, only
AmnioFix helps and only
when bovine serum is
used as a culture media.

17. ROS DCFDA Assay Results (SF vs PL vs FBS)
S
e
ru
m
-F
re
e
M
e
d
iu
m
A
m
n
io
F
ix
S
p
o
rts
M
e
d
B
io
D
R
e
s
to
re
F
lo
G
ra
ft
F
re
e
d
o
m
O
v
a
tio
n
R
e
p
a
ir
M
a
trix
C
o
m
p
le
te
C
u
ltu
re
M
e
d
iu
m
A
m
n
io
F
ix
S
p
o
rts
M
e
d
B
io
D
R
e
s
to
re
F
lo
G
ra
ft
F
re
e
d
o
m
O
v
a
tio
n
R
e
p
a
ir
M
a
trix
1
0
0
M
T
B
H
P
(+
)
C
o
n
tro
l
C
o
m
p
le
te
C
u
ltu
re
M
e
d
iu
m
(F
B
S
)
A
m
n
io
F
ix
S
p
o
rts
M
e
d
B
io
D
R
e
s
to
re
F
lo
G
ra
ft
F
re
e
d
o
m
O
v
a
tio
n
R
e
p
a
ir
M
a
trix
0
2 ,0 0 0
4 ,0 0 0
6 ,0 0 0
8 ,0 0 0
1 0 ,0 0 0
2 0 ,0 0 0
4 0 ,0 0 0
R O S A c tiv ity
B a s e M e d ia C o m p a ris o n
FluorescenceIntensity(a.u.)
B o v in e S e ru mP la te le t L ysa teS e ru m -F re e
**
*** p < 0 .0 0 0 5
** p < 0 .0 0 5
* p < 0 .0 5
*
***
*
***
If the amniotic tissues
were helping the
MSCs, then ROS would
be lower than CCM
(Complete Culture
Medium). Here it is
generally the same or
higher.

18. Conclusions:
• The amniotic products tested:
• Don’t contain viable stem cells or other viable cells once they’re
thawed per the manufacturer's recommendations.
• Do likely contain some active growth factors that can have a
positive proliferation effect on MSCs, but those GFs in this
experiment look like they are less effective than those found in
platelets.
• Don’t generally help the metabolic activity of MSCs.
• Don’t generally help MSCs show less oxidative stress.