Amniotic Stem Cell Products Testing Interventional Orthopedics Foundation

Do Amniotic Products Have Viable Stem Cells or Can They Help Autologous Stem Cells?

[All slides are protected by copyright-Interventional Orthopedics Foundation-2015]

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”.

Typical communications from amniotic vendors:

Provider Web-sites…

Overall Results of Viability Testing:

Flowable Amniotic ProductCells

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?

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 NoFloGraft Freedom No No

Ovation Cellular Repair Matrix Yes NoPalinGen Sport Flow No No

48 Hours (Pre-Wash)


BioD Restore (1:10 Dilution in CCM)




48 Hours (Pre-Wash)


FloGraft Freedom (1:10 Dilution in CCM)




48 Hours (Pre-Wash)


Ovation Cellular Repair Matrix (1:10 Dilution in CCM)




Adhered object exhibits cellular morphology

48 Hours (Pre-Wash)


PalinGen Flow Sport (1:10 Dilution in CCM)




Can Amniotic Products Help Human MSCs?

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

Complete Culture Medium (CCM)

hMSC Morphology (CCM has 16.5% PL)

PicoGreen Assay Results (SF vs PL)

Serum-Free

Med

ium

AmnioFix Sports

Med

BioD Res

tore

FloGraft F

reedom

Ovatio

n Rep

air M

atrix

Complete C

ulture

Medium

AmnioFix Sports

Med

BioD Res

tore

FloGraft F

reedom

Ovatio

n Rep

air M

atrix

0

100

200

300

400

500

dsDNA Quantification (PicoGreen)

Fluo

resc

ence

Inte

nsity

(a.u

.) Platelet LysateSerum-Free

***

***

*** p < 0.0005** p < 0.005* p < 0.05

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.

AlamarBlue Assay Results (SF vs PL)

Complete C

ulture

Medium (S

F)

AmnioFix Sports

Med

BioD Restore

FloGraft F

reedom

Ovatio

n Repair

Matr

ix

Complete C

ulture

Medium

AmnioFix Sports

Med

BioD Restore

FloGraft F

reedom

Ovatio

n Repair

Matr

ix0

200

400

600

800

1000

Metabolic Activity (AlamarBlue)

Fluo

resc

ence

Inte

nsity

(a.u

.)

Platelet LysateSerum-Free

**

***

***

* *

*** p < 0.0005** p < 0.005* p < 0.05

*

If the amniotic tissues were helping the MSCs, then metabolic activity would be higher than CCM (Complete Culture Medium). Here it is generally lower.

AlamarBlue Assay Results (SF vs PL vs FBS)

Complete C

ulture

Medium (S

F)

AmnioFix Sports

Med

BioD Restore

FloGraft F

reedom

Ovatio

n Rep

air M

atrix

Complete C

ulture

Medium

AmnioFix Sports

Med

BioD Res

tore

FloGraft F

reedom

Ovatio

n Rep

air M

atrix

Complete C

ulture

Medium (F

BS)

AmnioFix Sports

Med

BioD Res

tore

FloGraft F

reedom

Ovatio

n Rep

air M

atrix

0

200

400

600

800

1000

Alamar BlueBase Media Comparison

Fluo

resc

ence

Inte

nsity

(a.u

.)

Bovine SerumPlatelet LysateSerum-Free

**

***

***

* *

**

****

*** p < 0.0005** p < 0.005* p < 0.05*

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.

ROS DCFDA Assay Results (SF vs PL vs FBS)

Serum-Free

Med

ium

AmnioFix Sports

Med

BioD Res

tore

FloGraft F

reedom

Ovatio

n Repair

Matr

ix

Complete C

ulture

Medium

AmnioFix Sports

Med

BioD Restore

FloGraft F

reedom

Ovatio

n Rep

air M

atrix

100

M TBHP (+) C

ontrol

Complete C

ulture

Medium (F

BS)

AmnioFix Sports

Med

BioD Res

tore

FloGraft F

reedom

Ovatio

n Rep

air M

atrix

0

2,000

4,000

6,000

8,000

10,00020,000

40,000

ROS ActivityBase Media Comparison

Fluo

resc

ence

Inte

nsity

(a.u

.)

Bovine SerumPlatelet LysateSerum-Free

**

*** p < 0.0005** p < 0.005* p < 0.05

*

***

*

***

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.

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.

Health & Medicine

Amniotic Stem Cell Products Testing Interventional Orthopedics Foundation