1. Autologous human plasma in stem cell culture and cryopreservation in the creation of a tissue-engineered vascular graft 
Ping Zhang, PhD, Aleksandra Policha, MD, Thomas Tulenko, PhD, Paul DiMuzio, MD 
Journal of Vascular Surgery 
Volume 63, Issue 3, Pages (March 2016)
DOI: /j.jvs
Copyright © 2016 Society for Vascular Surgery Terms and Conditions

2. Fig 1
Morphology and expansion of human adipose-derived stem cells (ASCs) cultured with autologous human plasma (HP) vs fetal bovine serum (FBS). A, Phase contrast micrographs (20×) of human ASCs (passage 1) grown in medium supplemented with autologous HP (ASC-HP, left) vs FBS (ASC-FBS, right) for 7 days, demonstrating no significant morphologic change. B, Growth curves of human ASCs cultured in medium supplemented with autologous HP vs FBS during 14 days (n = 10). C, Bar graph of doubling times of human ASCs generated from growth curves (n = 10) revealing the trend toward a shorter doubling time for cells grown in HP.
Journal of Vascular Surgery  , DOI: ( /j.jvs )
Copyright © 2016 Society for Vascular Surgery Terms and Conditions

3. Fig 2
Effect of autologous human plasma (HP) or fetal bovine serum (FBS) on endothelial differentiation of human adipose-derived stem cells (ASCs). A, Expression of mRNA levels of endothelial cell (EC) markers in ASCs. CD31 and von Willebrand factor (vWF) message expression quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR) demonstrates that substitution of the HP resulted in increased expression of these endothelial molecular markers, although not significantly (n = 6; average ± standard error). B, Immunofluorescence micrograph (20×) of ASCs cultured in EGM-2 supplemented with autologous HP vs FBS for 10 days revealing uptake of DiI-labeled acetylated low-density lipoprotein (acLDL) and human lectin under both conditions (red, LDL; green, lectin). ASCs cultured in M199 medium were used as undifferentiated controls. C, Phase contrast photomicrograph (20×) of the differentiated cells subsequently plated onto Matrigel for 12 hours demonstrating the formation of cord-like structures under both culture conditions (representative pictures from two experiments from two different donors).
Journal of Vascular Surgery  , DOI: ( /j.jvs )
Copyright © 2016 Society for Vascular Surgery Terms and Conditions

4. Fig 3
Creation of a tissue-engineered vascular graft (TEVG) composed of endothelial-differentiated adipose-derived stem cells (ASCs). Laser confocal photomicrographs (20×; CellTracker green) of the luminal surface of vascular grafts (decellularized human saphenous vein) seeded with ASCs and flow conditioned for 5 days (0-9 dyne). A confluent monolayer was achieved with cells cultured in EGM-2 supplemented with either autologous human plasma (HP) or fetal bovine serum (FBS). The micrographs are representative of three grafts created from three different donors.
Journal of Vascular Surgery  , DOI: ( /j.jvs )
Copyright © 2016 Society for Vascular Surgery Terms and Conditions

5. Fig 4
Proliferation potential and viability of cryopreserved adipose-derived stem cells (ASCs). ASCs differentiated toward endothelial cells (ECs) were cryopreserved in freezing solution (5% dimethyl sulfoxide [DMSO] plus 95% autologous human plasma [HP]) for 1 month. The thawed ASCs were then plated in fresh EGM-2 culture medium. A, Proliferation of HP-ASCs measured by MTT assay before and after cryopreservation reveals preserved proliferation capacity after thawing. Bars show the mean (+standard error of the mean [SEM]) (n = 3). B, Similarly, the proliferation of ASCs cultured in EGM-2 medium supplemented with autologous HP vs fetal bovine serum (FBS) after cryopreservation is preserved. Bars show the mean (+SEM) (n = 3). C, Viability assessment of frozen/thawed ASCs after exposure to different concentrations of DMSO. ASCs cryopreserved in HP or FBS freezing solution containing 5% or 10% DMSO for 2 weeks. Cell viability was determined by the exclusion of trypan blue. Bars show the mean (+SEM) (n = 4). D, Cell survival rate of long-term cryopreserved ASCs was directly measured after thawing by use of the trypan blue assay. The ASCs were stored in HP or FBS freezing solution containing 5% DMSO for short-term (2 weeks) or long-term (>1 year) cryopreservation. Bars show the mean (+SEM) (n = 4).
Journal of Vascular Surgery  , DOI: ( /j.jvs )
Copyright © 2016 Society for Vascular Surgery Terms and Conditions

6. Fig 5
Expression of endothelial cell (EC) markers and angiogenic potential of cryopreserved adipose-derived stem cells (ASCs). A, Real-time reverse transcription-polymerase chain reaction (RT-PCR) evaluating CD31, von Willebrand factor (vWF), and CD144 message expression in ASCs cultured in medium supplemented with autologous human plasma (HP) vs fetal bovine serum (FBS) before and after cryopreservation revealing preserved expression of these endothelial markers with the exception of the decrease observed in vWF and CD144 in cells cultured in HP. The bars show the mean (+standard error of the mean) (n = 3). B, Representative phase contrast photomicrographs (20×) of ASCs plated on Matrigel for 12 hours before and after cryopreservation revealing preserved ability to form cord-like structures (n = 3).
Journal of Vascular Surgery  , DOI: ( /j.jvs )
Copyright © 2016 Society for Vascular Surgery Terms and Conditions

7. Fig 6
Cryopreservation of tissue-engineered vascular graft (TEVG). A, Photograph of a TEVG composed of decellularized human greater saphenous vein seeded with adipose-derived stem cells (ASCs) (luminal surface) and flow conditioned with bioreactor for 5 days. B and C, Representative confocal micrographs (10×; CellTracker green) of the luminal surfaces of a TEVG seeded with ASCs cultured in EGM-2 supplemented with autologous human plasma (HP, B) vs fetal bovine serum (FBS, C) and subsequently cryopreserved for 10 days in dimethyl sulfoxide (DMSO)/autologous HP vs FBS freezing solution (left). Comparison with the luminal surface of the same graft before freezing (right) reveals preservation of a confluent luminal surface.
Journal of Vascular Surgery  , DOI: ( /j.jvs )
Copyright © 2016 Society for Vascular Surgery Terms and Conditions