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, and Paul DiMuzio, MD, Philadelphia, Pa Objective: Previous work demonstrated the effectiveness of autologous adipose-derived stem cells (ASCs) as endothelial cell (EC) substitutes in vascular tissue engineering. We further this work toward clinical translation by evaluating ASC function after (1) replacement of fetal bovine serum (FBS) with autologous human plasma (HP) in culture and (2) cryopreservation. Methods: Human ASCs and plasma, isolated from periumbilical fat and peripheral blood, respectively, were collected from the same donors. ASCs were differentiated in endothelial growth medium supplemented with FBS (2%) vs HP (2%). Proliferation was measured by growth curves and MTT assay. Endothelial differentiation was measured by quantitative polymerase chain reaction, assessment of acetylated low-density lipoprotein uptake, and cord formation after plating on Matrigel (BD Biosciences, San Jose, Calif). Similar studies were conducted before and after cryopreservation of ASCs and included assessment of cell retention on the luminal surface of a vascular graft. Results: ASCs expanded in HP-supplemented medium showed (1) similar proliferation to FBS-cultured ASCs, (2) consistent differentiation toward an EC lineage (increases in CD31, von Willebrand factor, and CD144 message; acetylated low-density lipoprotein uptake; and cord formation on Matrigel), and (3) retention on the luminal surface after seeding and subsequent flow conditioning. Cryopreservation did not significantly alter ASC viability, proliferation, acquisition of endothelial characteristics, or retention after seeding onto a vascular graft. Conclusions: This study suggests that (1) replacement of FBS with autologous HPda step necessary for the translation of this technology into human useddoes not significantly impair proliferation or endothelial differentiation of ASCs used as EC substitutes and (2) ASCs are tolerant to cryopreservation in terms of maintaining EC characteristics and retention on a vascular graft. (J Vasc Surg 2014;-:1-10.) Clinical Relevance: Autologous adipose-derived stem cells have been shown to be effective endothelial cell substitutes in the creation of tissue-engineered vascular grafts in previous in vitro and in vivo animal studies. This study evaluates two aspects of translating this work into human use, namely, use of autologous plasma instead of fetal bovine serum in cell culture (improving safety) and cryopreservation (improving accessibility).
Autologous blood vessels remain the “gold standard” conduits for small-diameter (1 month. Thawing was accomplished by rapidly warming the cuvettes, suspending the cells in EGM-2 culture medium, and centrifuging the samples to remove the DMSO. The thawed cells were then plated in fresh culture medium. Vascular grafts were cryopreserved by submersion in a cryotube filled with 5% DMSO plus 95% FBS vs HP. The cryotube was sequentially frozen at 20 C for 30 minutes, then at 80 C overnight in an ethanol-jacketed closed container, and finally stored in liquid nitrogen (196 C) for >2 weeks. Thawing was accomplished by placing the graft in a 37 C water bath, washing twice in warm EGM-2 culture medium, and incubating in EGM-2 at 37 C with 5% CO2 for 2 hours. MTT assay. To evaluate the proliferation of cryopreserved ASCs, a 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. Cells were plated
at 1 104 cells/well in 0.5 mL medium in a 48-well plate. At 1, 2, 3, and 5 days, the culture medium was replaced with a medium containing 0.005% MTT solution (500 mL Dulbecco’s modified Eagle’s medium þ 40 mL MTT). After 4 hours of incubation at 37 C, the medium was discarded, and the purple formazan crystals formed were dissolved in 200 mL DMSO. The absorbance of the plate was read on a microplate reader at 570 nm. Fresh cells were used as controls. Cell viability assay. Trypan blue vital dye exclusion was used to determine cell viability. Cells were stained with trypan blue solution (1:1; Sigma-Aldrich) for 1 minute and viewed with light microscopy on a hemacytometer. Viable (cells that excluded the dye) and nonviable cell counts were used to determine percentage cell viability. Statistical analysis. The data are presented as mean 6 standard error. Student t-test was used to compare continuous data. Significance was set at P < .05. RESULTS Morphology and proliferation. Exchange of autologous HP for FBS did not appear to alter cell morphology. After culture in medium supplemented with autologous HP for 7 days, ASCs displayed a spindle-shaped, fibroblast-like morphology similar to those cultured in FBS (Fig 1, A).
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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 ¼ 6; average 6 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).
This change did not adversely affect proliferation. Growth curves constructed for cells grown in both conditions were not significantly different during 14 days (Fig 1, B; P > .5 throughout); we did, however, note a trend toward a shorter cumulative population doubling time for ASCs cultured with autologous HP (1.68 6 0.37 vs 2.09 6 0.88; P ¼ .06; Fig 1, C). Endothelial differentiation. ASCs were cultured in EGM-2 plus autologous HP for 2 weeks. Commitment toward an endothelial lineage was measured by evaluating CD31, vWF, and VE-cadherin (CD144) mRNA transcript levels by quantitative PCR. As shown in Fig 2, A, ASCs cultured in differentiating medium plus autologous HP demonstrated a trend toward increased levels of both CD31 mRNA (0.05 6 0.03 vs 0.02 6 0.01; P ¼ .1) and vWF mRNA (0.36 6 0.18 vs 0.16 6 0.06; P ¼ .1) compared with ASCs differentiated in medium containing FBS. Expression of VE-cadherin was the same in ASCs cultured in FBS vs HP. We assessed endothelial differentiation by testing for the uptake of acLDL and for the formation of cord-like
structures after plating on Matrigel. After differentiation in EGM-2 plus HP vs FBS for 10 days, >90% of ASCs stained positive for both DiI-acLDL (red) and human lectin (green) (Fig 2, B) in both groups; conversely, ASCs cultured in M199 medium (undifferentiated control) exhibited no significant acLDL uptake. Further, ASCs formed cord-like structures 12 hours after plating onto Matrigel (Fig 2, C) under both culture conditions; conversely, cells cultured in M199 medium (undifferentiated control) failed to form cords on Matrigel. Together, these results suggest that the substitution of autologous HP for FBS does not significantly alter endothelial differentiation by ASCs. Creation of a TEVG. To investigate the effect of exchanging FBS for autologous HP on the creation of a stem cell TEVG, we seeded the luminal surface of a decellularized human vein with ASCs cultured in EGM-2 plus autologous HP vs FBS. The grafts were flow conditioned by linearly increasing shear force within the lumen from 0 to 9 dynes during 5 days. Using confocal microscopy to evaluate cell seeding and retention, we observed complete
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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.
coverage of the luminal surface with cells aligned in the direction of flow (Fig 3), suggesting that the switch to autologous HP did not have a deleterious effect on the graft creation process. Cryopreservation of ASCs. The effect of cryopreservation on the proliferation of ASCs cultured in autologous HP was evaluated. We did not observe any significant change in morphology and proliferation of ASCs of the same cell line before or after a period of cryopreservation (Fig 4, A). Similarly, we found no significant difference in proliferation of cryopreserved ASCs cultured in medium supplemented with autologous HP vs FBS (Fig 4, B). The effect of cryopreservation on cell viability was evaluated by trypan blue exclusion. Fresh ASC cultures excluded the dye equally in both HP (96.3% 6 2.9%) and FBS (96.5% 6 0.58%; P > .05; Fig 4, C). After cryopreservation with 5% DMSO solutions, we did not observe any significant change in viability in HP (86.3% 6 5.3%) vs FBS (84.9% 6 4.8%; P > .05); nearly identical results were obtained after cryopreservation with 10% DMSO (Fig 4, C). In addition, we evaluated viability after long-term cryopreservation in 5% DMSO plus HP vs FBS (Fig 4, D). After 1 year of cryopreservation, we observed 83.5% 6 2.8% cell viability in the HP group vs 80.9% 6 5.6% in the FBS group (P > .05). The effect of cryopreservation on the expression of endothelial markers was also evaluated (Fig 5, A). After cryopreservation, mRNA expression of CD31 was unchanged compared with nonfrozen cells from both cell culture conditions. We did observe a trend toward decreasing vWF and CD144 mRNA in ASCs cultured in HP after cryopreservation (vWF: 1.09 6 0.23 vs 0.47 6 0.26, P ¼ .1; CD144: 0.01 6 0.003 vs 0.003 6 0.002; P ¼ .2); this trend was not observed in ASCs cultured in FBS. The effect of cryopreservation on cord formation was also tested (Fig 5, B). Twelve hours after plating onto Matrigel, both fresh and cryopreserved ASCs cultured in autologous HP formed cord-like structures that were grossly indistinguishable.
Cryopreservation of TEVGs. Finally, we evaluated the effect of cryopreservation on cell retention on the luminal surface of seeded grafts. TEVGs were constructed by seeding ASCs on the luminal surface of decellularized human saphenous vein, flow conditioned, and subsequently cryopreserved in 5% DMSO plus 95% autologous HP vs FBS for 10 days (Fig 6, A). After 2 weeks in storage at 196 C, the grafts were thawed and viewed with confocal microscopy. We found no significant loss of luminal coverage in TEVGs created and stored with autologous HP (Fig 6, B); similar results were obtained for control FBS-cryopreserved grafts (Fig 6, C). DISCUSSION We have previously investigated the use of autologous adult stem cells derived from adipose tissue for use as vascular cell substitutes to create a TEVG.7,11-13,21 The aim of this study was to evaluate the effect of replacing the commonly used medium component FBS with autologous HP on ASCs with regard to their use as EC substitutes. The major findings of this study suggest that this change does not adversely affect ASC isolation, proliferation, commitment toward an endothelial lineage, and adherence to the luminal surface of a vascular graft. Further, studies evaluating the effect of cryopreservation on ASC function and adherence, including the substitution of HP for FBS within the freezing solution, suggest that this technique does not significantly alter the stability of endothelial characteristics acquired by the stem cells or their adherence to the TEVG. Several clinical trials using mesenchymal stem cells for tissue engineering/regenerative medicine purposes have used FBS as a culture supplement.22,23 However, to optimize safety, replacement of the FBS with human serum/ plasma derivatives (such as human AB serum, platelet-rich plasma, and human platelet lysate) has been studied and suggests equal or higher proliferation and multilineage differentiation of bone marrow-derived mesenchymal stem cells24,25 and ASCs.26,27 Similarly, successful expansion
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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 (þstandard error of the mean [SEM]) (n ¼ 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 (þSEM) (n ¼ 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 (þSEM) (n ¼ 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 (þSEM) (n ¼ 4).
and differentiation of these cell lines have been demonstrated in serum-free media.28 To our knowledge, this is the first report evaluating the effect of autologous HP, an easily obtained blood component, on the proliferation and endothelial differentiation of ASCs. We hypothesized that this medium change would not adversely affect ASC isolation and proliferation. FBS is a known growth factor, and such a change could have proved deleterious.29 Throughout this study, the isolation and expansion of ASCs from more than 10 different donors of adipose tissue proved successful, independent of medium supplement (data not shown). The overall morphology of the stem cells grown in autologous HP was not grossly different from that of the cells grown in the standard medium. Others have observed the acquisition of a spindle-shaped appearance of bone marrowe derived mesenchymal stem cells, suggesting that such a change indicated increased motility.30 Growth curves constructed during 14 days demonstrated a trend toward a shorter doubling time for cells grown in autologous HP, suggesting a benefit to this change. This would be attractive clinically, as the large number of cells required for therapy could be achieved more rapidly.31
This study demonstrated that ASCs cultured in EGM-2 supplemented with autologous HP expressed endothelial molecular markers, suggesting commitment toward ECs. Platelet EC adhesion molecule 1 (CD31), vWF, and VE-cadherin (CD144) are considered reliable markers for ECs.32 Our previous work demonstrated that undifferentiated ASCs did not express these molecular markers and revealed their acquisition by the stem cells after culture in EGM-2 medium supplemented with FBS.7,12,13 Compared with cells grown in standard differentiating medium, cells grown in medium supplemented by HP appeared to have increased expression, although this did not reach significance. We acknowledge that a limitation of this study is that we did not test our markers at the protein level; however, our previous work uniformly revealed that upregulation of EC markers at the message level was followed by upregulation at the protein level.7,12,13 Also examined were the functional characteristics of acLDL uptake and angiogenic potential. Uptake of acLDL is considered a functional phenotype of ECs, as is the formation of capillary-like structures on plating on Matrigel demonstrating ability to participate in angiogenesis.33 Previously, we demonstrated that undifferentiated ASCs do
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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 (þstandard error of the mean) (n ¼ 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 ¼ 3).
not possess either characteristic; subsequent differentiation in EGM-2 media, however, resulted in the acquisition of both these EC characteristics.7,12,13 Whereas we did not quantify the results, we consistently found similar uptake of acLDL and cord formation by ASCs grown in EGM-2 media supplemented by either HP or FBS. Further, cells cultured under both conditions formed cords after plating onto Matrigel, suggesting the acquisition of angiogenic characteristics. Consistent with this finding, Ishikawa and Asahara34 reported that human endothelial progenitor cells cultured in autologous serum displayed angiogenic characteristics similar to those grown in FBS. In both circumstances, change to autologous HP continues to support the endothelial differentiation of ASCs at two important functional levels similar to that observed with EGM-2 media supplemented with FBS; we do, however, acknowledge that this study is limited in that we did not formally quantify acLDL uptake or cord formation. We originally chose to evaluate the change from FBS to autologous HP on a 1:1 basis. We did not attempt to optimize the medium with autologous HP for any of the several properties that were tested. This may represent a limitation to the current study and an opportunity for further research. In addition, we did not attempt to evaluate the consistency of autologous HP in achieving these results. This may become important, given that such technology may be used in patients with significant comorbidities and advanced age. We have previously reported the acquisition of endothelial characteristics by ASCs after culture in differentiating media, including the positive effects of shear forces.7,12,13 Subsequently, we reported the use of these cells to line the
luminal surface of a readily available natural vascular tissue scaffold.12,13 Each of these previous studies, including those testing the graft in vivo,12,13 used FBS to culture and to differentiate the ASCs toward ECs. This study demonstrates the successful creation of this novel TEVG with use of medium containing autologous HP rather than FBS. No significant effect on the establishment of a confluent monolayer of ASCs on the graft was observed, including resistance to shear forces after flow conditioning. In sum, it appears that the medium component substitution does not alter the creation of our TEVG. Evaluation of the functional characteristics of the ASC neointima created with autologous HP requires future testing in vivo. The second phase of this study evaluated the effect of cryopreservation on the maintenance of endothelial markers within the differentiated ASCs as well as gross preservation of the intimal layer of ASCs within the TEVG. Our standard “freezing” solution previously used a combination of 5% DMSO and 95% FBS; as such, we investigated cryopreservation along with a 1:1 exchange of the FBS for autologous HP. Other investigators used a higher concentration of DMSO (10%-20%)35; however, to increase cell recovery and function while avoiding DMSO toxicity,36 we chose to evaluate preservation at the 5% concentration. The results of our study suggest that the differentiated ASCs remain viable and continue to express endothelial molecular and functional markers. Whereas CD31 message remained unchanged, we did observe a trend toward decreased vWF and VE-cadherin message in cells frozen in HP that was not seen in FBS. These overall satisfactory results were achieved in our traditional solution (DMSO/FBS) as well
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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.
as in that using autologous HP. The additional studies comparing 5% vs 10% DMSO in both HP and FBS did reveal satisfactory cell viability in both the short and long term. These results support those of others suggesting that the lower concentration of DMSO (5%) may be as effective as 10% for preserving cells.37 We evaluated a simple method for cryopreserving the vascular graft. Previous work by others suggested that optimal EC survival rates were obtained within arterial segments cryopreserved with a cooling speed of 1 C/min, storage at 145 C, and slow thawing.38 However, Pegg et al39 observed that arteries that were slowly rewarmed showed a greater loss of endothelial function than those rapidly rewarmed. Given that our routine practice in cryopreservation of cells was to use a rapid thawing process, we
chose to test this method. Our method for cryopreservation and storage of ASC-seeded TEVG segments at 196 C followed by thawing done rapidly in a 37 C water bath preserves the confluent neointimal layer of stem cells. We acknowledge that our evaluation took place after a convenient, randomly selected time period (10 days) and that these results may not translate to longer periods of cryopreservation. Further, although we did assess the viability of cultured ASCs after cryopreservation, we did not directly assess the same for cells adherent to the TEVG. CONCLUSIONS We evaluated the effect of replacing FBS with autologous HP on adult stem cell function in the context of using them as EC substitutes. The results suggest that this change,
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designed to make the culture system safer and acceptable to governing agencies, was not deleterious to cell proliferation or differentiation. Further, we evaluated this change in the context of cryopreserving the cells and found satisfactory preservation of these endothelial markers with 5% DMSO combined with 95% autologous HP. Our proposed cryopreservation method, which uses a rapid thaw technique, was successful in maintaining the confluent neointimal layer of stem cells differentiated toward an endothelial phenotype. AUTHOR CONTRIBUTIONS Conception and design: PD, PZ, TT, AP Analysis and interpretation: PD, PZ, TT Data collection: PD, PZ, AP Writing the article: PD, PZ Critical revision of the article: PD Final approval of the article: PD, PZ, TT, AP Statistical analysis: PD, PZ Obtained funding: PD Overall responsibility: PD REFERENCES 1. Seifu DG, Purnama A, Mequanint K, Mantovani D. Small-diameter vascular tissue engineering. Nat Rev Cardiol 2013;10:410-21. 2. Ravari H, Kazemzade GH, Saied Modaghegh MD, Khashayar P. Patency rate and complications of polytetrafluoroethylene grafts compared with polyurethane grafts for hemodialysis access. Ups J Med Sci 2010;115:245-8. 3. Roll S, Muller-Nordhorn J, Keil T, Scholz H, Eidt D, Greiner W, et al. Dacron vs. PTFE as bypass materials in peripheral vascular surgeryd systematic review and meta-analysis. BMC Surg 2008;19:8-22. 4. Lamm P, Juchem G, Milz S, Schuffenhauer M, Reichart B. Autologous endothelialized vein allograft a solution in the search for small-caliber grafts in coronary artery bypass graft operations. Circulation 2001;18: I108-14. 5. Nemeno-Guanzon JG, Lee S, Berg JR, Jo YH, Yeo JE, Nam BM, et al. Trends in tissue engineering for blood vessels. J Biomed Biotechnol 2012;2012:956345. 6. L’Heureux N, Dusserre N, Marini A, Garrido S, de la Fuente L, McAllister T. Technology insight: the evolution of tissue-engineered vascular graftsdfrom research to clinical practice. Nat Clin Pract Cardiovasc Med 2007;4:389-95. 7. Zhang P, Moudgill N, Hager E, Tarola N, Dimatteo C, McIlhenny S, et al. Endothelial differentiation of adipose-derived stem cells from elderly patients with cardiovascular disease. Stem Cells Dev 2011;20: 977-88. 8. Harris LJ, Abdollahi H, Zhang P, McIlhenny S, Tulenko T, DiMuzio PJ. Differentiation of adult stem cells into smooth muscle for vascular tissue engineering. J Surg Res 2009:1-9. 9. Cao Y, Sun Z, Liao L, Mend Y, Han Q, Zhao RC. Human adipose tissueederived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo. Biochem Biophys Res Commun 2005;332:370-9. 10. Miranville A, Heeschen C, Sengenes C, Curat CA, Busse R, Boulomie A. Improvement of postnatal neovascularization by human adipose tissueederived stem cells. Circulation 2004;110:349-55. 11. DiMuzio P, Fischer L, McIlhenny S, DiMatteo C, Golesorhki N, Grabo D, et al. Development of a tissue-engineered bypass graft seeded with stem cells. Vascular 2006;14:338-42. 12. Fischer LJ, McIlhenny S, Tulenko T, Golesorkhi N, Zhang P, Larson R, et al. Endothelial differentiation of adipose-derived stem cells: effects of endothelial cell growth supplement and shear force. J Surg Res 2009;152:157-66.
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13. McIlhenny SE, Zhang P, Tulenko T, Comeau JA, Fernandez S, Policha A, et al. eNOS transfection of adipose-derived stem cells yields bioactive nitric oxide production and improved results in vascular tissue engineering. J Tissue Eng Regen Med 2013 Jan 14. [Epub ahead of print]. 14. Spees JL, Gregory CA, Singh H, Tucker HA, Peister A, Lynch PJ, et al. Internalized antigens must be removed to prepare hypoimmunogenic mesenchymal stem cells for cell and gene therapy. Mol Ther 2004;9: 747-56. 15. Le Blanc K, Samuelsson H, Lonnies L, Sundin M, Ringden O. Generation of immunosuppressive mesenchymal stem cells in allogeneic human serum. Transplantation 2007;84:1055-9. 16. Dahl JA, Duggal S, Coulston N, Millar D, Melki J, Shahdadfar A, et al. Genetic and epigenetic instability of human bone marrow mesenchymal stem cells expanded in autologous serum or fetal bovine serum. Int J Dev Biol 2008;52:1033-42. 17. Sun X, Gan Y, Tang T, Zhang X, Dai K. In vitro proliferation and differentiation of human mesenchymal stem cells cultured in autologous plasma derived from bone marrow. Tissue Eng Part A 2008;14:391-400. 18. Tonti GA, Mannello F. From bone marrow to therapeutic applications: different behaviour and genetic/epigenetic stability during mesenchymal stem cell expansion in autologous and fetal bovine sera? Int J Dev Biol 2008;52:1023-32. 19. Cleary MA, Geiger E, Grady C, Best C, Naito Y, Breuer C. Vascular tissue engineering: the next generation. Trends Mol Med 2012;18: 394-404. 20. Martin ND, Schaner PJ, Tulenko TN, Shapiro IM, Dimatteo CA, Williams TK, et al. In vivo behavior of decellularized vein allograft. J Surg Res 2005;129:17-23. 21. McIlhenny SE, Hager ES, Grabo DJ, DiMatteo C, Shapiro IM, Tulenko TN, et al. Linear shear conditioning improves vascular graft retention of adipose-derived stem cells by upregulation of the a5b1 integrin. Tissue Eng Part A 2010;16:245-55. 22. Bang OY, Lee JS, Lee PH. Gang Lee. Autologous mesenchymal stem cell transplantation in stroke patients. Ann Neurol 2005;57:874-82. 23. Ren GW, Chen XD, Dong FP, Li WZ, Ren XH, Zhang YY, et al. Concise review: mesenchymal stem cells and translational medicine: emerging issues. Stem Cells Transl Med 2012;1:51-8. 24. Nimura A, Muneta T, Koga H, Mochizuki T, Suzuki K, Makino H, et al. Increased proliferation of human synovial mesenchymal stem cells with autologous human serum: comparisons with bone marrow mesenchymal stem cells and with fetal bovine serum. Arthritis Rheum 2008;58:501-10. 25. Gruber R, Karreth F, Kandler B, Fuerst G, Rot A, Fischer MB, et al. Platelet-released supernatants increase migration and proliferation, and decrease osteogenic differentiation of bone marrowederived mesenchymal progenitor cells under in vitro conditions. Platelets 2004;15: 29-35. 26. Schallmoser K, Bartmann C, Rohde E, Reinisch A, Kashofer K, Stadelmeyer E, et al. Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells. Transfusion 2007;47:1436-46. 27. Kocaoemer A, Kern S, Klüter H, Bieback K. Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue. Stem Cells 2007;25:1270-8. 28. Dromard C, Bourin P, André M, De Barros S, Casteilla L, PlanatBenard V. Human adipose derived stroma/stem cells grow in serumfree medium as floating spheres. Exp Cell Res 2011;317:770-80. 29. Lindroos B, Aho KL, Kuokkanen H, Räty S, Huhtala H, Lemponen R, et al. Differential gene expression in adipose stem cells cultured in allogeneic human serum versus fetal bovine serum. Tissue Eng Part A 2010;16:2281-94. 30. Kobayashi T, Watanabe H, Yanagawa T, Tsutsumi S, Kayakabe M, Shinozaki T, et al. Motility and growth of human bone-marrow mesenchymal stem cells during ex vivo expansion in autologous serum. J Bone Joint Surg Br 2005;87:1426-33. 31. Baksh D, Song L, Tuan RS. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med 2004;8:301-16.
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32. Maruyama N, Kokubo K, Shinbo T, Hirose M, Kobayashi M, Sakuragawa N, et al. Hypoxia enhances the induction of human amniotic mesenchymal side population cells into vascular endothelial lineage. Int J Mol Med 2013;32:315-22. 33. Planat-Bernard V, Silvestre JS, Cousin B, Andre M, Nibbelink M, Tamarat R, et al. Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives. Circulation 2004;109:656-63. 34. Ishikawa M, Asahara T. Endothelial progenitor cell culture for vascular regeneration. Stem Cells Dev 2004;13:344-9. 35. Mugishima H, Harada K, Chin M, Suzuki T, Takagi K, Hayakawa S, et al. Effects of long-term cryopreservation on hematopoietic progenitor cells in umbilical cord blood. Bone Marrow Transplant 1999;23: 395-6.
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36. Davis JM, Rowley SD, Braine HG, Piantadosi S, Santos GW. Clinical toxicity of cryopreserved bone marrow graft infusion. Blood 1990;75: 781-6. 37. Liseth K, Abrahamsen JF, Bjorsvik S, Grottebo K, Bruserud O. The viability of cryopreserved PBPC depends on the DMSO concentration and the concentration of nucleated cells in the graft. Cytotherapy 2005;7:328-33. 38. Song YC, Pegg DE, Hunt CJ. Cryopreservation of the common carotid artery of the rabbit: optimization of dimethyl sulfoxide concentration and cooling rate. Cryobiology 1995;32:405-21. 39. Pegg DE, Wusteman MC, Boylan S. Fractures in cryopreserved elastic arteries. Cryobiology 1997;34:183-92. Submitted Jul 22, 2014; accepted Oct 11, 2014.
Autologous blood vessels remain the “gold standard” conduits for small-diameter (1 month. Thawing was accomplished by rapidly warming the cuvettes, suspending the cells in EGM-2 culture medium, and centrifuging the samples to remove the DMSO. The thawed cells were then plated in fresh culture medium. Vascular grafts were cryopreserved by submersion in a cryotube filled with 5% DMSO plus 95% FBS vs HP. The cryotube was sequentially frozen at 20 C for 30 minutes, then at 80 C overnight in an ethanol-jacketed closed container, and finally stored in liquid nitrogen (196 C) for >2 weeks. Thawing was accomplished by placing the graft in a 37 C water bath, washing twice in warm EGM-2 culture medium, and incubating in EGM-2 at 37 C with 5% CO2 for 2 hours. MTT assay. To evaluate the proliferation of cryopreserved ASCs, a 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. Cells were plated
at 1 104 cells/well in 0.5 mL medium in a 48-well plate. At 1, 2, 3, and 5 days, the culture medium was replaced with a medium containing 0.005% MTT solution (500 mL Dulbecco’s modified Eagle’s medium þ 40 mL MTT). After 4 hours of incubation at 37 C, the medium was discarded, and the purple formazan crystals formed were dissolved in 200 mL DMSO. The absorbance of the plate was read on a microplate reader at 570 nm. Fresh cells were used as controls. Cell viability assay. Trypan blue vital dye exclusion was used to determine cell viability. Cells were stained with trypan blue solution (1:1; Sigma-Aldrich) for 1 minute and viewed with light microscopy on a hemacytometer. Viable (cells that excluded the dye) and nonviable cell counts were used to determine percentage cell viability. Statistical analysis. The data are presented as mean 6 standard error. Student t-test was used to compare continuous data. Significance was set at P < .05. RESULTS Morphology and proliferation. Exchange of autologous HP for FBS did not appear to alter cell morphology. After culture in medium supplemented with autologous HP for 7 days, ASCs displayed a spindle-shaped, fibroblast-like morphology similar to those cultured in FBS (Fig 1, A).
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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 ¼ 6; average 6 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).
This change did not adversely affect proliferation. Growth curves constructed for cells grown in both conditions were not significantly different during 14 days (Fig 1, B; P > .5 throughout); we did, however, note a trend toward a shorter cumulative population doubling time for ASCs cultured with autologous HP (1.68 6 0.37 vs 2.09 6 0.88; P ¼ .06; Fig 1, C). Endothelial differentiation. ASCs were cultured in EGM-2 plus autologous HP for 2 weeks. Commitment toward an endothelial lineage was measured by evaluating CD31, vWF, and VE-cadherin (CD144) mRNA transcript levels by quantitative PCR. As shown in Fig 2, A, ASCs cultured in differentiating medium plus autologous HP demonstrated a trend toward increased levels of both CD31 mRNA (0.05 6 0.03 vs 0.02 6 0.01; P ¼ .1) and vWF mRNA (0.36 6 0.18 vs 0.16 6 0.06; P ¼ .1) compared with ASCs differentiated in medium containing FBS. Expression of VE-cadherin was the same in ASCs cultured in FBS vs HP. We assessed endothelial differentiation by testing for the uptake of acLDL and for the formation of cord-like
structures after plating on Matrigel. After differentiation in EGM-2 plus HP vs FBS for 10 days, >90% of ASCs stained positive for both DiI-acLDL (red) and human lectin (green) (Fig 2, B) in both groups; conversely, ASCs cultured in M199 medium (undifferentiated control) exhibited no significant acLDL uptake. Further, ASCs formed cord-like structures 12 hours after plating onto Matrigel (Fig 2, C) under both culture conditions; conversely, cells cultured in M199 medium (undifferentiated control) failed to form cords on Matrigel. Together, these results suggest that the substitution of autologous HP for FBS does not significantly alter endothelial differentiation by ASCs. Creation of a TEVG. To investigate the effect of exchanging FBS for autologous HP on the creation of a stem cell TEVG, we seeded the luminal surface of a decellularized human vein with ASCs cultured in EGM-2 plus autologous HP vs FBS. The grafts were flow conditioned by linearly increasing shear force within the lumen from 0 to 9 dynes during 5 days. Using confocal microscopy to evaluate cell seeding and retention, we observed complete
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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.
coverage of the luminal surface with cells aligned in the direction of flow (Fig 3), suggesting that the switch to autologous HP did not have a deleterious effect on the graft creation process. Cryopreservation of ASCs. The effect of cryopreservation on the proliferation of ASCs cultured in autologous HP was evaluated. We did not observe any significant change in morphology and proliferation of ASCs of the same cell line before or after a period of cryopreservation (Fig 4, A). Similarly, we found no significant difference in proliferation of cryopreserved ASCs cultured in medium supplemented with autologous HP vs FBS (Fig 4, B). The effect of cryopreservation on cell viability was evaluated by trypan blue exclusion. Fresh ASC cultures excluded the dye equally in both HP (96.3% 6 2.9%) and FBS (96.5% 6 0.58%; P > .05; Fig 4, C). After cryopreservation with 5% DMSO solutions, we did not observe any significant change in viability in HP (86.3% 6 5.3%) vs FBS (84.9% 6 4.8%; P > .05); nearly identical results were obtained after cryopreservation with 10% DMSO (Fig 4, C). In addition, we evaluated viability after long-term cryopreservation in 5% DMSO plus HP vs FBS (Fig 4, D). After 1 year of cryopreservation, we observed 83.5% 6 2.8% cell viability in the HP group vs 80.9% 6 5.6% in the FBS group (P > .05). The effect of cryopreservation on the expression of endothelial markers was also evaluated (Fig 5, A). After cryopreservation, mRNA expression of CD31 was unchanged compared with nonfrozen cells from both cell culture conditions. We did observe a trend toward decreasing vWF and CD144 mRNA in ASCs cultured in HP after cryopreservation (vWF: 1.09 6 0.23 vs 0.47 6 0.26, P ¼ .1; CD144: 0.01 6 0.003 vs 0.003 6 0.002; P ¼ .2); this trend was not observed in ASCs cultured in FBS. The effect of cryopreservation on cord formation was also tested (Fig 5, B). Twelve hours after plating onto Matrigel, both fresh and cryopreserved ASCs cultured in autologous HP formed cord-like structures that were grossly indistinguishable.
Cryopreservation of TEVGs. Finally, we evaluated the effect of cryopreservation on cell retention on the luminal surface of seeded grafts. TEVGs were constructed by seeding ASCs on the luminal surface of decellularized human saphenous vein, flow conditioned, and subsequently cryopreserved in 5% DMSO plus 95% autologous HP vs FBS for 10 days (Fig 6, A). After 2 weeks in storage at 196 C, the grafts were thawed and viewed with confocal microscopy. We found no significant loss of luminal coverage in TEVGs created and stored with autologous HP (Fig 6, B); similar results were obtained for control FBS-cryopreserved grafts (Fig 6, C). DISCUSSION We have previously investigated the use of autologous adult stem cells derived from adipose tissue for use as vascular cell substitutes to create a TEVG.7,11-13,21 The aim of this study was to evaluate the effect of replacing the commonly used medium component FBS with autologous HP on ASCs with regard to their use as EC substitutes. The major findings of this study suggest that this change does not adversely affect ASC isolation, proliferation, commitment toward an endothelial lineage, and adherence to the luminal surface of a vascular graft. Further, studies evaluating the effect of cryopreservation on ASC function and adherence, including the substitution of HP for FBS within the freezing solution, suggest that this technique does not significantly alter the stability of endothelial characteristics acquired by the stem cells or their adherence to the TEVG. Several clinical trials using mesenchymal stem cells for tissue engineering/regenerative medicine purposes have used FBS as a culture supplement.22,23 However, to optimize safety, replacement of the FBS with human serum/ plasma derivatives (such as human AB serum, platelet-rich plasma, and human platelet lysate) has been studied and suggests equal or higher proliferation and multilineage differentiation of bone marrow-derived mesenchymal stem cells24,25 and ASCs.26,27 Similarly, successful expansion
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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 (þstandard error of the mean [SEM]) (n ¼ 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 (þSEM) (n ¼ 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 (þSEM) (n ¼ 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 (þSEM) (n ¼ 4).
and differentiation of these cell lines have been demonstrated in serum-free media.28 To our knowledge, this is the first report evaluating the effect of autologous HP, an easily obtained blood component, on the proliferation and endothelial differentiation of ASCs. We hypothesized that this medium change would not adversely affect ASC isolation and proliferation. FBS is a known growth factor, and such a change could have proved deleterious.29 Throughout this study, the isolation and expansion of ASCs from more than 10 different donors of adipose tissue proved successful, independent of medium supplement (data not shown). The overall morphology of the stem cells grown in autologous HP was not grossly different from that of the cells grown in the standard medium. Others have observed the acquisition of a spindle-shaped appearance of bone marrowe derived mesenchymal stem cells, suggesting that such a change indicated increased motility.30 Growth curves constructed during 14 days demonstrated a trend toward a shorter doubling time for cells grown in autologous HP, suggesting a benefit to this change. This would be attractive clinically, as the large number of cells required for therapy could be achieved more rapidly.31
This study demonstrated that ASCs cultured in EGM-2 supplemented with autologous HP expressed endothelial molecular markers, suggesting commitment toward ECs. Platelet EC adhesion molecule 1 (CD31), vWF, and VE-cadherin (CD144) are considered reliable markers for ECs.32 Our previous work demonstrated that undifferentiated ASCs did not express these molecular markers and revealed their acquisition by the stem cells after culture in EGM-2 medium supplemented with FBS.7,12,13 Compared with cells grown in standard differentiating medium, cells grown in medium supplemented by HP appeared to have increased expression, although this did not reach significance. We acknowledge that a limitation of this study is that we did not test our markers at the protein level; however, our previous work uniformly revealed that upregulation of EC markers at the message level was followed by upregulation at the protein level.7,12,13 Also examined were the functional characteristics of acLDL uptake and angiogenic potential. Uptake of acLDL is considered a functional phenotype of ECs, as is the formation of capillary-like structures on plating on Matrigel demonstrating ability to participate in angiogenesis.33 Previously, we demonstrated that undifferentiated ASCs do
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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 (þstandard error of the mean) (n ¼ 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 ¼ 3).
not possess either characteristic; subsequent differentiation in EGM-2 media, however, resulted in the acquisition of both these EC characteristics.7,12,13 Whereas we did not quantify the results, we consistently found similar uptake of acLDL and cord formation by ASCs grown in EGM-2 media supplemented by either HP or FBS. Further, cells cultured under both conditions formed cords after plating onto Matrigel, suggesting the acquisition of angiogenic characteristics. Consistent with this finding, Ishikawa and Asahara34 reported that human endothelial progenitor cells cultured in autologous serum displayed angiogenic characteristics similar to those grown in FBS. In both circumstances, change to autologous HP continues to support the endothelial differentiation of ASCs at two important functional levels similar to that observed with EGM-2 media supplemented with FBS; we do, however, acknowledge that this study is limited in that we did not formally quantify acLDL uptake or cord formation. We originally chose to evaluate the change from FBS to autologous HP on a 1:1 basis. We did not attempt to optimize the medium with autologous HP for any of the several properties that were tested. This may represent a limitation to the current study and an opportunity for further research. In addition, we did not attempt to evaluate the consistency of autologous HP in achieving these results. This may become important, given that such technology may be used in patients with significant comorbidities and advanced age. We have previously reported the acquisition of endothelial characteristics by ASCs after culture in differentiating media, including the positive effects of shear forces.7,12,13 Subsequently, we reported the use of these cells to line the
luminal surface of a readily available natural vascular tissue scaffold.12,13 Each of these previous studies, including those testing the graft in vivo,12,13 used FBS to culture and to differentiate the ASCs toward ECs. This study demonstrates the successful creation of this novel TEVG with use of medium containing autologous HP rather than FBS. No significant effect on the establishment of a confluent monolayer of ASCs on the graft was observed, including resistance to shear forces after flow conditioning. In sum, it appears that the medium component substitution does not alter the creation of our TEVG. Evaluation of the functional characteristics of the ASC neointima created with autologous HP requires future testing in vivo. The second phase of this study evaluated the effect of cryopreservation on the maintenance of endothelial markers within the differentiated ASCs as well as gross preservation of the intimal layer of ASCs within the TEVG. Our standard “freezing” solution previously used a combination of 5% DMSO and 95% FBS; as such, we investigated cryopreservation along with a 1:1 exchange of the FBS for autologous HP. Other investigators used a higher concentration of DMSO (10%-20%)35; however, to increase cell recovery and function while avoiding DMSO toxicity,36 we chose to evaluate preservation at the 5% concentration. The results of our study suggest that the differentiated ASCs remain viable and continue to express endothelial molecular and functional markers. Whereas CD31 message remained unchanged, we did observe a trend toward decreased vWF and VE-cadherin message in cells frozen in HP that was not seen in FBS. These overall satisfactory results were achieved in our traditional solution (DMSO/FBS) as well
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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.
as in that using autologous HP. The additional studies comparing 5% vs 10% DMSO in both HP and FBS did reveal satisfactory cell viability in both the short and long term. These results support those of others suggesting that the lower concentration of DMSO (5%) may be as effective as 10% for preserving cells.37 We evaluated a simple method for cryopreserving the vascular graft. Previous work by others suggested that optimal EC survival rates were obtained within arterial segments cryopreserved with a cooling speed of 1 C/min, storage at 145 C, and slow thawing.38 However, Pegg et al39 observed that arteries that were slowly rewarmed showed a greater loss of endothelial function than those rapidly rewarmed. Given that our routine practice in cryopreservation of cells was to use a rapid thawing process, we
chose to test this method. Our method for cryopreservation and storage of ASC-seeded TEVG segments at 196 C followed by thawing done rapidly in a 37 C water bath preserves the confluent neointimal layer of stem cells. We acknowledge that our evaluation took place after a convenient, randomly selected time period (10 days) and that these results may not translate to longer periods of cryopreservation. Further, although we did assess the viability of cultured ASCs after cryopreservation, we did not directly assess the same for cells adherent to the TEVG. CONCLUSIONS We evaluated the effect of replacing FBS with autologous HP on adult stem cell function in the context of using them as EC substitutes. The results suggest that this change,
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designed to make the culture system safer and acceptable to governing agencies, was not deleterious to cell proliferation or differentiation. Further, we evaluated this change in the context of cryopreserving the cells and found satisfactory preservation of these endothelial markers with 5% DMSO combined with 95% autologous HP. Our proposed cryopreservation method, which uses a rapid thaw technique, was successful in maintaining the confluent neointimal layer of stem cells differentiated toward an endothelial phenotype. AUTHOR CONTRIBUTIONS Conception and design: PD, PZ, TT, AP Analysis and interpretation: PD, PZ, TT Data collection: PD, PZ, AP Writing the article: PD, PZ Critical revision of the article: PD Final approval of the article: PD, PZ, TT, AP Statistical analysis: PD, PZ Obtained funding: PD Overall responsibility: PD REFERENCES 1. Seifu DG, Purnama A, Mequanint K, Mantovani D. Small-diameter vascular tissue engineering. Nat Rev Cardiol 2013;10:410-21. 2. Ravari H, Kazemzade GH, Saied Modaghegh MD, Khashayar P. Patency rate and complications of polytetrafluoroethylene grafts compared with polyurethane grafts for hemodialysis access. Ups J Med Sci 2010;115:245-8. 3. Roll S, Muller-Nordhorn J, Keil T, Scholz H, Eidt D, Greiner W, et al. Dacron vs. PTFE as bypass materials in peripheral vascular surgeryd systematic review and meta-analysis. BMC Surg 2008;19:8-22. 4. Lamm P, Juchem G, Milz S, Schuffenhauer M, Reichart B. Autologous endothelialized vein allograft a solution in the search for small-caliber grafts in coronary artery bypass graft operations. Circulation 2001;18: I108-14. 5. Nemeno-Guanzon JG, Lee S, Berg JR, Jo YH, Yeo JE, Nam BM, et al. Trends in tissue engineering for blood vessels. J Biomed Biotechnol 2012;2012:956345. 6. L’Heureux N, Dusserre N, Marini A, Garrido S, de la Fuente L, McAllister T. Technology insight: the evolution of tissue-engineered vascular graftsdfrom research to clinical practice. Nat Clin Pract Cardiovasc Med 2007;4:389-95. 7. Zhang P, Moudgill N, Hager E, Tarola N, Dimatteo C, McIlhenny S, et al. Endothelial differentiation of adipose-derived stem cells from elderly patients with cardiovascular disease. Stem Cells Dev 2011;20: 977-88. 8. Harris LJ, Abdollahi H, Zhang P, McIlhenny S, Tulenko T, DiMuzio PJ. Differentiation of adult stem cells into smooth muscle for vascular tissue engineering. J Surg Res 2009:1-9. 9. Cao Y, Sun Z, Liao L, Mend Y, Han Q, Zhao RC. Human adipose tissueederived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo. Biochem Biophys Res Commun 2005;332:370-9. 10. Miranville A, Heeschen C, Sengenes C, Curat CA, Busse R, Boulomie A. Improvement of postnatal neovascularization by human adipose tissueederived stem cells. Circulation 2004;110:349-55. 11. DiMuzio P, Fischer L, McIlhenny S, DiMatteo C, Golesorhki N, Grabo D, et al. Development of a tissue-engineered bypass graft seeded with stem cells. Vascular 2006;14:338-42. 12. Fischer LJ, McIlhenny S, Tulenko T, Golesorkhi N, Zhang P, Larson R, et al. Endothelial differentiation of adipose-derived stem cells: effects of endothelial cell growth supplement and shear force. J Surg Res 2009;152:157-66.
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36. Davis JM, Rowley SD, Braine HG, Piantadosi S, Santos GW. Clinical toxicity of cryopreserved bone marrow graft infusion. Blood 1990;75: 781-6. 37. Liseth K, Abrahamsen JF, Bjorsvik S, Grottebo K, Bruserud O. The viability of cryopreserved PBPC depends on the DMSO concentration and the concentration of nucleated cells in the graft. Cytotherapy 2005;7:328-33. 38. Song YC, Pegg DE, Hunt CJ. Cryopreservation of the common carotid artery of the rabbit: optimization of dimethyl sulfoxide concentration and cooling rate. Cryobiology 1995;32:405-21. 39. Pegg DE, Wusteman MC, Boylan S. Fractures in cryopreserved elastic arteries. Cryobiology 1997;34:183-92. Submitted Jul 22, 2014; accepted Oct 11, 2014.
