Development of an Operator-Independent Method for Seeding Tissue-Engineered Vascular Grafts

Udelsman, Brooks V.; Hibino, Narutoshi; Villalona, Gustavo A.; McGillicuddy, Edward A.; Nieponice, Alejandro; Sakamoto, Yuki; Matsuda, Shunji; Vorp, David A.; Shinoka, Toshiharu; Breuer, Christopher K.

doi:10.1089/ten.tec.2010.0581

Cited by 36 publications

(39 citation statements)

References 23 publications

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“…5 In the clinical arena, traditional methods of isolating BM-MNCs rely on density centrifugation, which is a timeconsuming and labor-intensive process with a high degree of operator variability. 4 The filter-based method was originally used for isolating MNCs from peripheral blood. 12 Due to its success, we applied this technique to our method for construction of TEVG and demonstrated feasibility in isolating MNCs from human bone marrow as an alternative to conventional density centrifugation by showing morphologic equivalence in neovessel formation in the TEVG implantation model.…”

Section: Discussionmentioning

confidence: 99%

“…The cellularity of the seeded scaffolds after overnight incubation was determined by measuring the DNA content with a PicoGreen detection assay (Molecular Probes, Eugene, OR). 4 …”

Section: Scaffoldsmentioning

confidence: 99%

“…However, current techniques for the construction of TEVG use density centrifugation as a means of isolating BM-MNCs and are limited by the risk of bacterial contamination, loss of cell viability, and need for a specialized clean room. 4 The development of simpler, more rapid, and operator-independent methods for manufacturing TEVG using closed disposable systems could overcome these disadvantages and facilitate more widespread use of TEVG products. We previously demonstrated the feasibility of a novel filtration system to isolate BM-MNCs from human bone marrow 5 and applied this technique to a closed disposable system for construction of TEVG by using an ovine model.…”

Section: Introductionmentioning

confidence: 99%

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Comparison of the Biological Equivalence of Two Methods for Isolating Bone Marrow Mononuclear Cells for Fabricating Tissue-Engineered Vascular Grafts

Kurobe

Tara

Maxfield

et al. 2015

Tissue Engineering Part C: Methods

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Our approach for fabricating tissue-engineered vascular grafts (TEVG), applied in the surgical management of congenital heart disease, is accomplished by seeding isolated bone marrow-derived mononuclear cells (BMMNCs) onto biodegradable scaffolds. The current method used for isolation of BM-MNCs is density centrifugation in Ficoll. This is a time-consuming, labor-intensive, and operator-dependent method. We previously demonstrated that a simpler, faster, and operator-independent method for isolating BM-MNCs using a filter elution technique was feasible. In this study, we compare the use of each technique to determine if the BMMNCs isolated by the filtration elution method are biologically equivalent to BM-MNCs isolated using density centrifugation. Scaffolds were constructed from a nonwoven poly(glycolic acid) fiber mesh coated with 50:50 poly(l-lactide-co-e-caprolactone) sealant. BM-MNCs were isolated from the bone marrow of syngeneic C57BL/6 mice by either density centrifugation with Ficoll or filtration (Ficoll vs. Filter), then statically seeded onto scaffolds, and incubated overnight. The TEVG were implanted in 10-week-old C57BL/6 mice (n = 23 for each group) as inferior vena cava interposition grafts and explanted at 14 days for analysis. At 14 days after implantation, there were no significant differences in graft patency between groups (Ficoll: 87% vs. Filter: 78%, p = 0.45). Morphometric analysis by hematoxylin and eosin staining showed no difference of graft luminal diameter or neointimal thickness between groups (luminal diameter, Ficoll: 620.3 -82.9 mm vs. Filter: 633.3 -131.0 mm, p = 0.72; neointimal thickness, Ficoll: 37.9 -7.8 mm vs. Filter: 37.9 -11.2 mm, p = 0.99). Histologic examination demonstrated similar degrees of cellular infiltration and extracellular matrix deposition, and endothelial cell coverage on the luminal surface, in either group. Macrophage infiltration showed no difference in the number of F4/80-positive cells or macrophage phenotypes between the two experimental groups (Ficoll: 2041 -1048 cells/mm 2 vs. Filter: 1887 -907.7 cells/mm 2 , p = 0.18). We confirmed the biological equivalence of BM-MNCs, isolated using either density centrifugation or filtration, for making TEVG.

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Section: Discussionmentioning

confidence: 99%

“…The cellularity of the seeded scaffolds after overnight incubation was determined by measuring the DNA content with a PicoGreen detection assay (Molecular Probes, Eugene, OR). 4 …”

Section: Scaffoldsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Comparison of the Biological Equivalence of Two Methods for Isolating Bone Marrow Mononuclear Cells for Fabricating Tissue-Engineered Vascular Grafts

Kurobe

Tara

Maxfield

et al. 2015

Tissue Engineering Part C: Methods

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“…More recently, stem cells from various sources have been exploited for vascular tissue engineering applications. Indeed, a variety of stem cell types including embryonic stem cells 7 , induced pluripotent stem cells (iPSCs) 8,9 , PiPSC 10,11 , bone marrow-derived mononuclear cells 12 , mesenchymal stem cells 13 , endothelial progenitor cells and adult vessel wallderived stem cell antigen-1 (Sca-1)+ stem/progenitor cells 14,15 have all been demonstrated to be capable of differentiation into either functional endothelial or smooth muscle cells in response to defined media and culture conditions. Furthermore, the unlimited self renewal capacity of the stem cells make them better candidates unlike mature endothelial and smooth muscle cells which can only divide for a finite number of times before undergoing growth arrest and senescence.…”

Section: Introductionmentioning

confidence: 99%

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Wong

Hong

Karamariti

et al. 2015

JoVE

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The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future. Video LinkThe video component of this article can be found at

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“…6 The use of an open technique increases the risk of contamination and introduces operator variability to the process of making the TEVG. Clean rooms are expensive to build and maintain and require significant manpower to operate in compliance with good manufacturing process standards.…”

mentioning

confidence: 99%

Comparison of a Closed System to a Standard Open Technique for Preparing Tissue-Engineered Vascular Grafts

Kurobe

Maxfield

Naito

et al. 2015

Tissue Engineering Part C: Methods

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We developed a prototype for a closed apparatus for assembling tissue-engineered vascular grafts (TEVGs) with the goal of creating a simple operator-independent method for making TEVGs to optimize safety and enable widespread application of this technology. The TEVG is made by seeding autologous bone marrowderived mononuclear cells onto a biodegradable tubular scaffold and is the first man-made vascular graft to be successfully used in humans. A critical barrier, which has prevented the widespread clinical adoption of the TEVG, is that cell isolation, scaffold seeding, and incubation are performed using an open method. To reduce the risk of contamination, the TEVG is assembled in a clean room. Clean rooms are expensive to build, complex to operate, and are not available in most hospitals. In this investigation, we used an ovine model to compare the safety and efficacy of TEVGs created using either a standard density centrifugation-based open method or the new filter-based closed system. We demonstrated no graft-related complications and maintenance of growth capacity in TEVGs created using the closed apparatus. In addition, the use of the closed system reduced the amount of time needed to assemble the TEVG by *50%. Adaptation of similar methodologies may facilitate the safe translation and the widespread use of other tissue engineering technologies.

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Development of an Operator-Independent Method for Seeding Tissue-Engineered Vascular Grafts

Cited by 36 publications

References 23 publications

Comparison of the Biological Equivalence of Two Methods for Isolating Bone Marrow Mononuclear Cells for Fabricating Tissue-Engineered Vascular Grafts

Comparison of the Biological Equivalence of Two Methods for Isolating Bone Marrow Mononuclear Cells for Fabricating Tissue-Engineered Vascular Grafts

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Comparison of a Closed System to a Standard Open Technique for Preparing Tissue-Engineered Vascular Grafts

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