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

Abstract

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.

Rational Design of an Improved Tissue-Engineered Vascular Graft: Determining the Optimal Cell Dose and Incubation Time

“…Using our current methodology based on isolating the BM-MNC using density centrifugation in Ficoll followed by a 2-h incubation approximately 268 ± 9 min are required to assemble the TEVG [4]. In an attempt to reduce the time required for TEVG assembly, we previously developed a closed disposable system using a filtration/elusion system to minimize time for the first two parts of the TEVG assembly, isolating BM-MNC and seeding [12]. Using the system, we could be able to drastically minimize the time for cell isolation and seeding without impacting the patency using a sheep model [11].…”

“…Assembling a TEVG used in our clinical trial involves a three step process, first, cell harvest and isolation, second, cell seeding onto the scaffold, and third, incubating the seeded graft for a period of time [4]. Previously we tried to reduce the time to assemble the TEVG by using a closed disposable system and were able to reduce the time for cell isolation and seeding without affecting TEVG patency [11,12]. However, to date, we have not studied the optimal incubation time after cell seeding onto the scaffold.…”

Effect of cell seeding on neotissue formation in a tissue engineered trachea

“…Aspirated bone marrow was then filtered through a 100-μm cell strainer (Falcon) and collected in sterile 50 mL conical tubes. The BM-MNC fraction was obtained from the filtered bone marrow aspirate with the Purecell Select System for Whole Blood MNC Enrichment (Pall Medical), following the manufacturer’s protocol as previously described [13,14]. The cell suspension was subsequently vacuum seeded onto the polymeric TETG scaffolds using a previously described vacuum seeding technique (Fig 2A) [14,15].…”

Toward a patient-specific tissue engineered vascular graft

“…We have previously characterized and determined the biological equivalency of a closed disposable filtration/elution system for BM-MNC enrichment. 33 – 35 Not only does this method accomplish BM-MNC enrichment in less than 1 h, use of multiple filters in parallel would permit the processing of greater bone marrow volumes which could be used to increase the seeded cell dose by providing a greater number of BM-MNCs in the preseeding solution. What has been unexplored to date, however, is the seeding of a scaffold with nonlinear geometry and nonconstant diameter.…”