Murielle Rémy-Zolghadri

ISOLATION AND CULTURE OF THE THREE VASCULAR CELL TYPES FROM A SMALL VEIN BIOPSY SAMPLE

SummaryThe availability of small-diameter blood vessels remains a significant problem in vascular reconstruction. In small-diameter blood vessels, synthetic grafts resulted in low patency; the addition of endothelial cells (EC) has clearly improved this parameter, thereby proving the important contribution of the cellular component to the functionality of any construct. Because the optimal source of cells should be autologous, the adaptation of existing methods for the isolation of all the vascular cell types present in a single and small biopsy sample, thus reducing patient’s morbidity, is a first step toward future clinical applications of any newly developed tissue-engineered blood vessel. This study describes such a cell-harvesting procedure from vein biopsy samples of canine and human origin. For this purpose, we combined preexisting mechanical methods for the isolation of the three vascular cell types: EC by scraping of the endothelium using a scalpel blade, vascular smooth muscle cells (VSMC), and perivascular fibroblasts according to the explant method. Once in culture, cells rapidly grew with the high level of enrichment. The morphological, phenotypical, and functional expected criteria were maintained: EC formed cobblestone colonies, expressed the von Willebrand factor, and incorporated acetylated low-density lipoprotein (LDL); VSMC were elongated and contracted when challenged by vasoactive agents; perivascular fibroblasts formed a mechanically resistant structure. Thus, we demonstrated that an appropriate combination of preexisting harvesting methods is suitable to isolate simultaneously the vascular cell types present in a single biopsy sample. Their functional characteristics indicated that they were suitable for the cellularization of synthetic prosthesis or the reconstruction of functional multicellular autologous organs by tissue engineering.

Review: The Self-Assembly Approach for Organ Reconstruction by Tissue Engineering

One must not forget that tissue engineering was first introduced as a life saving procedure for burn patients (1). The successful engraftment of autologous epidermal sheets was the first proof of concept of the powerful technology that we know today (2–4). However this very interesting initial approach fell into some disrepute because of perceived drawbacks and limitations (5, 6). The subsequent efforts in the field followed essentially three “schools” of thought. The first approach consists of the seeding of cells into various gels, which are then reorganized, by the incorporated cells (7–14). Alternatively, a second approach is to seed cells into a scaffold where they will thrive and secrete extracellular matrix (15–17). The scaffold materials are bioresorbable over a wide range of time periods depending on their chemical structures (18–25). A third approach is different since it uses the principle of a tissue template that allows, after implantation, the ingress of cells into the appropriately organized scaffold. Thus, these grafts are acellular and must stimulate the regenerative potential of the tissue wherever they are implanted (26–31). Our group has developed a different and original method for the reconstruction of soft tissues. It takes full advantage of the various intrinsic properties of cells when appropriately cultured. This entails particular media composition and appropriate mechanical straining of these three-dimensional structures.

Signal Transduction and Procoagulant State of Human Cord Blood—Progenitor-Derived Endothelial Cells after Interleukin-1α Stimulation

Isolation of endothelial progenitors from human umbilical cord blood generated great hope in vascular tissue engineering. However, before clinical use, progenitor derived endothelial cells (PDECs) have to be compared with mature endothelial cells (ECs). The aim of this study was to explore the behavior of PDECs exposed to a proinflammatory cytokine (interleukin-1α; IL-1α) according to the mitogen-activated protein (MAP) kinase and nuclear factor (NF)-κB signal transduction pathways as well as procoagulant activity (PCA). CD34+ mononuclear cells were isolated using magnetic beads, cultured, and compared with human saphenous vein ECs (HSVECs). PDECs express endothelial markers: CD31, VE-cadherin, von Willebrand factor, KDR, and incorporate acetylated low-density lipoprotein (Dil-Ac-LDL). IL-1α similarly activates c-Jun N-terminal protein kinase (JNK) and p38 pathways in HSVECs and PDECs, whereas extracellular signal-related kinase (ERK)1/2 phosphorylation is lower in PDECs than in HSVECs. Low ERK1/2 phospho...

Recent optimization of a tissue engineered blood vessel: the LOEX experience

Abstract Creating a blood vessel by tissue engineering is one of the most demanding goals in tissue engineering. Our laboratory developed, using the self-assembly approach, the first completely biological tissue engineered blood vessel (TEBV) constituted of living human cells in the absence of any synthetic or exogenous material. The phenotypic and functional variations of smooth muscle cell (SMC) are of paramount importance in TEBV reconstruction. Thus, the phenotype and extracellular matrix (ECM) production of SMC were studied along the whole sequence of TEBV production. The functional and mechanical properties can be greatly enhanced by active cell orientation in the ECM. Accordingly, the method of preparing living tissue engineered sheets was modified to obtain an optimal alignment of SMC before rolling them into a tubular form. These results have allowed us to create a better TEBV.