Volume 13 Number 5 May 1991

MICROVESSEL DERIVED ENDOTHELIAL CELL ISOLATION, ADHERENCE, AND MONOLAYER FORMATION FOR VASCULAR GRAFTS Transplantation of endothelium onto the blood flow surface of a small-diameter vascular conduit (e.g. polymeric graft or damaged native artery) to partially reproduce the antithrombogenic lining of a naturally occurring intima is a logical concept. In spite of this logic, endothelial cell transplantation has failed to make a signilicant transition from the animal laboratory to the clinical arena. In part, this transition has not occurred because of issues associated with clinical feasibility and reproducibility as well as clinical efficacy as measured by a graft patency benefit. Issues such as the type and source of the endothelial cell, the dynamics of creating the monolayer, the durability of the subsequently created monolayer, and ultimately the long-term function of this monolayer in humans must be addressed sequentially. We have chosen to use microvessel-derived endothelial cells (MVECs) as our source of endothelial cells.’ In studies that now include over 400 human MVEC isolations, we have found that more than lo6 cells can be reproducibly isolated per gram of fat over a 45minute time period. There are many important aspects to this isolation including the devices used and the enzymes (e.g. collagenase) used for cell dissociation. The source of fat used for MVEC isolation is a critical element toward successful endothelial cell transplantation. Although adipocytes and endothelium are the predominant cell type in most adipose tissues, we have observed a significant variability in the presence of other cell types (i.e., mesothelium, fibroblasts, pericytes) in different fat types.* As a summary of these studies, we have observed that human subcutaneous fat is composed predominantly of adipocytes and MVECs, making this source our source of choice. Other fat sources, particularly omentum, have a much higher ratio of other cell types. One could concentrate efforts to completely characterize these cell types and to eliminate “unwanted cells” from the cell isolate. We have taken the approach that the in vivo performance of the subsequently created monolayer is a more important indicator of a successful cell isolation. Ultimately this is the only valid option because we do not know what an acceptable mix of cell types would be and can only judge that based upon in vivo results. A second issue relates to monolayer creation and durability. Our in vitro studies have established that endothelial cells can rapidly attach to a surface and resist detachment in high shear fields as produced by the rotating disc model3 These studies have been extended to real-time video studies that have confirmed these rapid attachment dynamics. They have also highlighted the fact that the rate limiting step in covering a surface with cells appears to be delivery of cells to the surface, not actual attachment and spreading, which occurs over a 15-minute or less period. To improve the rate-limiting step, we have conceptually

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defined the graft as a porous “filter” and used filtration to deposit the cells on the graft surface as the suspending culture medium is forced through the graft interstices.4 On expanded polytetrafluoroethylene (ePTFE) this has produced a twofold increase in attached cells when compared to gravity alone acting over a 24-hour period. Other variables that can be optimized in this process and include protein type in the suspending medium and process temperature, with 37” C being optimal. It is reasonable to ask whether cells that have attached to the graft surface are the same cells that are seen on the graft surface after exposure to flow in the animal. Nabel et al.’ and Wilson et al6 have addressed this question by genetic labeling of seeded cells and observing the marker on the cells 1 month later. We have used an alternative approach of fluorescently labeling the cells at the time of isolation with the dye, PKH26-GL (Zynaxis), which attaches to cellular lipid membranes and remains visible for at least 6 months. This method has the benefit that it requires minimal ex-vivo manipulation or culture of the cells, whereas genetic labeling requires extensive in vitro culture and cell selection. Using this technique in animals, we have observed that the cells lining the graft at 3 weeks demonstrate fluorescent intensity and labeled cell number very similar to that observed at the time of cell labeling and monolayer formation. This provides direct evidence that the initially attached cells are directly related to the subsequently developed flow surface at 3 weeks. There are many unanswered questions about MVEC isolation and monolayer formation. However, the methods that we have developed are currently reproducible and promising enough that we have tested them in laboratory animals and humans. The animal model that we have used is the carotid artery interposition graft in the dog.7 Paired 4 mm diameter, 6 cm length ePTFE end-to-end grafts are implanted with MVECs on one graft and no MVECs on the opposite graft. Animals are given aspirin and persantine for the first 4 weeks only, and patency is serially assessed by duplex ultrasonography followed by excision and histologic evaluation. MVEC treatment of the graft produces a cell-lined surface on the graft that is associated with minimal hyperplasia at both the anastomotic areas and the midgraft region. We have observed an antithrombogenic lining at 3 weeks (the earliest time for our explants), and this layer was stable for MVEC treated grafts implanted for periods as long as 12 months. It is more important to note that patency has been significantly better with MVEC treatment. In one paired study, 9/l 1 MVEC treated 4 mm grafts remained patent, whereas l/11 nontreated grafts remained patent (p

Microvessel derived endothelial cell isolation, adherence, and monolayer formation for vascular grafts.

MVECs can be isolated from animal and human fat in quantities and in a pure enough form to produce a cell-lined vascular graft. In animal studies graf...
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