Bruce E. Jarrell

Collagenase lot selection and purification for adipose tissue digestion

Crude Clostridial collagenase (CCC) remains the most widely used enzyme for the digestion of tissues prior to cell isolation and culture. CCC contains numerous components in addition to specific collagenases and proteases. A chronic problem associated with CCC is significant lot variability which occurs with respect to the ability of different lots of CCC to digest tissue. We have evaluated numerous commercially available samples of CCC for their ability to digest human liposuction-derived SC fat. Digestion capacity was evaluated as the ability to release endothelial cells from fat as well as the ability of isolated cells to adhere to tissue culture plastic. A significant variation in digestion efficacy between lots of collagenase was observed. We subsequently purified CCC using a partial purification method with dialysis and centrifugation as well as a complete purification, using liquid chromatography, to remove all nonspecific proteases. While partially purified collagenase retained digestion capacity, pure collagenase exhibited reduced digestion capacity. Maximum digestion was achieved with pure collagenase when trypsin was added. The use of completely purified collagenase with trypsin is advantageous where all components in the enzyme digestion mixture must be known.

Endothelial Cell Transplantation onto Polymeric Arteriovenous Grafts Evaluated Using a Canine Model

Prosthetic arteriovenous grafts (AVG) placed for hemodialysis access fail in humans due to the thrombogenicity of the flow surface and development of cellular intimal hyperplasia, particularly at the venous anastomosis. The poor patency rates of prosthetic AVG result in significant morbidity and mortality in dialysis patients. Consequently, investigators have been evaluating methods to improve the patency of prosthetic grafts by examining endothelial cell transplantation as a means of creating an antithrombogenic lining on artificial polymers. A canine model was developed to study the effects of cell transplantation of autologous, fat-derived microvessel endothelial cells (MVEC) onto the luminal surface of expanded polytetrafluoroethylene (ePTFE) grafts. Microvessel endothelial cells were isolated from falciform ligament fat, with each dog receiving its own endothelial cells. Isolated cells were subsequently placed into the lumen of the graft (4 mm by 20 cm ePTFE). The graft lumen was pressurized to 5 pounds per square inch (psi) resulting in the partial denucleation of the graft, due to the flow of buffer into the interstices of the graft, and the forced deposition of cells onto the luminal surface. Animals were maintained on aspirin and persantine during the implant phase. During the implant phase, grafts were evaluated by both duplex ultrasound and magnetic resonance angiography (MRA). At explant, gross observation of the sodded grafts revealed a glistening white flow surface with no evidence of thrombosis. Morphologic and scanning electron microscopic evaluations revealed the presence of a cellular lining on the luminal flow surface that exhibited characteristics of antithrombogenic endothelial cells. Midgraft samples were evaluated by immunocytochemistry and indicated that cells on the luminal surface react positively with antibodies to von Willebrand factor. Results from this study demonstrate that the canine model provides an excellent method of studying the effects of MVEC sodding on the thrombogenicity and hyperplastic response of prosthetic arteriovenous graft.

A porcine model for adipose tissue-derived endothelial cell transplantation.

The transplantation of endothelial cells represents a technology which has been suggested for applications ranging from improvement in function of implanted vascular devices to genetic therapy. The use of microvascular endothelial cell transplantation has seen increased use both in animal studies as well as clinical use. This report describes our techniques for the isolation and establishment of initial cultures of microvascular endothelial cells derived from porcine fat. A variety of anatomic sites within the pig were evaluated to determine the appropriateness of different sources of fat for endothelial cell isolation. The properitoneal fat was determined to be optimal due to the predominance of endothelium in this tissue and the ease of isolation of microvascular endothelium following collagenase digestion. The study of endothelial cell transplantation in the porcine model is now possible using the methods described for adipose tissue-derived micro vessel endothelial cell isolation.

Cultured and Immediately Procured Endothelial Cells: Current and Future Clinical Applications

Great progress has been made in the last several years in our ability to culture human endothelial cells. In addition, techniques to immediately procure and utilize these cells have also been developed. The purpose of this paper is to present an overview of the current and potential uses of these cells in both vascular and nonvascular conditions. It is likely that endothelial cells will be used in a variety of applications in the near future. Immediately procured and cultured cells will be used to resurface vascular prosthetic grafts. They may also be used on the surface of vessels following procedures such as balloon angioplasty or atherectomy. In addition, they may be placed upon the surface of implantable devices such as expandable stents. Through the mechanism of genetic engineering, these cells may be modified to produce proteins, which may modify thrombogenicity and perhaps decrease the rate of recurrent stenosis by influencing cellular hyperplasia. Genetically modified endothelial cells also have great potential in nonvascular disease. Their contact with circulating blood makes them an ideal cell for production of proteins to correct systemic conditions such as the insulin deficiency found in diabetes mellitus. The application of endothelial cell biology in both vascular and nonvascular science represents one of the most exciting fields of research active today.

National Transplantation Pregnancy Registry: Outcomes of Pregnancies Fathered by Male Transplant Recipients

Previous reports of male renal transplant recipients who have fathered pregnancies total 71 pregnancies. All report greater than 80% live births and none reported complications or malformations in the offspring. Only eleven pregnancies were fathered by renal transplant recipients ma intained on cyclosporine A (Sandimmune).

Tissue engineering: Vascular system

While major advances have been made in the field of clinical vascular surgery, a confounding problem remains that small diameter (< 6 mm) vascular grafts continue to exhibit poor, clinically unacceptable long term patency. The conspicuous complications which lead to this poor performance are the occurrence of intimal hyperplasia and thrombosis. The basic inherent problem with these prosthetic grafts, that is grafts constructed of synthetic polymeric materials (e.g. ePTFE and Dacron), is the relative lack of biocompatibility of these polymeric materials. Numerous solutions have been hypothesized, developed and tested toward improvement in polymeric graft function. Some of these solutions are provided in Table 1.