In situ cannulation, microgrid follow-up and low-density plating provide first passage endothelial cell masscultures for in vitro lining

Abstract

A rapid and reliable harvest and culture technique was developed to provide a sufficient number of autologous endothelial cells for the confluent in vitro lining of cardiovascular prostheses. Enzymatic endothelial cell detachment was achieved by the in situ application of collagenase to short vessel segments. This harvest technique resulted in a complete lack of contaminating smooth muscle cells in all of 124 cultures from nonhuman primates and 13 cultures from human adults. The use of a microgrid technique enabled the daily in situ quantification of available endothelial cells. To assess ideal plating densities after passage the population doubling time was continuously related to the cell density. Surprisingly, a low plating density of 1.5 X 10(3) endothelial cells/cm2 achieved 43% shorter cell cycles than the usual plating density of 1.0 X 10(4) endothelial cells/cm2. Moreover, low density plating enabled mass cultures after one single cell passage, thereby reducing the cell damaging effect of trypsin. When the growth characteristics of endothelial cells from five anatomically different vessel sites were compared, the external jugular vein--which would be easily accessible and dispensable in each patient--proved to be an excellent source for endothelial cell cultures. By applying in situ administration of collagenase, low density plating and microgrid follow-up to adult human saphenous vein endothelial cells, 14,000,000 first passage endothelial cells--sufficient for the in vitro lining of long vascular prostheses--were obtained 26.2 days after harvest. (95% confidence interval:22.3 to 32.2 days).

Clinical in vitro endothelialization of femoropopliteal bypass grafts: An actuarial follow-up over three years

“…After a single passage at preconfluence from the six-well plate into two 162 cm 2 culture flasks (Costar), a microgrid technique enabled the daily in situ quantification of available endothelial cells. 15 The culture medium consisted of medium 199 (JRH Biosciences, Lenexa, Kan.), 180 J.Lg/ml preservativefree heparin (Novo Industries NS, New York, N. Y.…”

Improving the Clinical Patency of Prosthetic Vascular and Coronary Bypass Grafts: The Role of Seeding and Tissue Engineering

“…There are currently two strategies in seeding of grafts: two‐stage and single‐stage (23,39). Two‐stage seeding is the conventional and successful technique, and this is discussed below.…”

“…In two‐stage seeding, the ECs are extracted and undergo a prolonged period of cell culture in the laboratory in order to increase the cell numbers before seeding. The cells are normally cultured for 2–4 weeks (39). The main source of ECs in two‐stage seeding is a vein or occasionally an artery.…”

Imbalance between the endothelial cell‐derived contracting factors prostacyclin and angiotensin II and nitric oxide/cyclic GMP in human primary varicosis

“…Segments (∼4 cm) of the inguinal saphenous vein or umbilical vein and their endothelial cells were harvested as described previously (Zilla et al , 1990). Cells were suspended in Medium 199 containing human serum (10%) and endothelial cell growth supplement (75 mg l −1 ) and divided among three wells predated with human fibronectin (∼2 cm 2 each).…”