Adult human endothelial cell enzymatic harvesting

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Adult human endothelial cell enzymatic harvesting John B. Sharefkin, M.D., Harold E. Van Wart, Ph.D., David F. Cruess, Ph.D., Robert A. Albus, M.D., Elliot M. Levine, Ph.D. Journal of Vascular Surgery Volume 4, Issue 6, Pages 567-577 (December 1986) DOI: 10.1016/0741-5214(86)90171-0 Copyright © 1986 Society for Vascular Surgery and North American Chapter, International Society for Cardiovascular Surgery Terms and Conditions

Fig. 1 Scanning electron micrographs of human saphenous veins perfusion-fixed at their approximate in situ length and diameter after being transported to the laboratory in the same manner as veins used by us for enzymatic endothelial cell (EC) harvest. Most scanning micrographs showed significant EC loss after vein dissection (A). Only four fields showed well-preserved EC layers (B) suitable for plotting counts to estimate in situ EC density (C); these four samples had a mean EC density of 1.3 ± 0.2 (SD) × 105 EC/cm2. (Original magnification × 1000.) Journal of Vascular Surgery 1986 4, 567-577DOI: (10.1016/0741-5214(86)90171-0) Copyright © 1986 Society for Vascular Surgery and North American Chapter, International Society for Cardiovascular Surgery Terms and Conditions

Fig. 2 Eleven aliquots of harvested ECs suspended in complete medium were allocated into 10 replicate microwell cultures and into one microcentrifuge tube for immediate nuclear counting. Total initial suspension volume (TISV) of approximately 1.2 ml was measured for each harvest in microliters to within 2% for calculations. Wells used for culture were surrounded by a buffer zone of wells filled with calcium- and magnesium-free phosphate-buffered saline solution (dark shading) to prevent evaporative losses. Each day's well count was used to calculate the number of cells the harvest would have yielded from a culture that plated the entire TISV at the same density as the 10 microwells. Journal of Vascular Surgery 1986 4, 567-577DOI: (10.1016/0741-5214(86)90171-0) Copyright © 1986 Society for Vascular Surgery and North American Chapter, International Society for Cardiovascular Surgery Terms and Conditions

Fig. 3 A, Light micrograph of first passage culture 13 days after cell harvest with crude collagenase. B and C, Immunofluorescent stain for factor VII—related antigen (B) and fluorescent stain for uptake of acetylated low-density lipoprotein (C) of fifth passage culture 34 days after harvest with partially purified enzyme. Journal of Vascular Surgery 1986 4, 567-577DOI: (10.1016/0741-5214(86)90171-0) Copyright © 1986 Society for Vascular Surgery and North American Chapter, International Society for Cardiovascular Surgery Terms and Conditions

Fig. 4 Growth of primary endothelial cell (EC) cultures in a trial of crude collagenase, basement membrane lysis activity (BMLA)-equivalent partially purified collagenase, and sham harvest solution with no enzyme. The difference between the optical count of ovoid endothelial nuclei (day 0) and plated count 24 hours later is the estimated plating efficiency of harvested cells. Horizontal dotted line shows the number of cells that would have been counted had 100% of the estimated ECs present in situ been harvested with 100% plating efficiency. The mean count of plated cells, which was lowest on day 3, was chosen as the best estimate of the number of harvested ECs capable of survival and subsequent logarithmic growth on the fibronectin-coated dishes (inset). Data points for crude (dark squares) and partially purified collagenase (circles) showed no significant difference on any day (p ⩾ 0.11 all days) but sham harvests with no enzyme (diamonds) showed significantly fewer cells on all days. Journal of Vascular Surgery 1986 4, 567-577DOI: (10.1016/0741-5214(86)90171-0) Copyright © 1986 Society for Vascular Surgery and North American Chapter, International Society for Cardiovascular Surgery Terms and Conditions

Fig. 5 Degradation of human fibronectin by harvesting enzymes was measured by enzymelinked immunosorbent assay after exposure of fibronectin-coated microwells to enzyme-containing solutions for 15 minutes at 37° C. Increasing y axis values connote increased loss of activity. Dark circles: reference standard of purified crystalline trypsin in medium 199. Dark squares: medium 199 with 0.5% BSA and crude CLS II collagenase. Open circles: medium 199 with 0.5% BSA and partially purified collagenase derived from the same lot of crude material. Arrows connect BMLA-equivalent concentrations of crude and pure enzyme preparations. Data points are means of eight replicate wells; error bars show 1 standard deviation. Journal of Vascular Surgery 1986 4, 567-577DOI: (10.1016/0741-5214(86)90171-0) Copyright © 1986 Society for Vascular Surgery and North American Chapter, International Society for Cardiovascular Surgery Terms and Conditions