1. In situ cannulation, microgrid follow-up and low-density plating provide first passage endothelial cell masscultures for in vitro lining 
Peter Zilla, MD *, Roland Fasol, MD *, Uta Dudeck, MD *, Susanne Siedler, Petra Preiss, PhD *, Teddy Fischlein, MD **, Werner Müller- Glauser, PhD ‡, Gaby Baitella, PhD ‡, David Sanan, PhD ‡, John Odell, FRCS *, Bruno Reichart, MD * 
Journal of Vascular Surgery 
Volume 12, Issue 2, Pages (August 1990)
DOI: / (90)90106-K
Copyright © 1990 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

2. Fig. 1
In situ vessel cannulation for EC harvest: After careful touch-fee dissection, the vessel segment is ligated on both ends and stretched between the ligatures. After half-circumferential vessel incisions, two free-flow plastic cannulas—containing a 3-way tap—are tied in. The proximal 3-way tap is connected to an intravenous infusion line to collect the flushing solutions at its end. After a gentle pulsatile flush with 20 ml incomplete medium (serum and growth-factor free) from distally, the first two thirds of 10 ml 0.1% collagenase solution (containing 0.5% human serum albumin) are flushed through the vessel before the proximal 3-way tap is closed and the vessel is extended under gentle pressure with the remaining collagenase solution. After excision the cannulated and collagenase filled vessel is transferred to the cell laboratory in a thermos container with 300 ml Hank's balanced salt solution at 37° C.
Journal of Vascular Surgery  , DOI: ( / (90)90106-K)
Copyright © 1990 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

3. Fig. 2
Ocular microgrid (1 cm2) for the continuous quantitative follow-up of EC cultures. A, Determination of partial cell coverage ‘p’: In four arbitrarily selected areas only those of the 36 grid intersections are counted which subtend ECs. The total number of counted intersections from the four areas is then divided by 144, which is the total number of intersections from four grids. The quotient is ‘p’, representing a number smaller than one. ‘p’ equal 1 therefore stands for confluence. B, Determination of cell density: By use of an objective lens and an ocular lens with a magnification of × 10 (tubus factor 1.0), the 1 cm2 ocular rid (A-B-C-D) covers a flask area of 10 μm2. In 10 randomized areas either the whole grid (A-B-C-D) was counted if ‘p’ was <0.2 or the four corner squares (A-E-F-G) if ‘p’ was >0.2. Cells that lay only partially within the grid field were only counted at the left top margins (if ‘p’ <0.2: D-A-B; if ‘p’ >0.2: G-A-E). Since the whole grid covers 10 μm2 and one corner square covers 4 × 106 μm2 of flask area, the final number of counted cells has to be multiplied by 10 (‘p’ < 0.2) or 62.5 (‘p’ > 0.2) to provide values for ECs/cm2.
Journal of Vascular Surgery  , DOI: ( / (90)90106-K)
Copyright © 1990 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

4. Fig. 3
Correlation of cell density and population doubling time (PDT): Second passage baboon ECs (ext. jug. vein), plated at low density (1.5 × 103/cm2) [▴----▴] and high density (1.0 × 104/cm2) [●----●] are counted every 24 hours for the determination of the PDT. Shortly after low density plating ECs enter a stage of rapid proliferation during which they reach their shortest PDT of 21.9 ( ) hours at 5.0 × 103 ECs/cm2. From this cell density on the duration of doubling cycles steadily increases. High density plating shows a significantly longer PDT at 1.0 × 104 EC/cm2; thereafter the PDT curves of both groups merge at a cell density of 1.5 × 104 ECs/cm2. Since the PDT at this stage is already as long as 37.3 ( ) hours, high-density plating never reaches the short PDTs of low-density plating. Confluence is reached at 3.45 × 104 ECs/cm2 in the low-density group and at 3.39 × 104 ECs/cm2 in the high-density group. When low-density plating is applied to first passage adult human saphenous vein EC (AHSVEC) [■----■] the shortest PDT of 19.9 (16.7—23.8) hours is again reached at a cell density of 5.0 × 103 ECs/cm2. Because of the exponential increase of PDT between 8.0 × 104 ECs/cm2 and 1.0 × 105 EC/cm2, AHSVEC cultures are terminated at 8.0 × 104 ECs/cm2. In these cultures confluence is reached at 3.61 × 104 ECs/cm2.
Journal of Vascular Surgery  , DOI: ( / (90)90106-K)
Copyright © 1990 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

5. Fig. 4
Time course of EC proliferation obtained from 13 adult human saphenous vein EC cultures. After cell harvest with the in situ cannulation technique, primary cultures were grown in 9.6 cm2 wells. Fifty percent of the culture medium was replaced twice weekly, and the partial cell coverage (‘p’) of the culture well was performed every 24 hours. At ‘p’ = 0.8 primary cultures were passaged to 175 cm2 culture flasks. This stage was reached on day 10.3 (0.1 to 12.3). After low-density plating (1.7 × 103 ECs/cm2) microgrid cell counts were performed every 24 hours to determine the cell density and to calculate the total number of available cells. Again only 50% of the culture medium was replaced twice weekly. For both, primary and first passage AHSVEC, the culture medium contained 75 μg/ml endothelial cell growth supplement and 30% fetal calf serum. Cultures were terminated at a cell density of 8.0 × 104 ECs/cm2, which equals a total number of 14 million AHSVEC. This number of first passage ECs was reached on day 26.2 (22.3 to 32.2).
Journal of Vascular Surgery  , DOI: ( / (90)90106-K)
Copyright © 1990 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

6. Fig. 5
Evidence for the purity of EC cultures harvested by in situ cannulation. A, Phasecontrast microscopy and B, immunohistochemical visualization of α-actin filaments in a venous smooth muscle cell culture, serving as control (×950). C, Negative immunohistochemical staining of α-actin and D, entirely positive uptake of fluorescence labeled acetylated low density lipoprotein in adult human saphenous vein EC (×950).
Journal of Vascular Surgery  , DOI: ( / (90)90106-K)
Copyright © 1990 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions