Biotransformation of diazepam in a clinically relevant flat membrane bioreactor model using primary porcine hepatocytes.

In vitro biotransformation of drug using commercial culture medium with serum may not be the ideal culture medium for clinical application in extracorporeal bioartificial liver support (BAL) systems. In these systems, patient's blood or plasma is plumbed to primary hepatocytes within a seeded bioreactor, creating interaction between plasma and seeded hepatocytes. To address this situation, we investigated the biotransformation potential of diazepam in primary porcine hepatocytes with a flat membrane bioreactor (FMB); we used human plasma exposure and serum-free media in organotypical double gel culture model for long-term culture. We investigated diazepam clearance and all major metabolites of diazepam, such as oxazepam, temazepam, and desmethyldiazepam, in conventional single gel and organotypical sandwich models and compared them to the FMB model. Diazepam elimination was higher in double gel cultures with exposure to both SF 3 medium conditions and plasma, when compared to the single gel model in a Petri dish. It was observed that in the FMB, diazepam elimination was stable at about 3 pg/h/cell in plasma and SF 3 exposure. Oxazepam synthesis in the bioreactor was approximately one quarter less than in the Petri dish, but there were no differences between N-desmethyldiazepam and temazepam synthesis in double gel culture. In the flat membrane bioreactor, there was no decrease in the biotransformation of diazepam in plasma exposure compared with the control group. Our results suggest that this plasma exposure bioreactor may offer a useful approach in clinical use of extracorporeal BAL, as well as for drug metabolite investigation into toxicological research.

Preclinical characterization of primary porcine hepatocytes in a clinically relevant flat membrane bioreactor.

Using primary porcine hepatocytes, artificial extracorporeal liver support (AEL) is a therapy that carries out the liver functions of liver failure patients until their own organs have been regenerated or until whole organ transplantation. Significant variation exists with regard to current bioreactor designs for AEL, and they may not reflect the in vivo architecture of the liver since each individual hepatocyte has its own direct contact with blood plasma for oxygen and nutrient supply and detoxification. The present study, based on our flat membrane bioreactor (FMB), aimed at in vivo liver architecture and to meet authentic clinical levels of human plasma exposure. Since many existing preclinical AELs are based on commercial culture medium with or without nonhuman serum, they may not authentically reflect the clinical situation in human patients, and little research has been done on human plasma exposure in in vitro culture-based bioreactors. To address this situation, herein we examined liver-specific functions such as albumin secretion, urea synthesis, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), cell membrane stability by lactate dehydrogenase (LDH) test and ammonia clearance by using human plasma and serum-free medium in long-term culture of primary porcine hepatocytes to show the potential of our clinically relevant FMB. We observed that the organotypical double-gel (DG) culture is superior to conventional collagen-coated single-gel (SG) cultures. The performance of liver-specific functions by the FMB has long-term stability with intact cell morphology for up to 20 days under both plasma exposure and serum-free media. Our three focus points (long-term culture that correlates with the generation time of spontaneous regeneration, high-density culture, organotypical culture model using human plasma) may provide valuable clinical clues for AEL.

Quality control after total arterial revascularisation: multislice computer tomography cannot replace coronary angiography.

INTRODUCTION: Coronary angiography is regarded as the gold standard in evaluating graft patency, Multi-slice CT (MSCT) which enable rapid imaging of cardiac structures, including coronary arteries may be a less invasive technique. Therefore in our institution a prospective pilot study was performed combining these procedures. PATIENTS AND METHODS: Starting from July 2004 a study was done with 13 patients. They received TAR using composite left internal thoracic artery (LITA) and left radial artery (RA) as T-graft. Intra-operative angiography was performed in these patients to confirm graft patency. Follow-up control (9-21 months) was performed with exercise Ergometry and 64 slides MSCT. RESULT: Mean procedure time for intra-operative angiography was 13.7 +/- 7.3 min and mean fluoroscopy time was 6.2 +/- 4.6 min. In one patient, RA-marginal artery side to side anastomoses was stenosed and had to be revised. And in another, there was a kinking of the LITA and was corrected. At follow-up, exercise ergometry showed no signs of angina or ECG-changes in all patients. MSCT showed occluded radial artery grafts in two patients. In two other patients interpretation was difficult due to resolution reasons. In all patients the LITA graft was patent. CONCLUSION: The intra-operative graft angiography can be performed in patients undergoing TAR easily. MSCT can be used for post-operative less-invasive angiography with limitations in patients with small graft/coronary diameters and arrhythmias. However, this study shows that an interdisciplinary cooperation is a new possibility toward quality control during and after TAR.

Total arterial revascularization and concomitant aortic valve replacement.

The safety of total arterial revascularization with a left internal thoracic artery-radial artery T-graft was evaluated in patients with at least two-vessel coronary artery disease and aortic valve stenosis requiring concomitant aortic valve replacement. From June 2001 to January 2005, 18 patients underwent aortic valve replacement and total arterial revascularization, while 101 had aortic valve replacement and conventional grafting. By matching age, sex, left ventricular ejection fraction, and number of distal anastomoses, 1:2 matched groups were generated: 15 patients with a left internal thoracic-radial artery T-graft, and 30 with left internal thoracic artery and additional vein grafts. Aortic cross clamp and cardiopulmonary bypass times were similar in both groups. There were no significant differences in postoperative data between the groups. Early mortality was 0% in the T-graft group and 2% in those with conventional grafts. Follow-up ranged from 2 to 50 months. Event-free survival was 100% in the T-graft group and 90% in the conventional graft group. Total arterial grafting with a left internal thoracic-radial artery T-graft can be performed in selected patients with aortic valve stenosis requiring simultaneous aortic valve replacement.

Anastomotic obstruction after magnetic vascular coupling after minimally invasive direct coronary artery bypass grafting.

We report a 61-year-old man who required reoperation 8 months after minimally invasive direct coronary artery bypass grafting after magnetic vascular coupling due to a symptomatic subtotal obstruction at the anastomotic site. It was also determined that the patient had been noncompliant in following the prescribed postoperative antiplatelet therapy.

A multifunctional bioreactor for three-dimensional cell (co)-culture.

Investigation of cell abilities to growth, proliferation and (de)-differentiation in a three-dimensional distribution is an important issue in biotechnological research. Here, we report the development of a new bioreactor for three-dimensional cell culture, which allows for co-cultivation of various cell types with different culture conditions in spatial separation. Preliminary results of neonatal rat cardiomyocyte cultivation are shown. Isolated neonatal rat cardiomyocytes were cultured in spatial separated bioreactor compartments in recirculating medium on a biodegradable fibrin matrix for 2 weeks. Glucose, lactate, and lactate dehydrogenase (LDH), pO2, pCO2, and pH levels were monitored in the recirculated medium, daily. Morphological characterization of matrix and cells was assessed by hematoxylin and eosin staining, and MF-20 co-immunostaining with 4',6-diamidino-2-phenylindole (DAPI). Cell viability was determined by LIVE/DEAD staining before cultivation and on day 3, 7, and 14. The optimized seeding density in the matrix was 2.0 x 10(7) cells retaining cellular proportions over the cell culture period. The bioreactor allows the maintenance of physiologic culture conditions with aerobic cell metabolism (low release of lactate, LDH), a high oxygen tension (pO2-183.7 +/- 18.4 mmHg) and physiological pH values (7.4 +/- 0.02) and a constant level of pCO2 (43.1 +/- 2.9) throughout the experimental course. The cell viability was sufficient after 2 weeks with 82 +/- 6.7% living cells. No significant differences were found between spatial separated bioreactor compartments. Our novel multifunctional bioreactor allows for a three-dimensional culture of cells with spatial separation of the co-cultured cell groups. In preliminary experiments, it provided favorable conditions for the three-dimensional cultivation of cardiomyocytes.

Magnetic Vascular Port in minimally invasive direct coronary artery bypass grafting.

BACKGROUND: Minimally invasive direct coronary artery bypass grafting (MIDCAB) is a well-established operative procedure. However, it is technically demanding and is therefore somewhat underused. We evaluated the clinical and angiographic outcome of patients undergoing a MIDCAB procedure with the Ventrica Magnetic Vascular Port (MVP) system. METHODS AND RESULTS: A Ventrica MVP system was used in 10 of 11 selected MIDCAB patients. The system consists of 6 magnetic clips, with 3 clips forming a set. One magnetic clip set is positioned at the arteriotomy of the target artery and of the bypass graft using a preloaded delivery system. These ports then form an anastomosis by magnetic coupling. The mean age of the 10 patients (6 male) was 60.3+/-11.0 years. Three patients had an angiogram at the time of discharge and 8 returned for a 6-month angiogram. The total procedure time was 128.2+/-12.2 minutes. The mean anastomotic time was 199 seconds. The mean ischemic time during the anastomosis was 146+/-146 seconds. There were no in-hospital complications and no device-related adverse events. All 3 predischarge and all 8 6-month angiograms showed patent anastomoses. CONCLUSIONS: The magnetic vascular port facilitates the MIDCAB procedure significantly and reduces the ischemic time during the anastomosis. This minimally invasive procedure has the potential to be an alternative to percutaneous transluminal coronary angioplasty and stenting in proximal left anterior descending (LAD) stenosis. It may expand the acceptance of hybrid procedures in which a left internal mammary artery (LIMA)-to-LAD graft optimally supplies the anterior wall and the septum while the circumflex and right coronary artery may be treated interventionally.

Magnetic vascular coupling for distal anastomosis in coronary artery bypass grafting: a multicenter trial.

OBJECTIVE: The hand-sewn anastomosis is the "gold standard" for performing coronary artery bypass grafts. However, performing a hand-sewn anastomosis is more demanding and time-consuming when used in less invasive approaches such as small access, totally endoscopic or beating heart surgery. In conjunction with attempts to reduce the surgical trauma of coronary artery bypass grafts by using these less invasive approaches, alternative methods for constructing distal anastomoses should be explored. These data report on predischarge angiographic findings and 30-day clinical follow up of patients who have received a new distal anastomotic device. METHODS: In a multicenter trial, 32 patients (mean age: 65 +/- 9 years; 85% men) requiring multivessel coronary artery bypass surgery had 1 of the anastomoses performed using a novel anastomotic technology. The Magnetic Vascular Positioner System was used in 1 of the bypass grafts and the other bypasses were completed by conventional hand-sewn technique. The Magnetic Vascular Positioner System consists of 4 magnetic, gold-plated implants and 2 delivery devices that facilitate the creation of a functional end-to-side anastomosis. A predischarge angiogram was performed to evaluate graft patency. RESULTS: There were no device-related major adverse events. The application of the Magnetic Vascular Positioner device was successful in 32 of 41 cases (78%). Nine patients were intended for treatment but did not receive the Magnetic Vascular Positioner System. In 5 of the cases the coronary artery was too small; 1 case had a posterior wall plaque in the target artery; and 3 patients had a nonhemostatic anastomosis after coupling of the port and were subsequently converted to hand-sewn anastomoses. The median total Magnetic Vascular Positioner anastomotic time was 137 seconds with a range from 65 to 370 seconds. Overall patency rate of the Magnetic Vascular Positioner anastomosis was 93.5% versus 91.7% (P = not significant) in hand-sewn grafts. One patient (3.1%) died due to low cardiac output but had patent grafts at autopsy. One myocardial infarction (3.1%) occurred the day after a percutaneous transluminal coronary angioplasty of a hand-sewn graft. One prolonged mechanical ventilation (3.1%) was required because of pneumonia and adult respiratory distress syndrome. CONCLUSIONS: Magnetic vascular coupling in coronary surgery is safe and effective and has acceptable early patency rates. This new technique may facilitate beating heart and minimally invasive coronary artery bypass grafts.