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.
Division of Thoracic and Cardiovascular Surgery, Hannover Medical School, Carl-Neuberg-Str. 1, Hannover 30625, Germany. lichtenberg@thg.mh-hannover.de
Artur
A
Lichtenberg
Goekhan
G
Dumlu
Thorsten
T
Walles
Michael
M
Maringka
Stefanie
S
Ringes-Lichtenberg
Arjang
A
Ruhparwar
Heike
H
Mertsching
Axel
A
Haverich
D000818:Animals; D000831:Animals, Newborn; D019149:Bioreactors; D002245:Carbon Dioxide / Q000032:analysis; D002452:Cell Count; D018929:Cell Culture Techniques / Q000295:instrumentation*; D002454:Cell Differentiation; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured / Q000166:cytology / Q000378:metabolism; D018920:Coculture Techniques / Q000295:instrumentation*; D003470:Culture Media; D017077:Culture Media, Conditioned / Q000737:chemistry; D004734:Energy Metabolism; D004867:Equipment Design; D006863:Hydrogen-Ion Concentration; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D010100:Oxygen / Q000032:analysis; D010313:Partial Pressure; D011673:Pulsatile Flow; D051381:Rats; D017208:Rats, Wistar; D023822:Tissue Engineering / Q000295:instrumentation*
D003470:Culture Media; D017077:Culture Media, Conditioned; D002245:Carbon Dioxide; D010100:Oxygen