Macroporous microcarriers provide a large amount of surface area and space for growth of animal cells in the interior of beads. It has been observed that the cell distribution in the interior of microcarriers is uneven. In some cases, the pores appear to be unoccupied. Question thus arises as to whether all the pores are open to the surface of the beads from which cells enter and proliferate. To examine the accessibility and geometrical features of the pores confocal laser scanning microscopy (CLSM) was used to optically section the beads. Fluorescein isothiocyanate (FITC) was used to stain the microcarriers and dialkyl indocarbocyanines (DiIC 18)to stain the 3T3 CRE BaG 2 cells for time lapse observation of cell behavior in the interior. Individual bead was optically sectioned at a 3um step size. Three dimensional image was reconstructed by combining the sections fo r examination of the openness as well as the connectivity of the pores. To further evaluate the geometry of the pores, the solid structure of the beads was removed from the reconstructed three dimensional images to allow for direct visualization of the p ores. A wide variety of geometry was observed, some were interconnected while others segregated. In general the surface of the pore was relatively smooth and should be suitable for cell movement within the pores. However, sometime sharp bends on the inter nal surface were observed and the ability of cells to cross such barrier is dubious. Through time lapse optical sectioning cell division in the interior was observed. In general, cells moved little after initial attachment. The results suggest that the initial cell distribution on microcarriers is critical for optimal performance of the cultivation. This confocal microscopic technique for direct evaluation of pore structure of macroporous beads is noninvasive and requires minimal sample preparation. It can easily be adapted for in situ observation of cell behaviors. It may also find application for s tructural assessment for many biomedical materials.
Confocal Images of Microcarriers
 | Bancel, S. and Hu W-S. Topographical imaging of macroporous microcarriers. (1995) K. Funatsu et al (eds.), Aminal Cell Technology: Basic & Applied Aspects, Vol. 8, 89-94. |
| Bancel, S., and Hu, W.-S.(1996) Confocal laser scanning microscopy examination of cell distribution in macroporous microcarrieris. Biotechnol. Progress., 12, 398-402. |
| Bancel, S., Hu, W.-S. (1996) Topographical imaging of macroporous microcarriers using laser scanning confocal microscopy. J. Ferm. Bioeng., 81, 437-444. |
| Hu, W-S., Meier, J. and Wang, D.I.C. (1985) A mechanistic analysis of the inoculum requirement for the cultivation of mammalian cells on microcarriers. Biotechnol. Bioeng. 27, 585-595. |
| Hu, W-S. and Wang, D.I.C. (1985) Serial propagation of mammalian cells on microcarriers. Biotechnol. Bioeng. 27, 1466-1476. |
| Frame, K.K. and Hu, W-S. (1985) Oxygen uptake of mammalian cells in microcarrier culture: Response to changes of glucose concentration. Biotechnol. Lett., 7, 147-152. |
| Himes, V.B. and Hu, W-S. (1987) Attachment and growth of mammalian cells on microcarriers with different ion exchange capacities. Biotechnol. Bioeng. 29, 1155-1163 |
| Tao, T-Y., Ji, G-Y. and Hu, W-S. (1987) Human fibroblastic cells attach to controlled-charge and gelatin-coated microcarriers at different rates. J. Biotechnol. 6, 9-12. |
| Hu, W-S. and Wang, D.I.C. (1987) Selection of microcarrier diameter for the cultivation of mammalian cells on microcarriers. Biotechnol. Bioeng. 30, 548-555 |
| Tao, T-Y., Ji, G-Y. and Hu, W-S. (1988) Serial propagation of mammalian cells on gelatin-coated microcarriers. Biotechnol. Bioeng. 32, 1037-1052 |
| Smiley, A.L., Hu, W-S., and Wang, D.I.C. (1989) Production of human immune interferon by recombinant mammalian cells cultivated on microcarriers. Biotechnol. Bioeng. 33, 1182-9 |
| Nikolai, T.J., Peshwa, M.V., Goetghebeur, S. and Hu, W-S. (1991) Improved microscopic observation of mammalian cells on microcarriers by fluorescent staining. Cytotechnology, 5, 141-146. |
| Nikolai, T.J. and Hu, W-S. (1992) Cultivation of mammalian cells on macroporous microcarriers. Enzyme Microb. Technol., 14, 203-208. |
| Lim, H-S., Han, B-K., Kim, J-H., Peshwa, M.V. and Hu, W-S., (1992) Spatial distribution of mammalian cells grown on macroporous microcarriers with improved attachment kinetics, Biotechnol. Progr., 8, 486-493. |
| Kim, J.-H., Lim, H.-S., Han, B.-K., Peshwa, M.V., and Hu, W.-S., (1992) Characterization of cell growth and improvement of attachment kinetics on macroporous microcarriers, In: Animal Cell Technology: Basic & Applied Aspects, Ed. H. Murakam i, S. Shirahata and H. Tachibana, Kluwer Pub. pp. 77-80, Proceedings, Japanese Association of Animal Cell Culture Technology, annual meeting, Fukuoka, Japan. |
| Giard, D.J., Hu, W-S., Wang, D.I.C., Detachment of anchorage-dependent cells from microcarriers - by treatment with proteolytic enzyme e.g. trypsin pronase collagenase or proteinase-K . Patent Assignee: M.I.T.; #WO 8601531; Date: March 13, 1986. |
Viral vectors have become an invaluable tool for gene transfer in the recent years. Although many viruses have important, unique characteristics, the adenovirus is one of the best studied DNA viruses and has gained popularity as possible treatment for a number of illnesses. Additionally, its strong promoters enable recombinant proteins to be manufactured in large quantities in mammalian cells. When a foreign gene is inserted concurrently with a gene for GFP, the detection of infected cells is made possible by non-invasive fluorescent microscopy.
A human kidney epithelial cell line (293) was established that contains a segment of the adenovirus DNA that is required for replication. Genes for recombinant proteins can be inserted in place of these segments, and the replication-defective adenovirus can be manufactured in 293 cells. This adenovirus is beneficial for gene therapy, since the virus cannot replicate in vivo.
Experiments were performed to study GFP-Adenovirus infection in various 293 cell cultures. The 293 cells were grown on microcarriers, microspheres, and adapted for growth as aggregates in suspension. Differences in the morphology and cellular interaction of these cultures can be observed. Studies aimed to determine if these variations have an impact on virus infection. It was found that the cells grown in microsphere cultures produced the most virus particles.
The number of virus particles was determined by a novel titration
procedure. Serial dilutions of the virus were used to infect
HepG2 cells
in which the virus cannot replicate. A fluorescent plate reader was used
to determine the amount of fluorescence in each dilution. A standard
virus solution with a known virus concentration was compared to unknown
samples, and the number of virus particles was calculated.
293 cells on Sephadex microspheres
Last updated: May 6, 1998
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