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Self-assembling Fmoc dipeptide hydrogel for in situ 3D cell culturing.

Liebmann T, Rydholm S, Akpe V, Brismar H - BMC Biotechnol. (2007)

Bottom Line: Degradation improved the removal of hydrogel from the microstructures, permitting reuse of the analysis platforms.Self-assembling diphenylalanine derivative hydrogel provided a method to dramatically reduce the typical difficulties of microculture formation.Effective generation of patterned 3D cultures will lead to improved cell study results by better modeling in vivo growth environments and increasing efficiency and specificity of cell studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell Physics, Department of Applied Physics, Royal Institute of Technology, S-106 91 Stockholm, Sweden. liebmann@kth.se

ABSTRACT

Background: Conventional cell culture studies have been performed on 2D surfaces, resulting in flat, extended cell growth. More relevant studies are desired to better mimic 3D in vivo tissue growth. Such realistic environments should be the aim of any cell growth study, requiring new methods for culturing cells in vitro. Cell biology is also tending toward miniaturization for increased efficiency and specificity. This paper discusses the application of a self-assembling peptide-derived hydrogel for use as a 3D cell culture scaffold at the microscale.

Results: Phenylalanine derivative hydrogel formation was seen to occur in multiple dispersion media. Cells were immobilized in situ within microchambers designed for cell analysis. Use of the highly biocompatible hydrogel components and simplistic procedures significantly reduced the cytotoxic effects seen with alternate 3D culture materials and microstructure loading methods. Cells were easily immobilized, sustained and removed from microchambers. Differences in growth morphology were seen in the cultured cells, owing to the 3-dimentional character of the gel structure. Degradation improved the removal of hydrogel from the microstructures, permitting reuse of the analysis platforms.

Conclusion: Self-assembling diphenylalanine derivative hydrogel provided a method to dramatically reduce the typical difficulties of microculture formation. Effective generation of patterned 3D cultures will lead to improved cell study results by better modeling in vivo growth environments and increasing efficiency and specificity of cell studies. Use of simplified growth scaffolds such as peptide-derived hydrogel should be seen as highly advantageous and will likely become more commonplace in cell culture methodology.

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Related in: MedlinePlus

Microchip platform and Perfusion System. Left image is the microchip platform designed for a confocal microscope stage. Right image is the tubing configuration for continuous perfusion that was performed via a syringe pump and the tubing configuration presented here. Holes in cover-plate (right) coincide with inlet and outlet holes in the microchannels.
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Figure 10: Microchip platform and Perfusion System. Left image is the microchip platform designed for a confocal microscope stage. Right image is the tubing configuration for continuous perfusion that was performed via a syringe pump and the tubing configuration presented here. Holes in cover-plate (right) coincide with inlet and outlet holes in the microchannels.

Mentions: Mechanical reversing was performed by applying sheer forces to a stable gel. Large samples were destabilized by vortex or pipette mixing. Environmental pH changes were also used to reverse assembled gel stability. NaOH (0.5M) was added to elevate the matrix solution above pH 9. Enzyme reversing was performed with proteinase K (Sigma-Aldrich). Reversing in microstructures was performed by actively applying a flow of reversing solution: 0.05 mg/ml proteinase K, 1.0 wt% SDS, pH between 9 and 10, and temperature control at 37°C. Microchambers were all flanked by parallel channels (Figure 4) where the enzyme reversing solution was perfused. Precision pumps were fitted to drive the solution from a syringe, through flexible tubing, into the inlets of the parallel microchannels, and out through the outlets. The microchip stage and perfusion tubing setup is shown in Figure 10.


Self-assembling Fmoc dipeptide hydrogel for in situ 3D cell culturing.

Liebmann T, Rydholm S, Akpe V, Brismar H - BMC Biotechnol. (2007)

Microchip platform and Perfusion System. Left image is the microchip platform designed for a confocal microscope stage. Right image is the tubing configuration for continuous perfusion that was performed via a syringe pump and the tubing configuration presented here. Holes in cover-plate (right) coincide with inlet and outlet holes in the microchannels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2235856&req=5

Figure 10: Microchip platform and Perfusion System. Left image is the microchip platform designed for a confocal microscope stage. Right image is the tubing configuration for continuous perfusion that was performed via a syringe pump and the tubing configuration presented here. Holes in cover-plate (right) coincide with inlet and outlet holes in the microchannels.
Mentions: Mechanical reversing was performed by applying sheer forces to a stable gel. Large samples were destabilized by vortex or pipette mixing. Environmental pH changes were also used to reverse assembled gel stability. NaOH (0.5M) was added to elevate the matrix solution above pH 9. Enzyme reversing was performed with proteinase K (Sigma-Aldrich). Reversing in microstructures was performed by actively applying a flow of reversing solution: 0.05 mg/ml proteinase K, 1.0 wt% SDS, pH between 9 and 10, and temperature control at 37°C. Microchambers were all flanked by parallel channels (Figure 4) where the enzyme reversing solution was perfused. Precision pumps were fitted to drive the solution from a syringe, through flexible tubing, into the inlets of the parallel microchannels, and out through the outlets. The microchip stage and perfusion tubing setup is shown in Figure 10.

Bottom Line: Degradation improved the removal of hydrogel from the microstructures, permitting reuse of the analysis platforms.Self-assembling diphenylalanine derivative hydrogel provided a method to dramatically reduce the typical difficulties of microculture formation.Effective generation of patterned 3D cultures will lead to improved cell study results by better modeling in vivo growth environments and increasing efficiency and specificity of cell studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell Physics, Department of Applied Physics, Royal Institute of Technology, S-106 91 Stockholm, Sweden. liebmann@kth.se

ABSTRACT

Background: Conventional cell culture studies have been performed on 2D surfaces, resulting in flat, extended cell growth. More relevant studies are desired to better mimic 3D in vivo tissue growth. Such realistic environments should be the aim of any cell growth study, requiring new methods for culturing cells in vitro. Cell biology is also tending toward miniaturization for increased efficiency and specificity. This paper discusses the application of a self-assembling peptide-derived hydrogel for use as a 3D cell culture scaffold at the microscale.

Results: Phenylalanine derivative hydrogel formation was seen to occur in multiple dispersion media. Cells were immobilized in situ within microchambers designed for cell analysis. Use of the highly biocompatible hydrogel components and simplistic procedures significantly reduced the cytotoxic effects seen with alternate 3D culture materials and microstructure loading methods. Cells were easily immobilized, sustained and removed from microchambers. Differences in growth morphology were seen in the cultured cells, owing to the 3-dimentional character of the gel structure. Degradation improved the removal of hydrogel from the microstructures, permitting reuse of the analysis platforms.

Conclusion: Self-assembling diphenylalanine derivative hydrogel provided a method to dramatically reduce the typical difficulties of microculture formation. Effective generation of patterned 3D cultures will lead to improved cell study results by better modeling in vivo growth environments and increasing efficiency and specificity of cell studies. Use of simplified growth scaffolds such as peptide-derived hydrogel should be seen as highly advantageous and will likely become more commonplace in cell culture methodology.

Show MeSH
Related in: MedlinePlus