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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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Cell proliferation/motility. Some migration is noticeable as COS 7 cells shift in or out of the focal plane. Most of the original cells are seen to maintain their initial position and new cells are present (indicated by arrows). Scale bar represents 25 μm. The left image is after 4 days of incubation, and the right image is after 5 days of incubation.
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Figure 7: Cell proliferation/motility. Some migration is noticeable as COS 7 cells shift in or out of the focal plane. Most of the original cells are seen to maintain their initial position and new cells are present (indicated by arrows). Scale bar represents 25 μm. The left image is after 4 days of incubation, and the right image is after 5 days of incubation.

Mentions: An initial 3D growth study demonstrating cultivation of Chondrocytes in preassembled (as opposed to in situ assembly) Fmoc diphenylalanine hydrogels reported that limited or no net proliferation was seen after the first three days of cultivation, while measurable growth was evident after 7 days [6]. We performed additional growth studies with all of our cell strains. Over the first few days of culturing, relatively little growth was detected (none in most test samples) while cell viability remained. Viability was verified by administering calcein-AM to the hydrogel culture. Viable cells were observable after calcein-AM uptake and internal modification to fluorescent calcein. Figure 7 presents an example of cell proliferation and/or migration in an in situ assembled hydrogel plug. This comparison of identical planes within the microchamber is between 4 and 5 days of incubation, for left and right images respectively. A majority of the cells remain in a fixed position, while some migration is seen with cells shifting in and out of the focal plane. There is a positive net change in cell number density shown between the images, resulting from a net flow into the focal plane and/or growth of new cells. Additional testing should be performed to differentiate the cell density change owing to migration from that of proliferation.


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

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

Cell proliferation/motility. Some migration is noticeable as COS 7 cells shift in or out of the focal plane. Most of the original cells are seen to maintain their initial position and new cells are present (indicated by arrows). Scale bar represents 25 μm. The left image is after 4 days of incubation, and the right image is after 5 days of incubation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Cell proliferation/motility. Some migration is noticeable as COS 7 cells shift in or out of the focal plane. Most of the original cells are seen to maintain their initial position and new cells are present (indicated by arrows). Scale bar represents 25 μm. The left image is after 4 days of incubation, and the right image is after 5 days of incubation.
Mentions: An initial 3D growth study demonstrating cultivation of Chondrocytes in preassembled (as opposed to in situ assembly) Fmoc diphenylalanine hydrogels reported that limited or no net proliferation was seen after the first three days of cultivation, while measurable growth was evident after 7 days [6]. We performed additional growth studies with all of our cell strains. Over the first few days of culturing, relatively little growth was detected (none in most test samples) while cell viability remained. Viability was verified by administering calcein-AM to the hydrogel culture. Viable cells were observable after calcein-AM uptake and internal modification to fluorescent calcein. Figure 7 presents an example of cell proliferation and/or migration in an in situ assembled hydrogel plug. This comparison of identical planes within the microchamber is between 4 and 5 days of incubation, for left and right images respectively. A majority of the cells remain in a fixed position, while some migration is seen with cells shifting in and out of the focal plane. There is a positive net change in cell number density shown between the images, resulting from a net flow into the focal plane and/or growth of new cells. Additional testing should be performed to differentiate the cell density change owing to migration from that of proliferation.

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