3D Tissue Engineering

=Edite Figueiras: SPIM for 3D tissue engineering imaging=

In Tissue Engineering (TE) research field there is a need of 3D non-destructive imaging techniques capable of rendering images over time to investigate cells differentiation and proliferation; dynamics of the cell behaviors; cells interactions with each other and with the biomaterial; etc. In this project I want to answer to the questions: Can I use SPIM to image TE based in hydrogels? Can the samples be prepared inside FEP tubes? How deep I can image inside the gel without loosing resolution?

The inlfuence of the FEP tubes
I imaged TE constructs of Gellan Gum with adipose stem cells inside and outside FEP tubes. The images are less blurry when the tube is not used, but Gellan Gum also affects the images.

TE constructs of alginate with neurons
Rat primary cortical neurons derived and encapsulated in alginate hydrogel, maintained in culture for 5 days, stained with calcein AM (Invitrogen) and ﬁxed in 4% formaline.

Samples fixed in FEP tubes with 0.5 mm
'''Image parameters
 * Dimensions (um): x- 487; y - 487; z - 341
 * Objective: 10X, NA = 1
 * Light Sheet thickness: 4.77 um
 * Illumination wavelenght: 488 nm







Samples fixed in FEP tubes with 0.2 mm
'''Image parameters
 * Dimensions (um): x- 438; y - 438; z - 838
 * Objective: 10X, NA = 1
 * Light Sheet thickness: 4.52 um
 * Illumination wavelenght: 488 nm







'''Conclusions From the images obtained, it can be clearly seen that better quality images were obtained with the thinner FEP tube. In the videos of the stacks we can also clearly see that there is a degradation of the images with the increasing of the depth (this videos start from the deepest image).

'''Acknowledgements I want to thank you to my collaborators that provide me the samples I imaged during this course, mainly, Gemma Palazzolo, Janne Koivisto and Jyrki Sivula. I also want to thank to the organizers and all the Instructors of the EMBO practical course.