Poster K19 - Cell Biology Techniques for Microscopic Visualization of 2D and 3D Biological Systems

Thursday, October 24, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Co-Author(s)

EM

Elsa Mazari-Arrighi

Post-Doctorate Researcher
Laboratoire Photonique, Numérique et Nanosciences, United States
AB

Adeline Boyreau

Engineer
Laboratoire Photonique, Numérique et Nanosciences, United States

Primary Investigator(s)

PN

Pierre Nassoy

Research Director
Laboratoire Photonique Numérique et Nanosciences, United States

Introduction: Three-dimensional culture systems have been gaining traction due to their ability to more accurately recapitulate bodily tissues and functions, increasing overall physiological relevance. Because of this, there has become a higher demand for the visualization and characterization techniques used in two-dimensional culture to be optimized and translated for use within three-dimensional culture systems. Immunostaining procedures are an elementary but powerful technology because of the technique’s ability to reliably mark key parts of the tissues for visualization. Additionally, gene editing methods are being employed not only for regenerative applications in medicine but also to increase fluorescent imaging of cells in more intricate model systems. In this work, we seek to use several staining markers often used in the visualization and characterization of cell types used in vascular engineering and optimize it for use in 3D culture. We also attempt to use lentivirus driven transduction of induced pluripotent stem cells within a 3D culture system to examine how the 3D culture system may help increase the transduction of the hard-to-transfect iPSCs.

Materials and

Methods: Smooth muscle cells (SMC), Human dermal microvascular endothelial cells (HDMEC) and Human dermal lymphatic endothelial cells (HDLEC) were obtained from a cell line and cultured to reach the appropriate confluency. They were then harvested using the appropriate volume of trypsin and seeded into an 8 well chamber slide. 4% paraformaldehyde was used to fix the cells and a solution of FBS, Triton X-100 and Tween-20 was used to simultaneously permeabilize and block the cells. The antibodies marking for the protein ZO-1 was applied to both endothelial cell types. KI-67 was also stained for at two different concentrations for the HDLECs. 3D vesseloid structures were bio-fabricated using a pre-established technique and the same process was completed but with longer incubation times. They were stained for ZO-1 as well. Both the 2D and 3D samples were imaged using a spinning disk fluorescent microscope.
Lentiviruses were produced via standard virus fabrication techniques. Plasmid DNA coding for the virus’ components were introduced into human embryonic kidney (HEK) cells which produced the viruses. The viruses were then collected and concentrated. 3D culture of induced pluripotent stem cells (iPSCs) was bio-fabricated using a pre-established technique. A 3D culture of MCF-10 cells which have been known to be able to be successfully transduced was produced and used as a negative control. The produced viruses were introduced into the encapsulated cell system at a concentration proportionate to cell count. Once the transduction was completed, imaging for fluorescence was done on days 2 and 5 using an inverted microscope.

Results, Conclusions, and Discussions: Immunostaining for KI-67 in HDLECs were able to successfully show which cells had the capacity for continued proliferation and which did not. As this was done with two different concentrations, we were able to optimize the concentration for 3D structures, both saving resources and avoiding antibody clumping and difficulty of diffusion throughout the 3D model. ZO-1 staining of HDLECs and HDMECs in both 2D and 3D showed an inverse relationship between cell type in culture type, and presence of ZO-1. In 2D culture, ZO-1 presence was mainly in the cell membrane for HDLECs, reliably staining the tight junctions between cells. In HDMECs within 2D culture, there is ZO-1 presence in the cell membrane with the tight junctions but there’s also a continued ZO-1 presence within the cytoplasm. The inverse is true for these same cells in vesseloid 3D culture. For HDLECs, the ZO-1 presence is mainly in the cytoplasm while in HDMECs there is some cytoplasmic presence but the ZO-1 staining is largely in the tight junctions within the membrane. This reveals how the bio-fabrication process affects protein expression and localization within these cells and has larger implications of how this protein that is characteristic of cell-cell interactions may function in more physiologically relevant 3D models.
The transduction of encapsulated MCF-10s only succeeded for cells where the alginate shell surrounding it was sheared open. This reveals that the alginate shell, while increasing cell-matrix and cell-cell interactions, had prohibited the virus from contacting the cells. This idea was supported by the results of the experimental iPSC group where there was minimal capsule shearing and no transduction. Due to alginate pore sizes being on the lower nanoscale, this may be an issue of the mesh size. Following this experiment, there is motivation to examine partial dissolution of the alginate shell to increase pore size as well as complete dissolution of the alginate shell to fully rely on the cell-cell interactions of the 3D structure. These approaches examine pore size based restrictions and following these results, we can begin looking into possible charge based restrictions between the alginate shell and the lentiviral vectors.

Acknowledgements (Optional): Iacocca International Internship Program (IIIP)
Laboratoire Photonique, Numérique et Nanosciences (LP2N)
Lehigh University
BioImaging OptoFluidics (BioF)
Centre National de la Recherche Scientifique (CNRS)
University of Bordeaux