1. A novel hydrogel based culture of endothelial cells to study the effect of Dengue
nonstructural 1 (NS1) protein in vitro
Preksha Jain, Shobha Gangodkar, Kanjaksha Ghosh ,Atanu Basu
Electron Microscopy & Pathology Group
National Institute of Virology (ICMR), Pune

3. Abstract
In vitro, three dimensional (3D) cell culture models represent a more accurate, intermediate
platform between simplified 2D culture model and complex and expensive in vivo models.
Hydrogels are water swollen cross-linked polymeric structures, which are ideal to use as 3D
culture scaffold because of their ability to mimic the in vivo physiological environment. In the
present work, we examined the susceptibility of a human sinusoidal endothelial cell line SK
HEP1 to create an in vitro model to study Dengue virus (DENV) nonstructural protein (NS1)
interactions with endothelial cells. Growth of SK HEP 1 cells in hydrogels lead to generation
of three kinds of clump morphology, small, medium and large. The SKHEP 1 cells could be
maintained for 10-11 days in 3D hydrogel environment, which is normally 2-3 days in normal
2D cultures. Exposure of DENV nonstructural protein (5 ug) to SK HEP1 cells showed
inhibitory effect on growth of clump morphology with Dengue virus 2 and 3 nonstructural
protein being more cytopathic to cells in comparison to Dengue virus 1 and 4 NS 1 protein. In
summary, this assay may prove useful in further understanding of the mechanisms by which
DENV NS1 protein adversely affects endothelial cell function. Moreover, this assay has the
potential for screening possible inhibitors of NS1 protein in therapeutic development.

4. Background
In vitro, three dimensional (3D) cell culture models represent a more accurate, intermediate
platform between simplified 2D culture model and complex and expensive in vivo models.
Hydrogels are water swollen cross-linked polymeric structures, which are ideal to use as 3D
culture scaffold because of their ability to mimic the in vivo physiological environment. In
the present work, we examined the susceptibility of a human sinusoidal endothelial cell line
SK HEP1 to create an in vitro model to study Dengue virus (DENV) nonstructural protein
(NS1) interactions with endothelial cells.

5. Material and Methods
Hydromatrix peptide cell culture scaffold (Sigma) was used for the set up of hydrogel assay.
Briefly, approximately 10,000 cells (SK HEP1) were mixed with hydrogel (0.75%) and
Dengue virus nonstructural protein 1 (5ug) of serotype 1,2, 3, 4 (Abcam) and assay was set
up according to manufacture’s instructions. Colony count was scored manually after 24, 48
and 72 hrs. under the light microscope.

6. Results
Growth of SK HEP 1 cells in hydrogels lead to generation of three kinds of clump morphology,
small, medium and large. The SK HEP1 cells could be maintained for 10-11 days in 3D hydrogel
environment, which is normally 2-3 days in normal 2D cultures. Exposure of Dengue
nonstructural protein 1 to SK HEP1 cells showed inhibitory effect on growth of clump
morphology with Dengue virus 2 and 3 NS1 protein being more cytopathic to cells in comparison
to Dengue virus 1 and 4 NS 1 protein

7. Conclusion
In summary, this assay may prove useful in further understanding of the mechanisms by
which DENV NS1 protein adversely affects endothelial cell function. Moreover, this assay
has the potential for screening possible inhibitors of NS1 protein in therapeutic development.

8. Acknowledgement
I would like to thank the Director National Institute of Virology and staff of Electron
Microscopy and Histopathology Group; Mrs. Shobha gangdodkar, Mr. R.M. Kolahpure, Ms.
Sharda Prasad, Mrs. Nitali Tadkalkar for their support.

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10. Figure Legends- Effect of dengue nonstructural protein (NS1) on endothelial cells (SK
HEP 1) in hydrogel environment. (a) Clump morphology analysis of SK HEP 1 cells
after Dengue NS1 24hrs. exposure. (b) Clump morphology analysis of SK HEP 1 cells
after Dengue NS1 48 hrs. exposure (c) SK HEP 1 in Hydrogel after 24 hrs. Control and
Dengue NS1 (1,2,3,4) exposed (d) SK HEP1 in Hydrogel after 48 hrs. Control and
Dengue NS1 (1,2,3,4) exposed