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Analyzing Cell-Scaffold Interaction through Unsupervised 3D Nuclei Segmentation
size around 2−3 µm mostly likely are fragment, and should factors can directly impact cell adhesion, proliferation
be clipped to prevent further analysis from becoming and migration. A good balance between the diffusion
skewed by such outliers. Under 95% confidence, the of nutrients and removal of waste within the scaffold
average nuclei size on day 1 is about 9.22 µm on PCL-10-D construct can lead to ideal cell proliferation and migration.
and 9.18 µm on PCL-20-D scaffolds. This corresponds to The above analysis method provides an intuitive tool
the reported cell diameter of A459 which is about 10.59 µm to optimize scaffold design for specific cell types and
from transmission electron microscopy images . Besides, develop appropriate cell culture protocols.
[33]
no obvious size difference was identified under varied
surface morphology during cell adhesion on day 1. 5. Ongoing research issues and future
On day 3, the difference of the average nuclei size perspectives
was less than 4% when culturing on the PCL-10-D and
PCL-20-D scaffolds. It is worth mentioning that more With the aid of the unsupervised ML method AD-GAN,
nuclei with the size of 10 µm were observed on the PCL- we have successfully segmented nuclei and performed
20-D scaffold, and more nuclei with the size of 9 µm were cell-scaffold interaction analysis using CLSM images
observed on the PCL-10-D scaffold. The slightly larger from scaffold-based cell culture. Nevertheless, some
nuclei size found on PCL-20-D scaffold may be induced ongoing issues need to be further researched.
by rougher surface. Overall, this quick screening method 5.1. CLSM image quality
would facilitate scaffold design modification for better
cell culture performance. Of course, further studies and The effective laser penetration in scaffolds is the key to
more data are expected for detailed analysis. obtaining high-quality CLSM images. To a large extent,
the quality of collected CLSM images is determined
4.2. NIH-3T3 cell proliferation analysis by the level of the scaffolds’ transparency and optical
CLSM images from culturing NIH-3T3 cells on PCL uniform. The fabricated scaffolds’ porosity is usually
scaffold on days 3 and 6 as shown in (Figure 13A around 80 – 90% with interconnected pores, while
and B) were collected to study cell proliferation. most of biopolymer materials used for fibrous scaffold
Building on the segmentation results, the corresponding fabrication such as PCL, are nontransparent. In addition,
heat maps with nuclei density distribution are shown in most of the scaffolds collected from the cell culture
(Figure 13C and D), respectively. Cells exhibited different process have closely packed cell clusters. Thus, the
proliferation and migration characteristics in the porous CLSM technology can only visualize scaffold regions
network with the pore size of 100 µm. On day 3, NIH-3T3 within a smaller depth and subsurface which are not
nuclei had proliferated along fibers and clustered in some obstructed by fibers or cell clusters. Cells in deeper pores
pores. On day 6, the pores were fully filled with nuclei and of the scaffolds or below fibers are not visible. This may
there were no clear migration directions as in (Figure 13D), lower the effectiveness of nuclei segmentation and cell-
since NIH-3T3 cells used neighboring cells as support to scaffold interaction analysis. A possible solution is to
cross pores. The number of nuclei under varied size is develop transparent biopolymer scaffold materials for
plotted in Figure 14. Although NIH-3T3 cells were of better CLSM visualization capability.
irregular size and difficult to count , the majority of nuclei In addition, confocal microscopes have anisotropic
[34]
were found ellipsoid with the size ranging 9-10 µm and the spatial sampling frequency, which has particularly low
number of nuclei doubled from day 3 to day 6. resolution along Z-axis. One common workaround
Fibrous scaffolds can be designed in terms of size, is to apply a simple trilinear interpolation for spatial
morphology, surface roughness and complexity. These normalization to ensure the same resolution of each axis.

A B C D

Figure 13. NIH-3T3 cell proliferation analysis on poly-E-caprolactone scaffold with pore size of 100 µm. (A and B) confocal laser scanning
microscopy images of cell culture on days 3 and 6. (C and D) Heatmap of cell distribution in (A) and (B).

178 International Journal of Bioprinting (2022)–Volume 8, Issue 1