Optical projection tomography as a quantitative tool for analysis of cell morphology and density in 3D hydrogels

Abstract

Assessing cell morphology and function, as well as biomaterial performance in cell cultures, is one of the key challenges in cell biology and tissue engineering (TE) research. In TE, there is an urgent need for methods to image actual three-dimensional (3D) cell cultures and access the living cells. This is difficult using established optical microscopy techniques such as wide-field or confocal microscopy. To address the problem, we have developed a new protocol using Optical Projection Tomography (OPT) to extract quantitative and qualitative measurements from hydrogel cell cultures. Using our tools, we demonstrated the method by analyzing cell response in three different hydrogel formulations in 3D with 1.5 mm diameter samples of: gellan gum (GG), gelatin functionalized gellan gum (gelatin-GG), and Geltrex. We investigated cell morphology, density, distribution, and viability in 3D living cells. Our results showed the usability of the method to quantify the cellular responses to biomaterial environment. We observed that an elongated morphology of cells, thus good material response, in gelatin-GG and Geltrex hydrogels compared with basic GG. Our results show that OPT has a sensitivity to assess in real 3D cultures the differences of cellular responses to the properties of biomaterials supporting the cells.

Murine cerebral organoids develop network of functional neurons and hippocampal brain region identity

“…To assess the volume of the organoids, we measured the 3D structures by performing a 3D optical projection tomography (OPT) reconstruction using in-house build system ( Belay et al., 2021 ) ( Figure 1 C and Videos S1 and S2 ). In line with the maximum diameter measures, this analysis showed a higher organoid volume during the induction phase (0.063 ± 0.008 mm 3 ) compared to the differentiation stage (0.023 ± 0.007 mm 3 ) ( Figure 1 C).…”

“…In-house-built 3D imaging system for Optical Projection Tomography (OPT) ( Belay et al., 2021 ) was used to image and characterize the 3D structures of the organoids. Brightfield OPT was used to capture the 360-degree structure of the organoids.…”

Three‐dimensional tissue volume generation in conventional brightfield microscopy

“…The idea of 3D optical tissue slicing is not novel. It has been presented by different research groups elsewhere (Belay et al, 2021; Dragomir et al, 2008; Du et al, 2020; Fernandez‐Gonzalez et al, 2002; Liu et al, 2019; Magee et al, 2015; Sabdyusheva Litschauer et al, 2020; Sands et al, 2006; Sharpe, 2003; Zhai et al, 2020; Zhang et al, 2020). Although previous studies have presented promising methodologies, they have specific limitations.…”

“…This technology has been used for many applications during the last 20 years ranging from studying the anatomy of embryos (Sharpe, 2003) to visualizing the morphology of cells (Belay et al, 2021). Another alternative for 3D tissue volume reconstruction has been presented for whole slide imaging stacks using registration algorithms (Paknezhad et al, 2020).…”

“…Another alternative for 3D tissue volume reconstruction has been presented for whole slide imaging stacks using registration algorithms (Paknezhad et al, 2020). However, the technologies that have been presented in literature (Belay et al, 2021; Davis et al, 2019; Du et al, 2020; Liu et al, 2019; Magsam et al, 2018; Nguyen et al, 2017; Prunskaite‐Hyyrylainen, 2019; Sharpe, 2003; Vallejo Ramirez et al, 2019; Zhang et al, 2020) a/ require the sample to be fully rotated around the light source, b/ necessitate the design of a special instrument devoted for this purpose and c/ have limited application in clinical practice.…”