A platform for automated and label-free monitoring of morphological features and kinetics of spheroid fusion

Abstract

Spheroids are widely applied as building blocks for biofabrication of living tissues, where they exhibit spontaneous fusion toward an integrated structure upon contact. Tissue fusion is a fundamental biological process, but due to a lack of automated monitoring systems, the in-depth characterization of this process is still limited. Therefore, a quantitative high-throughput platform was developed to semi-automatically select doublet candidates and automatically monitor their fusion kinetics. Spheroids with varying degrees of chondrogenic maturation (days 1, 7, 14, and 21) were produced from two different cell pools, and their fusion kinetics were analyzed via the following steps: (1) by applying a novel spheroid seeding approach, the background noise was decreased due to the removal of cell debris while a sufficient number of doublets were still generated. (2) The doublet candidates were semi-automatically selected, thereby reducing the time and effort spent on manual selection. This was achieved by automatic detection of the microwells and building a random forest classifier, obtaining average accuracies, sensitivities, and precisions ranging from 95.0% to 97.4%, from 51.5% to 92.0%, and from 66.7% to 83.9%, respectively. (3) A software tool was developed to automatically extract morphological features such as the doublet area, roundness, contact length, and intersphere angle. For all data sets, the segmentation procedure obtained average sensitivities and precisions ranging from 96.8% to 98.1% and from 97.7% to 98.8%, respectively. Moreover, the average relative errors for the doublet area and contact length ranged from 1.23% to 2.26% and from 2.30% to 4.66%, respectively, while the average absolute errors for the doublet roundness and intersphere angle ranged from 0.0083 to 0.0135 and from 10.70 to 13.44°, respectively. (4) The data of both cell pools were analyzed, and an exponential model was used to extract kinetic parameters from the time-series data of the doublet roundness. For both cell pools, the technology was able to characterize the fusion rate and quality in an automated manner and allowed us to demonstrate that an increased chondrogenic maturity was linked with a decreased fusion rate. The platform is also applicable to other spheroid types, enabling an increased understanding of tissue fusion. Finally, our approach to study spheroid fusion over time will aid in the design of controlled fabrication of “assembloids” and bottom-up biofabrication of living tissues using spheroids.

Recapitulating the Drifting and Fusion of Two-Generation Spheroids on Concave Agarose Microwells

“…Spheroids undergo multiple levels of self-assembly in the process of fusion. The size of the agarose-based micro-well is 2 mm in diameter and 2 mm in depth, thus having a larger volume than the agarose micro-wells used in previous studies [33,37,38]. The larger size of the agarose micro-well makes it possible to precisely pipette the same amount of cell suspension into each well, ensuring cell number uniformity between the wells and the reproducibility of different batches.…”

“…The larger size of the agarose micro-well makes it possible to precisely pipette the same amount of cell suspension into each well, ensuring cell number uniformity between the wells and the reproducibility of different batches. The previous studies focused on the fusion of spheroids that were direct contact with each other [14,19,26,28,33,39]. However, in this work, the larger micro-well was adopted to investigate the interaction between two generations of cells for the first time, observing the fusion of the contacted spheroids and the migration behavior of the 2nd generation cells before they contacted the 1st generation cells.…”

“…Liu et al established scaffold-free airway tubes with predefined shapes by assembling individual airway organoids of different sizes using multi-Organoid patterning and fusion [29]. Those works were conducted by generating spheroids on platforms, such as hanging drops [25,30], agarose-filled micro-well plates [31], non-adhesive microplates [26], pipette tips [32] or agarose microarrays [24,33]. Then two or more spheroids were put together to achieve physical contact, and the subsequent merging or fusion process of the spheroids was monitored.…”

The influence of spheroid maturity on fusion dynamics and micro-tissue assembly in 3D tumor models

“…Kim et al used multi-cell type cell spheroids as building blocks to form 3D vascularized micro-tissue [22]. Spheroids could be placed on a non-adherent substrate, such as agarose mold [23][24][25], low-attachment plate [26], and hanging drops [27,28], and allowed to contact each other to achieve the assembling. In addition, microtissue assembly has been successfully achieved in various patterns, ranging from concave or tubular microwells to more complex toroid or honeycomb shapes [18,25,29,30].…”

Robotics-driven manufacturing of cartilaginous microtissues for the bio-assembly of skeletal implants

“…Monitoring of microtissue cultures and their morphometric quality profiles can be done non-invasively through imaging. Software is already available to segment and analyse microtissues in hydrogel microwells and floating cultures, even enabling selection of desirable microtissues and studying their fusion kinetics (13)(14)(15)(16)(17). Commercial microwell systems are typically produced from PCL or PDMS in different microwell shapes, which interact with light, creating complex backgrounds in brightfield images.…”