Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2020 Jul 6;10:11071. doi: 10.1038/s41598-020-67960-0

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Spheroids form spontaneously in hanging drops. (A) Schematic representation of the spheroid seeding tool used throughout this work. Spheroids in hanging drops or cytotoxic drugs are seeded onto an inverted cell culture plate at various volumes using a peristaltic pump and a high precision x–y–z robot arm. A CCD camera monitors the spheroid quality and saves the images to a database. (1) Reservoir for cell suspension, (2) Peristaltic pump, (3) Nozzle for DOD droplet generation, (4) Droplet of homogenous shape and volume, (5) CCD camera, (6) Database, (7) Detection of shape and conformational alterations. (B) Representative images of spheroids of mIMCD3, HCT116, HEK293T or HEp2 cell lines forming spontaneously in 30 µl medium droplets containing 1 × 10⁶ cells/ml of trypsinised cells after 24 h. Size marker = 50 µm. (C) The volume of the spheroids consisting of mIMCD3 cells increases logarithmically depending on the droplet size/cell number. The volume was calculated measuring pixels of the captured images and is therefore set to arbitrary units [a.u.]. Cell concentration = 1 × 10⁶ cells/ml. Shown are means (horizontal line) ± s.d., n > 20. (D) Cell viability of mIMCD3 cells inside the spheroid after 48 h of incubation in spheroids of 30, 40 and 50 µl of volume. Cell viability was assessed by flow cytometry using Annexin-V counterstain. Cell concentration = 1 × 10⁶ cells/ml. Shown are individual survival rates (dots), means (horizontal line) ± s.d., n = 3. (E) Evaporation time of hanging drops (DMEM containing 10% FCS and 1% Pen/Strep) in a non-humid environment. Droplet sizes larger than 30 µl plateau at 8,500 s = 140 min. Shown are individual times (dots), means (horizontal line) ± s.d., n > 10. (F) The formation of hanging drops depends on an inversion of the carrier plate that holds the droplets. The larger the droplets are, the higher the probability that the droplets spontaneously trickle away during this process. Shown are percentages of spontaneously lost droplets on a plate bearing 80 droplets in three different experiments (dots), means (horizontal line) ± s.d., n = 3.