Automated spheroid generation drug application and efficacy screening using a deep learning classification: a feasibility study

摘要

Spheroids form spontaneously in hanging drops. ( ) 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. ( ) Representative images of spheroids of mIMCD3, HCT116, HEK293T or HEp2 cell lines forming spontaneously in 30 µl medium droplets containing 1 × 10 cells/ml of trypsinised cells after 24 h. Size marker = 50 µm. ( ) 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 × 10 cells/ml. Shown are means (horizontal line) ± s.d., n > 20. ( ) 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 × 10 cells/ml. Shown are individual survival rates (dots), means (horizontal line) ± s.d., n = 3. ( ) 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. ( ) 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.