HomeNovelA new feature for monitoring the enzymatic harvesting process of adherent cell cultures based on lensless imaging
A new feature for monitoring the enzymatic harvesting process of adherent cell cultures based on lensless imaging
December 23, 2022
A lensless imaging device (LFI).19mounted inside a standard cell culture incubator (37 °C, 5% CO2 and 95% humidity, Thermo Fisher Scientific) with a 3D printed stage for the 6-well plate holder (Fig. 1), was used for continuous monitoring of the cell harvesting process. As the acquisition speed might be a limiting factor due to the heating of the image sensor, the temperature response was monitored for a high acquisition speed, as described in Supplementary Fig. S1.
Human periosteum-derived mesenchymal stem cells (hPDCs) were isolated from periosteal biopsies of seven different donors, as described by Roberts et al..20. The procedures were approved by the Ethics Committee for Human Medical Research (KU Leuven) and the patient informed consent forms were obtained. The hPDC donors were expanded (5700 cells/cm2) at 37°C, 5% CO2 and 95% RH in Dulbecco’s GlutaMAX™ Modified Eagle High Glucose Medium (DMEM; Life Technologies, UK) containing 1 × 10-3 m sodium pyruvate and supplemented with 10% irradiated fetal bovine serum (HyClone FBS; Thermo Scientific, USA) and 1% antibiotic-antifungal (100 units/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg /ml of amphotericin B; Invitrogen). The medium was changed every 3-4 days. At all passages, cells were harvested with TrypLE™ Express 1× (Life Technologies, UK). In this study, all experiments and methods involving these cells were performed in accordance with relevant guidelines and regulations.
A collection assay involving different donors, cell densities, and collection solutions was performed. Seven different hPDC donors were seeded in 6-well plates (5700 cells/cm2, 5 wells per donor) in 3 ml of growth medium (DMEM-C), which was completely refreshed after 3 days. In order to study the effect of culture time (related to cell density and matrix formation) and the dilution of the enzyme solution on the shedding rate of each donor, cells were cultured for 3, 5 and 7 days and harvested enzymatically with a standard (day 3, 5 and 7) and diluted TrypLE Express solution (5x in PBS, day 5 and 7). The following protocol was used for each well: (i) Growth medium was aspirated and cells were washed 1× with phosphate-buffered saline (PBS, 1 mL). (ii) The LFI was calibrated to the cells (in PBS) and a time-lapse experiment was started with an interval of 20 s. (iii) PBS was aspirated and the “dry” well placed under the LFI. Reference images were taken before TrypLE was added. (iv) Between two acquisitions, 1 mL of TrypLE solution was added to the well plate (opened) and the incubator was closed. The collection process was monitored for 20 minutes and all images were acquired without plate coverage.
Feature engineering and image reconstruction
LFI phase and intensity images were reconstructed, equivalent to conventional phase-contrast and bright-field microscopy, respectively19. Different reconstruction parameters, such as the reconstruction method [3L or iterative phase recovery (IPR)], iteration count, IPR threshold (80-120), and focus level were varied for the intensity image to identify a new feature for measuring cell detachment. Based on the visual inspection, the following reconstruction parameters were chosen: temperature (37°C), clipping (no), phase rotation (auto). More specifically, the phase image was reconstructed using the 3L method (number of iterations: 5) and the intensity image with the IPR method (IPR threshold: 90, number of iterations: 10). For each dataset, the reconstruction depth was determined manually on cells attached around the image center. The raw image has been cropped with the following pixel parameters: [836:2236, 1198:2898].
With “man” and “auto” referring to the manual and automated extraction of the percentage of detached cell regions, respectively. For each culture time, absolute percent errors were represented as mean ± std.
For all conditions, the optimal time point to inhibit the reaction was determined by applying a fixed threshold of 92.5%. This threshold was confirmed by visual inspection of the detached cell cultures at the respective times of inhibition. Additionally, supplementary videos (4-7) were constructed to visualize the response to detachment and highlight the optimal time point for inhibition. In case the threshold was not reached within the 20 min period, the final percentage of detached cellular regions was indicated.