wild type hela  (ATCC)

Journal: Regenerative Therapy

Article Title: Development of a digital analysis system for a novel 3D culture-based colony formation to detect malignantly transformed cells in human cell-based therapeutic products

doi: 10.1016/j.reth.2025.101052

Figure Lengend Snippet: Transformed HeLa-green fluorescent protein (GFP) cells exhibit anchorage-independent proliferation and colony formation in a 3D culture environment with 0.03 % LA717, unlike surrounding MSCs. Representative whole-well and magnified views at 0, 24, 72, 120, 240, and 360 h. MSCs (20,000 cells/well) were co-cultured with a single HeLa-GFP cell in 200 μL of medium supplemented with 0.03 % LA717 in a low-adhesion 96-well plate. All cells were pre-labeled with CellVue Claret (CVC; pseudo-colored magenta). Bright-field (BF), GFP, CVC, and merged images are shown. Yellow arrows at 0, 24, 72, and 120 h indicate the HeLa-GFP cell/colony in both the GFP and CVC panels; later time points (240 and 360 h) are not arrowed for clarity. Under these conditions, MSCs remained dispersed and did not exhibit anchorage-independent proliferation, whereas the single HeLa-GFP cell proliferated and formed a distinct colony by 240–360 h. A stitched movie of the complete 360-h time-lapse (1-h intervals) is provided as the Supplementary Movie. Images were acquired using a CellVoyager CQ1 (Yokogawa Electric). The inner diameter of each well was 6.4 mm.

Article Snippet: A human cervical cancer cell line, wild-type HeLa (CCL-2, Lot 61647128), was obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Transformation Assay, Cell Culture, Labeling

Journal: Regenerative Therapy

Article Title: Development of a digital analysis system for a novel 3D culture-based colony formation to detect malignantly transformed cells in human cell-based therapeutic products

doi: 10.1016/j.reth.2025.101052

Figure Lengend Snippet: The colony-forming efficiency (CFE) of HeLa-GFP cells co-cultured with MSCs in LA717-supplemented medium under various culture conditions. (a) The CFE of HeLa-GFP cells was evaluated under four different concentrations of LA717 (0.03 %, 0.04 %, 0.05 %, and 0.06 %) in 200 μL of culture medium per well. HeLa-GFP cells (0.5 cells/well on average) were co-cultured with 20,000 MSCs per well in a 96-well plate ( n = 240 wells per condition) for 3 weeks without any medium changes or additions. CFE was calculated based on the number of wells containing colonies derived from HeLa-GFP cells, as determined by image analysis. Bar graphs represent the mean and standard deviation (SD) values from three independent experiments, and the colored lines show the individual results from each experiment. (b) The CFE of HeLa-GFP cells was evaluated under varying cell input amounts (λ = 0.125, 0.25, 0.5, and 1.0) in 200 μL of medium containing 0.03 % LA717 and 20,000 MSCs per well. HeLa-GFP cells were seeded into 840, 420, 240, and 120 wells, respectively, and cultured for 3 weeks. The CFE was calculated from the number of wells containing colonies detected by image analysis. Bars represent the mean and SD values of three independent experiments, and colored lines indicate the individual results. No colony formation was observed under control conditions (λ = 0; i.e., MSCs only, 60 wells). Detailed numerical data for each condition in (a) and (b) are provided in , respectively. The equation used to calculate CFE in (a) and (b) is described in , respectively. LA, LA717.

Article Snippet: A human cervical cancer cell line, wild-type HeLa (CCL-2, Lot 61647128), was obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Cell Culture, Derivative Assay, Standard Deviation, Control

Journal: Regenerative Therapy

Article Title: Development of a digital analysis system for a novel 3D culture-based colony formation to detect malignantly transformed cells in human cell-based therapeutic products

doi: 10.1016/j.reth.2025.101052

Figure Lengend Snippet: Fluorescence-based detection of colonies formed by single HeLa cells co-cultured with MSCs in LA717-supplemented medium. Representative images of colonies formed by a single HeLa-GFP or wild-type HeLa cell co-cultured with 20,000 MSCs per well for 3 weeks in 0.03 % LA717-supplemented medium (200 μL/well). Fluorescent staining markedly enhanced the accuracy of colony detection in image analysis. Images were acquired using the CellVoyager CQ1 confocal imaging cytometer (Yokogawa Electric). (a) A colony derived from a single HeLa-GFP cell. Live staining was performed by adding Hoechst 33342 (nucleus), MitoTracker Deep Red (mitochondria), or LysoTracker Red (lysosomes) to the culture medium and incubating the cells for a defined period. The GFP signal confirmed that the colony originated from HeLa-GFP cells, which was also clearly visualized with each dye. (b) A colony derived from a single wild-type HeLa cell. Live staining with Hoechst 33342 and Calcein-AM revealed strong fluorescence signals, indicating colony viability. (c) (Left) A wild-type live HeLa colony stained with Hoechst 33342 and LysoTracker Red. (Right) The same colony was imaged 14 days after fixation with 1 % paraformaldehyde (PFA). Fluorescence signals from Hoechst and LysoTracker staining were retained, indicating that this dye combination is compatible with fixation and long-term storage, allowing flexible scheduling of image acquisition and analysis. The inner diameter of each well was 6.4 mm.

Article Snippet: A human cervical cancer cell line, wild-type HeLa (CCL-2, Lot 61647128), was obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Fluorescence, Cell Culture, Staining, Imaging, Cytometry, Derivative Assay

Journal: Regenerative Therapy

Article Title: Development of a digital analysis system for a novel 3D culture-based colony formation to detect malignantly transformed cells in human cell-based therapeutic products

doi: 10.1016/j.reth.2025.101052

Figure Lengend Snippet: Detection sensitivity assessment of the D-LACF assay using MSCs spiked with HeLa-GFP cells. (a) Schematic overview of the sample preparation and culture process. Ten HeLa-GFP cells were isolated using a cell sorter and spiked into a suspension of 10 million MSCs to prepare a test sample containing 0.0001 % HeLa-GFP cells. The mixture was then aliquoted into 300 wells (60 wells × 5 plates) at 20,000 cells per well for D-LACF assay evaluation. (b) Summary of the number of GFP-positive colonies detected per run. Left: spiked condition (MSCs + HeLa-GFP), five independent runs. Right: MSC-only negative controls, three independent runs (all zero). (c) Representative plate maps from Test-1 showing all five 96-well plates; yellow boxes mark wells in which GFP-positive colonies were detected (confirmed by fluorescence and bright-field review). This figure was created using BioRender.com .

Article Snippet: A human cervical cancer cell line, wild-type HeLa (CCL-2, Lot 61647128), was obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Sample Prep, Isolation, Suspension, Fluorescence