ABSTRACT
The near-haploid human cell line HAP1 recently became a popular subject for CRISPR/Cas9 editing, since only one allele requires modification. Through the gene-editing service at Horizon Discovery, there are at present more than 7500 edited cell lines available and the number continuously increases. The haploid nature of HAP1 is unstable as cultures become diploid with time. Here, we demonstrated some fundamental differences between haploid and diploid HAP1 cells, hence underlining the need for taking control over ploidy status in HAP1 cultures prior to phenotyping. Consequently, we optimized a procedure to determine the ploidy of HAP1 by flow cytometry in order to obtain diploid cultures and avoid ploidy status as an interfering variable in experiments. Furthermore, in order to facilitate this quality control, we validated a size-based cell sorting procedure to obtain the diploid culture more rapidly. Hence, we provide here two streamlined protocols for quality controlling the ploidy of HAP1 cells and document their validity and necessity.
Live-cell phase holographic imaging analysis of haploid and diploid HAP1 cells show basic and specific ploidy-dependent differences. Ploidy-verified low-passage HAP1 haploids (2015 batch of C631, 1h) and their higher-passage diploid versions (1d) were seeded on laminin and monitored in a HoloMonitor M4 for 72 h with image acquisition every 15 min (A–E). (A) 2D representation of 3D holographic images at selected time points. The color bar indicates optical thickness or cell height information (z-values) for these 2D projected 3D images ranging from 0 µm (black) to 7.5 µm (yellow). Scale bar, 100 µm. (B) Morphological cell properties measured based on holographic images at 24 h (cell volume) or 48 h (cell area and optical thickness) post seeding. (C) Cell growth based on confluency measure of cell-covered area, relative to t=0. (D) Migration speed of random single-cell migration in culture measured from 24 to 48 h post seeding from two independent wells of haploid 1h (n=38) and diploid 1d (n=40) HAP1 cells. (E) Single-cell trajectory rose plots of the same cells as in D. For all bar charts, * P<0.05 and ** P<0.005 as determined with t-test (unpaired, two-tailed with Welch's correction) and error bars represent s.e.m. (only upper part shown).
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MATERIALS AND METHODS
Live-cell holographic imaging with HoloMonitor M4
Phase holographic imaging of live unlabeled cells was performed with the digital phase holographic imager HoloMonitor M4, as previously (Aksnes et al., 2018, Zhang et al., 2020). For experiments in Fig. 3, ploidy-verified haploid (C631, 2015, 1h) and diploid (C631, 2015, 1d) HAP1 CTRL cells were seeded in µ-dishes 35 mm high (ibidi 81156) precoated with laminin (11 µg/ml, 2 h). 50,000 cells were seeded per plate in 3 ml cell culture medium. The cells were incubated for 20 min at RT during initial cell attachment, before HoloLids were placed on each dish ensuring optimized imaging. Cells were imaged every 15 min with at least two different fields of view per dish. The holographic images were analyzed with HStudio software. Single cells were detected using the Auto-Otsu setting for background and minimum object size was set to 25. Based on the 3D holographic information, several cell morphological parameters were quantitatively measured. For the two independent experimental replicates in Fig. 4C and D, cells were seeded on laminin-coated wells in a µ-plate 24-well black (ibidi 82406). For experiments in Fig. 4C, ploidy-verified haploid (C631, 2015, 1h, internal passage 5/6) and diploid (C631, 2015, 1d_sort, internal passage 18/19) HAP1 control cells were used, and for experiments in Fig. 4D, ploidy-verified haploid (C631, 2019, 1.10h, passage 9/10) and diploid (C631, 2019, 1.10d_sort, passage 17/18) HAP1 control cells were used. Imaging and analysis was performed as above, except three fields from each of three different wells were imaged per cell line.
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