The morphological effect and rupturing dead trypan blue cells

Trypan blue has been the benchmark for staining dead cells to establish cell viability for a long time. The dye is excluded from membrane-intact live cells but can go through and concentrate in membrane-compromised dead cells, staining the cells a dark blue color.

In the past, there has been a general acceptance that trypan blue, without scientific support, is insufficient for cell viability below 80%. It was previously shown that trypan blue could modify the morphology of dead cells to a diffuse shape, which can result in over-estimation of viability.

In this article, an investigation of the origin of the dim and diffuse objects after trypan blue staining takes place. Using image and video acquisition techniques, the real-time transformation of cells into diffuse objects when stained with trypan blue is presented.

The same phenomenon was not noted when propidium iodide was used to stain cells. Moreover, the co-localization of trypan blue and propidium iodide is also demonstrated, confirming these diffuse objects are cells that contain nuclei.

The videos clearly indicate immediate cell rupturing post-contact with trypan blue. The formation of these diffuse objects was counted and monitored over time as cells died outside of the incubator.

It is hypothesized and demonstrated that rapid water influx may be causing the cells to rupture and dissolve. Due to the fact some dead cells seem to disappear after trypan blue staining, the final total may be under-counted, creating an over-estimation of cell viability.

This inaccuracy could influence and impede the outcomes of cellular therapies, which require precise measurements of immune cells that will be infused back into patients.

Cell viability measurement is one of the most crucial factors for cellular therapy. It is vital to measure the viability accurately for immune cell samples that will be re-introduced into patients.

Inaccurate cell viability measurement may cause poor efficacy or provoke unwanted autoimmune responses in patients undergoing therapeutic treatments.1-3 The U. S. Congress also acknowledged the great importance of cell counting and cell viability measurement standards in the 21st Century Cure Act for cell and gene therapy.

Cell counting and viability measurement assurance were noted as being a key opportunity for the development of standards in the workshop, “Synergizing Efforts in Standards Development for Cellular Therapies and Regenerative Medicine Products”, held by the U.S. Food and Drug Administration (FDA).

Under the guidance of the National Institute of Standards and Technology (NIST) and other stakeholders, numerous cell counting and cell viability measurement assurance tools were evaluated for enhancing the quality of cell therapy products.4

For well over a century, trypan blue (TB) dye has been used for cell viability measurement.5,6 Although issues, such as protein aggregation,7-9 a restricted counting time window10 and imprecise measurement when viability is less than 80%11–13 have been recorded, TB remains the standard viability dye of choice.

TB is an azo dye that possesses a molecular weight of 960 Da;6-14 it can concentrate in membrane-compromised dead cells but is expelled from membrane-impermeable live cells.15-16

Previously, it has been demonstrated that TB can produce morphological changes in dead or dying cell populations (forming dim and diffuse objects), and it was quantitatively shown that TB assays over-estimate viability when experimental samples fall below 80% viability.13

The previous study reflected on the identity of the diffuse objects in a TB-stained cell sample.13 Live (bright) and dead (dark, tight) cells are usually counted automatically using a brightfield cell counter or manually utilizing a hemacytometer.

However, the diffuse TB-positive objects are only slightly visible under a light microscope (S1 Fig) and are therefore often missed, potentially generating inaccurate viability measurements. There is typically a 10 to 15% viability difference between TB and nuclear fluorescent viability stains, with the subsequent method generating lower viability measurements.

This study investigates the formation of such dim and diffuse objects in TB-stained Jurkat cell samples in real-time videos that capture the staining process. Utilizing propidium iodide (PI), a fluorescent viability dye that stains the nuclei of membrane-compromised dead cells, it is demonstrated that these diffuse objects are PI-positive, verifying that these are dead cells.

The videos demonstrate morphological changes that take place in dead cells immediately after contact with TB. In particular, TB-positive cells expand and break apart, leading to diffuse morphology. Comparatively, PI staining neither induces morphological changes nor ruptures the cell body.

Additionally, as the cells die in an unfavorable environment, there is an increase in diffuse objects over time. The visual evidence captured in the experiments shows that TB can induce the formation of these diffuse objects and eventually lead to an over-estimation of viability, which may have considerable effects on downstream cellular therapy assays.

Materials and methods

Cell culture and reagent preparation

The Jurkat cell line (TIB-152, ATCC, Manassas, VA) was cultivated in RPMI 1640 (Gibco, Gaithersburg, MD) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (SigmaAldrich, St. Louis, MO). The Jurkat cell culture was held in an incubator at 37 ˚C with 5% CO2.

TB was acquired from BioVision (San Francisco, CA) and applied at 0.4%, 0.2%, and 0.1% working concentration. PI was purchased from Nexcelom Bioscience (Lawrence, MA) and used directly from the bottle.

Cellometer image cytometry instruments and disposable counting chamber

Cellometer image cytometry instruments (AutoT4, AutoM10, Vision 5X/10X) were utilized to for image and video capture of the morphological effects of TB and PI on dead cells.13 Cellometer AutoT4 and AutoM10 are bright field-only, automated cell counters with a color camera for imaging and video acquisition and 4 and 10X optical objectives.

Cellometer Vision are brightfield and fluorescent image cytometers with a 5X or 10X objective. They employ a monochromatic camera with two fluorescent channels: green (EX: 475 nm, EM: 527 nm) and red (EX: 540 nm, EM: 660 nm). The red channel was used to identify PI fluorescence.

The Nexcelom counting slide (CHT4-SD100) is comprised of two chambers that can each contain 20 μL. Each chamber has two small circular holes on the top, enabling the injection of cells through one and letting air escape out the other. After loading the chambers with the cell sample, the slide is fixed into the Cellometer system for image analysis and acquisition.

Initial visual observation of TB- or PI-stained Jurkat cells

A sample of Jurkat cells (10 mL) was acquired from cell culture and held at 4 ˚C for 2 days to let the cells die naturally. After 2 days, a 20-μL aliquot was retrieved and stained 1:1 with 0.4% TB or PI.

Half of the stained sample cells were pipetted into a counting chamber and imaged with the Cellometer AutoT4 and AutoM10 in brightfield. The images captured were qualitatively evaluated for cell morphology and population to distinguish between TB- and PI-stained Jurkat cells. The preliminary visual examination was performed 3 times.

Fluorescent image acquisition of TB and PI dual-stained Jurkat cells

To acquire bright field and fluorescent images of TB- and PI-stained Jurkat cells, the Cellometer Vision 10X was employed. First, a 20-μL aliquot of a cell sample kept at a temperature of 4 ˚C for 1 day was stained 1:1 with PI (20 μL) and subsequently pipetted into a counting chamber.

To establish the appropriate exposure time (3000 ms) for dead cells, it was imaged in both bright field and fluorescence. A sample stained with 0.4% TB was also imaged to scan for auto-fluorescence.

Finally, a sample (10 μL) was initially stained with PI (10 μL) and then stained with 0.4% TB (10 μL) immediately after. The dual-stained sample was then imaged under fluorescence and bright field to verify the presence of nuclei in the diffuse objects. The observation of the dual-stained Jurkat cells with TB and PI was performed twice.

Video acquisition of TB And PI staining dead or dying cells

Both AutoM10 and Cellometer AutoT4 instruments were physically opened to enable access to the stage that retains the counting slide. Jurkat cells that were stored at 4 ˚C for 1 day were pipetted into the counting chamber, and a small piece of Scotch tape was used to cover one of the two holes on the chamber.

Next, the counting chamber was placed under the objective lens for video capture. The Camtasia software suite (TechSmith, Okemos, MI) was arranged to capture the imaging screen. Subsequently, ~10 μL of 0.4% TB was pipetted onto the top of the open hole in the filled counting chamber, which facilitated slow diffusion of TB into the cell sample.

The counting slide setup is displayed in Fig 1. The slide was recorded for 1–10 minutes to demonstrate the effects of TB on dead Jurkat cells. The same process was then repeated for PI. The speed of the videos was increased using Adobe After Effect software (San Jose, CA). Video recording was performed twice for both TB and PI staining Jurkat cells.

Figure 1. Counting chamber setup for video acquisition. (a) A Nexcelom counting chamber is first filled with 20 μL of Jurkat cells. (b) A small piece of Scotch tape is then placed over the air escape hole to inhibit the flow of liquid in the chamber. (c) Next, a 10 μL aliquot of trypan blue is pipetted into the inlet hole to allow diffusion into the chamber. (d) The red square indicates the location where the interaction between Jurkat cells and trypan blue is recorded. Image Source: https://doi.org/10.1371/journal.pone.0227950.g001

Time-course analysis of naturally-dying Jurkat cell populations

A naturally-dying Jurkat cell sample was created by transferring 5 mL of cells (~4 x 106 cells/ mL) in one T25 flask and positioned into a bench-top drawer. The flask was held in the drawer at room temperature for the rest of the experiment.

A small aliquot (200 μL) of cells was taken out of the flask under sterile conditions at t = 0, 6, 12, 24, 48, 72, 96, and 168 hours, stained 1:1 with 0.4% TB (Sigma-Aldrich, St. Louis, MO) and then evaluated using the AutoT4 at n = 4.

The software was employed to establish the concentration and population percentages of the dead/dying cell population at various ranges of sizes (5–8, 8–12 and 12–30 μm). The experiment was performed three times.

Time-course analysis of naturally-dying mouse bulk splenocyte populations

A fresh mouse bulk splenocyte sample was donated by Christina A. Kuksin at the University of Massachusetts Amherst. The lysing protocol was initially conducted to lyse the red blood cells in the splenocyte sample.

Cells were spun down and resuspended in 1 mL of ACK lysing buffer (Life Technologies, Carlsbad, CA). After 5 minutes of incubation, the cells were then spun down again and resuspended in RPMI. The splenocytes were kept at room temperature and collected at 0, 33, and 50 hours.

The cells were stained 1:1 with 0.1% TB and evaluated on the Vision 5X at n = 4. Likewise, the software was used to establish the concentration and population percentages of the dead/dying cell population at various ranges of sizes (2–5, 5–9 and 9–30 μm). The experiment was performed only once due to the limited availability of mouse splenocytes.

Time-course analysis of naturally-dying PBMC populations

Frozen PBMCs were acquired from Stemcell Technologies (Vancouver, Canada). The PBMCs were defrosted and washed in accordance with manufacturer instructions. Following washing, the PBMCs were resuspended in 4 mL of RPMI media in a T25 flask and incubated overnight at 37 ˚C with 5% CO2.

Around 1 mL of the PBMCs was extracted and pipetted into a sterile T25 flask and kept at room temperature and then collected at 0, 24, 48, and 72 hours. The PBMCs were stained 1:1 with 0.2% TB and analyzed on the AutoT4 at n = 4.

Likewise, the software was employed to establish the concentration and population percentages of the dead/dying cell population at various ranges of sizes (3–9, 9–13, and 13–30 μm). The experiment was performed once.

Additionally, the AutoM10 was used to document the formation of the diffuse objects as previously described. The slide was recorded for 10 minutes to demonstrate the effects of TB on dead PBMCs. The video was sped up using Adobe After Effects.

Effects of buffer concentration on the cell rupturing phenomenon with TB

The Jurkat cells were resuspended in different concentrations of phosphate-buffered saline (PBS) to assess the effects of water influx in the hope of evaluating the potential cause of cell rupturing. One day-old Jurkat cells were gathered and split into seven 15-mL centrifuge tubes at 2 mL/ tube.

The samples were centrifuged and resuspended in 0.25, 1, 2, 4, 6, 8, or 10X concentrated PBS, as well as a control with media only. Subsequently, each sample was stained 1:1 with 0.2% TB, and images were captured directly using AutoM10.

The difference of light intensity of TBstained Jurkat cells and background (ΔIntensity = IntensityBackground − IntensityCell) were determined using ImageJ from the acquired images. The experiment was repeated by resuspending Jurkat cells in various concentrations of sucrose.

Two-day-old Jurkat cells were then gathered and separated into eight micro-centrifuge tubes at 400 μL/tube. The samples were centrifuged and re-suspended in 9.4, 18.8, 37.5, 75.0, 150.0, 300.0, 1500.0 or 3000 μM of sucrose. Next, staining of each sample was completed at a ratio of 1:1 with 0.2% TB, and images were captured immediately using AutoT4.

The light intensity of TB-stained Jurkat cells was captured using ImageJ from the acquired images. Both PBS and sucrose experiments were performed twice.

Video and image acquisition of TB effects on heat-killed Jurkat cells

Image and video acquisition on the AutoM10 was conducted for a heat-killed sample to show the differences with naturally dying cells. A fresh Jurkat cell sample was aliquoted (10 mL) into a 50-mL tube and then set on a hot plate into boiling water for 10 minutes.

Then, 1 mL of the heat-killed cells and 1 mL of fresh Jurkat cells were combined. The sample was pipetted into a counting chamber as previously detailed. Subsequently, ~10 μL of 0.4% TB was pipetted onto the open hole, and the slide was recorded for 10 minutes.

The final video was sped up once again using Adobe After Effects. Still image acquisition was conducted for the mixed Jurkat cells stained with TB for a final comparison. Cellometer Vision 10X was also employed for the capture of the mixed Jurkat cells double-stained with PI and TB. Video recording was performed twice for TB staining heat-killed Jurkat cells.

Results

Visual analysis of TB-stained Jurkat cell morphologies

The brightfield images that were captured of 2-day-old Jurkat cells using the AutoT4 and AutoM10 directly after TB staining demonstrated three distinct morphological populations (Fig 2). The cells that were bright, plump, and round were live cells not stained by TB. Cells that were blue, dark, and compact were dead, stained by TB.

Large, dimly lit and diffuse objects were possibly dead or dying cells that were sensitive to TB, as previously described.13 Jurkat cells stained with PI did not display the same morphological changes. Smaller cells with low bright field contrast were possibly dead, while round cells with thick membranes and bright centers were probability alive.

The imaging results verify the morphological effects of TB on cell samples and the lack of effects with PI. The AutoT4 at 4X magnification was used to initially capture images and videos of ruptured cells (Fig 2a).

The AutoM10 at 10X magnification was employed to boost the resolution and help distinguish more detailed features of the ruptured cells (Fig 2b). The 10X magnification demonstrated some bright material surrounding the diffuse objects, which mirrored the ruptured cell membrane.

The brightfield images captured using AutoT4 and AutoM10 demonstrated different background light intensities, which were reliant on the magnification of the system, and did not impede image analysis.

It is critical to note that the initial visual and quantification experiments used Jurkat cells (immortalized T lymphocytes) as a cell model for our experiments due to the fact these cells better mimic the cells used in cellular therapy.

Bright field images of 2-day-old Jurkat cells stained with trypan blue or propidium iodide. The cells were imaged using the Cellometer AutoT4 (top) or AutoM10 (bottom). Trypan blue-stained Jurkat cells were classified into three groups: live (white arrow), dead (black arrow), and diffuse (gray arrow). The diffuse objects at 10X magnification showed bright materials surrounding them, resembling ruptured cell membrane. Cells stained with propidium iodide did not exhibit the same diffuse morphology as trypan blue-stained cells.

Figure 2. Brightfield images of 2-day-old Jurkat cells stained with trypan blue or propidium iodide. The cells were imaged using the Cellometer AutoT4 (top) or AutoM10 (bottom). Trypan blue-stained Jurkat cells were classified into three groups: live (white arrow), dead (black arrow), and diffuse (gray arrow). The diffuse objects at 10X magnification showed bright materials surrounding them, resembling a ruptured cell membrane. Cells stained with propidium iodide did not exhibit the same diffuse morphology as trypan blue-stained cells. Image Source: https://doi.org/10.1371/journal.pone.0227950.g002

Visual proof that diffuse TB-positive objects are dead cells

To verify that dim and diffuse objects are dead cells, a test was performed to establish if these objects contain nuclear DNA.

First, it was shown that PI-stained dead cells fluoresce brightly at the set exposure time of 3000 milliseconds, and then demonstrated that TB-stained objects did not auto-fluoresce in the red channel at an equivalent exposure time, eradicating the uncertainty of fluorescent signals when cells are dual-stained with TB and PI (S2 Fig).

Finally, dual TB and PI staining demonstrated an overlap between the PI fluorescent signal and TB-positive objects, signaling that the diffuse TB-stained objects were, in fact, dead cells (Fig 3a).

Merged bright field and red fluorescence images demonstrate that PI-positive cells held a compact appearance and did not appear to display the diffuse morphology of TB-positive cells (Fig 3b), which indicates that PI does not have the same morphological effects as TB.

Bright field and fluorescent overlay images of 1-day-old Jurkat cells stained with propidium iodide with or without trypan blue. The cells were imaged using the Cellometer Vision 10X. (a) Co-localization of propidium iodide and trypan blue confirmed that dim and diffuse objects were dead cells. (b) Jurkat cells stained with propidium iodide retained cell membrane morphology.

Figure 3. Brightfield and fluorescent overlay images of 1-day-old Jurkat cells stained with propidium iodide with or without trypan blue. The cells were imaged using the Cellometer Vision 10X. (a) Co-localization of propidium iodide and trypan blue confirmed that dim and diffuse objects were dead cells. (b) Jurkat cells stained with propidium iodide retained cell membrane morphology. Image Source: https://doi.org/10.1371/journal.pone.0227950.g003

Video evidence of TB rupturing dead or dying Jurkat cells

The morphological transformation of 1-day old Jurkat cells was recorded during TB staining after it was verified that the TB-positive diffuse objects were all PI-positive. S1 Video (~30 seconds corresponding to 90 seconds in real time) demonstrated TB diffusing across the screen from bottom to top, which altered the cells to large diffuse objects (from ~8 to 26 μm) within 45 seconds after cooperating with TB.

S2 Video (~6 seconds at normal speed) demonstrated TB diffusing from top to bottom, which presented cell rupturing within ~2 seconds and disappearing toward the end. S3 Video (~30 seconds analogous to 10 minutes in real time) demonstrated TB-induced morphological changes in dead Jurkat cells at ~7 minutes.

Visually speaking, some dead cells were interpreted as dark and compact, while others were dim and diffuse. Finally, PI-staining of Jurkat cells is demonstrated in S4 Video (~30 seconds corresponding to 10 minutes in real-time), resulting in no noticeable morphological variations.

Formation of diffuse TB-stained Jurkat cell population over time

The morphological changes of TB-stained Jurkat cells were tracked over 168 hours to quantify any changes in the diffuse dead/dying cell concentration and population percentages. The time-dependent brightfield images of TB-stained Jurkat cells are displayed in Fig 4a, which exhibited the change in the number of diffuse cells over time.

The mean cell size results with standard deviation (Fig 4b) of TB-stained Jurkat cells displayed an increase in the diffuse population (12–30 μm) over the first 24 hours and progressively decreased over time (n = 4 cell samples).

Conversely, the cells that were counted (8–12 μm) demonstrated a nominal increase over time. Finally, a small size population (5–8 μm) demonstrated a considerable increase over time that depicted cell fragments and debris.

Accordingly, the TB-stained Jurkat cell concentrations increased over the168 hours (Fig 4c). Approximately 100–500 cells were counted to produce the cell size graphs.

Time-course morphological analysis of TB-stained Jurkat cells. (a) Bright field images of TB-stained Jurkat cells acquired by the AutoT4 showing diffuse objects. (b) Time-dependent size populations over 168 h, showing increase in diffuse objects. (c) Concentration changes for different size populations over time.

Figure 4. Time-course morphological analysis of TB-stained Jurkat cells. (a) Brightfield images of TB-stained Jurkat cells acquired by the AutoT4 showing diffuse objects. (b) Time-dependent size populations over 168 h, showing an increase in diffuse objects. (c) Concentration changes for different size populations over time. Image Source: https://doi.org/10.1371/journal.pone.0227950.g004

Formation of diffuse TB-stained mouse splenocyte population over time

The morphological changes of TB-stained mouse splenocytes were tracked over 50 hours to quantify any changes in the diffuse dead/dying cell concentration and population percentages. The time-dependent brightfield images of TB-stained mouse splenocytes are displayed in Fig 5a, which demonstrates the change in the number of diffuse cells over time.

The mean cell size results with standard deviation (Fig 5b) of TB-stained mouse splenocytes revealed a decrease in the diffuse population (9–30 μm) over the course of 50 hours (n = 4 cell samples).

Conversely, the typical cells counted (5–9 μm) and other fragmented cells (2–5 μm) displayed an increase over time. Accordingly, the TB-stained mouse splenocyte concentrations showed a slight increase over the 50 hours (Fig 5c). Around 100–500 cells were counted to produce the cell size graphs.

Time-course morphological analysis of TB-stained mouse splenocytes. (a) Bright field images of TB-stained mouse splenocytes acquired by the Vision 5X showing diffuse objects. (b) Time-dependent size populations over 50 h, showing increase in diffuse objects. (c) Concentration changes for different size populations over time.

Figure 5. Time-course morphological analysis of TB-stained mouse splenocytes. (a) Brightfield images of TB-stained mouse splenocytes acquired by the Vision 5X showing diffuse objects. (b) Time-dependent size populations over 50 h, showing an increase in diffuse objects. (c) Concentration changes for different size populations over time. Image Source: https://doi.org/10.1371/journal.pone.0227950.g005

Formation of diffuse TB-stained PBMC population over time

The morphological changes of TB-stained PBMCs were tracked for a period of 72 hours to measure any variations in the diffuse dead/dying cell concentration and population percentages. The time-dependent brightfield images of TB-stained PBMCs are displayed in Fig 6a, which shows the variation in the number of diffuse cells over time.

The mean cell size results with standard deviation (Fig 6b) of TB-stained PBMCs demonstrated a slight decrease in the standard and diffuse populations (9–30 μm) over a 72-hour time period (n = 4 cell samples). Conversely, the fragmented cells (3–9 μm) displayed high starting percentages and a decrease over time.

Accordingly, the TB-stained PBMC concentrations significantly increased over the 72 hours (Fig 6c). S5 Video demonstrates the morphological changes to the PBMC throughout TB staining. Around 100–200 cells were counted to generate the cell size graphs.

Time-course morphological analysis of TB-stained PBMCs. (a) Bright field images of TB-stained PBMCs acquired by the AutoT4 showing diffuse objects. (b) Time-dependent size populations over 72 h, showing increase in diffuse objects. (c) Concentration changes for different size populations over time.

Figure 6. Time-course morphological analysis of TB-stained PBMCs. (a) Brightfield images of TB-stained PBMCs acquired by the AutoT4 showing diffuse objects. (b) Time-dependent size populations over 72 h, showing an increase in diffuse objects. (c) Concentration changes for different size populations over time. Image Source: https://doi.org/10.1371/journal.pone.0227950.g006

Rupturing in the TB-based cells is a result of water influx

It was hypothesized that cell rupturing may be caused by higher osmotic pressure leading to a rapid influx of water. Various PBS concentrations had clear effects on TB staining (Fig 7a).

The use of 0.25X PBS produced increases in the number and size of ruptured cells. Similar to cells in medium, those incubated in 1X to 6X PBS had comparable diffuse morphology but with increased darkness.

The 8X and 10X PBS created precipitates, presumptively as a result of high salt concentration,17 but reduced the number and size of ruptured cells, with a considerable increase in darkness.

The PBS concentration-dependent darkness is displayed in Fig 7b, which demonstrates the reduction in light intensity as an increase in the PBS concentration occurs. These results strongly indicate that cells ruptured due to water influx during TB staining.

Similarly, when Jurkat cells were resuspended in different concentrations of sucrose, it was observed that the reduction in light intensity as the sucrose concentration increased (Fig 7c and 7d), signaling an increase in TB molecules in the dead/dying cells.

However, the light intensity reduced at the two highest sucrose concentrations. This could be a result of the density of the solution inhibiting proper mixing with TB.

Bright field images and trypan blue intensity analysis of aged Jurkat cells resuspended in different buffer concentrations. (a) Jurkat cells were resuspended in 0.25, 1, 2, 4, 6, 8, or 10X concentrated PBS, and imaged using the AutoM10. The white arrows indicated the change from diffuse to dark objects as PBS concentration increased, (b) which was also validated with ImageJ intensity measurement of the TB-stained cells. A significant precipitation formed when exposed to high salt concentrations with trypan blue. (c) Jurkat cells were resuspended in 9.4, 18.8, 37.5, 75.0, 150.0, 300.0, 1500.0, or 3000 μM of sucrose, and imaged using the AutoT4. Similarly, the bright field images showed increase in TB darkness as sucrose concentration increased, (d) which again was validated by the ImageJ intensity analysis.

Figure 7. Brightfield images and trypan blue intensity analysis of aged Jurkat cells resuspended in different buffer concentrations. (a) Jurkat cells were resuspended in 0.25, 1, 2, 4, 6, 8, or 10X concentrated PBS, and imaged using the AutoM10. The white arrows indicated the change from diffuse to dark objects as PBS concentration increased, (b) which was also validated with ImageJ intensity measurement of the TB-stained cells. Significant precipitation formed when exposed to high salt concentrations with trypan blue. (c) Jurkat cells were resuspended in 9.4, 18.8, 37.5, 75.0, 150.0, 300.0, 1500.0, or 3000 μM of sucrose, and imaged using the AutoT4. Similarly, the brightfield images showed an increase in TB darkness as sucrose concentration increased, (d) which again was validated by the ImageJ intensity analysis. Image Source: https://doi.org/10.1371/journal.pone.0227950.g007

Heat-killed dead cells present different TB staining behaviors

It was previously demonstrated that heat-killed Jurkat cells stained with TB did not express diffused morphology.13,18 Similarly, these cells demonstrated a completely different staining pattern than naturally dying cells.

In S6 Video, TB appeared to enter the dead cells and increased the darkness without rupturing the membranes (10X, ~30 seconds corresponding to 10 minutes in real-time). Cell size analysis shows that the change was less than 1 μm.

The final still images presented no expansion of TB-stained cells (Fig 8a), and PI fluorescence was emitted by dual-stained samples (Fig 8b). It is crucial to note that a mixture of live and heat-killed Jurkat cells was utilized to demonstrate morphological distinction in contrast to naturally dying cells when stained with trypan blue.

Bright field and fluorescent images of a 1:1 mixture of fresh and heat-killed Jurkat cells stained with trypan blue with or without propidium iodide. The cells were imaged using both AutoM10 and Vision 10X. (a) Unlike naturally dying cells, no membrane rupturing was observed, and heat-killed Jurkat cell morphology remained tight and dark when stained with trypan blue. (b) Co-localization of trypan blue and propidium iodide in heat-killed Jurkat cells.

Figure 8. Brightfield and fluorescent images of a 1:1 mixture of fresh and heat-killed Jurkat cells stained with trypan blue with or without propidium iodide. The cells were imaged using both AutoM10 and Vision 10X. (a) Unlike naturally dying cells, no membrane rupturing was observed, and heat-killed Jurkat cell morphology remained tight and dark when stained with trypan blue. (b) Co-localization of trypan blue and propidium iodide in heat-killed Jurkat cells. Image Source: https://doi.org/10.1371/journal.pone.0227950.g008

Discussion

Cellular therapies are key components in cancer treatments due to their efficacy and the 2017 FDA approval of two chimeric antigen receptor (CAR) T cell therapies.19 CAR T cell therapy necessitates gene editing of T cells from patients, expanding the culture, and infusing the final products back into the patients. Accurate cell counting is crucial to ensure the appropriate dosages are administered.

Crucially, cell products must have adequate viability to reduce the risk of an autoimmune response,1,2 where the FDA advises viability greater than 70% for cellular therapy products.20

It was hypothesized that transferring a significant amount of nonviable cells to the patients may pose a serious risk and provoke side effects such as cytokine release syndrome.21 Since 2012, two articles related to cell counting issues with TB staining have been published.

The first demonstrated that bright field counting with TB could overcount live peripheral blood mononuclear cells.22 The second publication showed that TB can cause morphological changes in dead or dying cells quantitatively, transforming them to dim and diffuse shapes for Jurkat cells and primary mouse splenocytes.13 The origin and identity of these objects have been left unanswered.

In this study, visual evidence of their formation was put forward, along with a potential explanation of why they form in the presence of TB. Biologists generally stain cells 1:1 with TB at concentrations ranging from 0.05% to 1%,23 which may result in inconsistencies in recognition of dead or dying cells, as we previously reported.13

Stained cells are seen under brightfield when the diffused objects have already formed and/or disappeared; thus they are not to be included in the dead cell count. Normally, diffuse objects were difficult to observe in microscopy images, leading to under-counting dead cells and over-estimating viability.

Conversely, the optical components in Cellometer instruments had the capacity to clearly image the dim and diffuse objects at different trypan blue concentrations and instruments. It was possible to set up the cell counting chamber and enable TB to diffuse in, so the interaction between cells and dye could be observed and recorded.

The videos clearly demonstrate that a number of cells stained with TB ruptured immediately and created the so-called diffuse objects (S1 and S2 Videos), while PI (S4 Video) did not provoke the same morphological changes (S3 Fig).

In Video 1–3, cells ruptured over various time frames; where some ruptured immediately after contacting TB, some in less than 20 seconds, and others ruptured more than 5 minutes later. This could be caused by the TB diffusion rate in the counting chamber.

PI staining demonstrates that the diffuse objects contained DNA, confirming that they were cells. The live-cell appearance was not influenced by TB or PI. Thus both methods generated comparable live cell counts as previously reported.13

The diffuse objects were quantified over time to show that as cells become unhealthy, they become more brittle, which enabled the TB to induce the formation of more diffuse cells. The Jurkat cells were first observed at high viability, and over the first 24 hours, the diffuse objects increased from 30–50%, which related to the images.

However, although the diffuse TB-stained cell concentration increased, the percentages did not continue to rise, which could be a result of the lack of whole intact cells in the samples readily affected by TB.

Conversely, the initial condition of the primary mouse splenocytes was already demonstrating large amounts of diffuse objects potentially caused by the preparation procedure and viability. Finally, primary human PBMCs first demonstrated a large number of fragmented cells and debris, which may necessitate more washing steps.

It was possible to observe the formation of large diffuse objects (S5 Video) over time. It is critical to note that Jurkat cells, primary mouse splenocytes, and PBMCs were chosen as the representation of immune cells typically used in the discovery phase of cell therapy.

Cell rupturing can be primarily attributed to the rapid water influx of water. Evaluation of whether osmotic pressure played a key role in cell rupturing in the presence of TB by resuspending cells in hypertonic, isotonic, and hypotonic buffers was conducted.

It is hypothesized that binding with TB swiftly increased the number of negatively charged residues on cytoplasmic proteins,16,24 which enticed positively charged ions such as sodium, and this produced high water influx due to osmotic pressure that ruptured the already fragile cell membrane and cytoskeleton.3,25–27

It was observed that an increase in darkness in dead/dying Jurkat cells as PBS concentration increased, which signaled an improvement in TB staining as a result of high salt content in the buffer. Additionally, the experiment was carried out using various concentrations of sucrose, which is an alternative method of changing the osmotic pressure of the buffer solution.

The results again demonstrated increased darkness in TB-stained Jurkat cells as sucrose concentration increased. However, the effects were not as impressive as with a salt solution, which signaled that the rupturing may be influenced more by the ionic strength of the media or buffer.

Interestingly, the heat-killed cells did not rupture by TB (S6 Video), which may be a result of the denaturing of membrane proteins when subjected to boiling, resulting in cell membrane hardening that can resist the influx of TB and water.28,29

Therefore, to reproduce cell conditions that can form diffuse objects after TB staining, the cells were allowed to die in an unfavorable environment. It was observed that the diffuse objects generally form at lower cell viabilities for immune cells.

Conversely, no similar morphological changes for typical cancer cell cultures were observed at high viabilities. It is of key importance that biologists select the most appropriate cell counting method for the intended application, especially for cellular therapy products such as CAR T, where cell number and viability significantly affect treatment efficacy.

Cells that rupture when first subjected to TB staining are hard to see under bright field imaging and can be under-counted, leading to an over-estimation of cell viability. It is recommended that fluorescent nuclear staining methods can help estimate cell viability more accurately.13,18,22,30–32

Future work will necessitate the development of a method to validate cell viability measurement, as well as determining different cell populations relating to the dying process to better improve the characterization of cell fitness and death.33

Supporting information

S1 Video. Bright field video captured with AutoT4 (4X, ~30-s time lapse corresponding to 90 s in real-time) of staining Jurkat cells with trypan blue. The Jurkat cell in the red circle immediately became diffuse after interacting with trypan blue. Numerous dead Jurkat cells were stained with trypan blue and transformed into diffuse objects. (MP4)

S2 Video. Bright field video captured with AutoM10 (10X, ~8 s in real-time) of staining Jurkat cells with trypan blue. The resolution was improved at 10X magnification. The Jurkat cells in the red circle immediately ruptured after contact with trypan blue. Many other cells clearly developed large and diffuse morphology. (MP4)

S3 Video. Bright field video captured with AutoM10 (10X, ~30-s time lapse corresponding to 10 min in real-time) of staining Jurkat cells with trypan blue. The Jurkat cells in the red circle again showed staining and morphological transformation after contact with trypan blue. The cells clearly expanded into large and diffuse shapes. (MP4)

S4 Video. Bright field video captured with AutoM10 (10X, ~30-s time lapse corresponding to 10 min in real-time) of Jurkat cells staining with propidium iodide. Unlike trypan blue, the video showed no morphological changes to cells staining with propidium iodide within 10 min shown in the red circle. (MP4)

S5 Video. Bright field video captured with AutoM10 (10X, ~25-s time lapse corresponding to 2 min in real-time) of staining PBMCs with trypan blue. The PBMCs in the red circle immediately ruptured after contact with trypan blue. (MP4)

S6 Video. Bright field video captured with AutoM10 (10X, ~30-s time lapse corresponding to 10 min in real-time) of staining heat-killed Jurkat cells with trypan blue. The red circle in the video indicates dead heat-killed Jurkat cells stained with trypan blue without rupturing the cell membrane. (MP4)

S1 Fig. Digitally captured brightfield image from a light microscope using the 10X objective. The zoomed image shows three populations: bright, round, and plump (white arrow, live-cell); blue, dark, and tight (black arrow, dead cell); and large, dim, and diffuse (gray arrow, ruptured dead cell). (TIF)

S2 Fig. Bright field and fluorescent images of 1-day-old Jurkat cells stained with trypan blue or propidium iodide and imaged using the Vision 10X. (a) Dead Jurkat cells stained with propidium iodide exhibited bright red fluorescence. (b) Dead Jurkat cells stained with trypan blue showed no background signal. (TIF)

S3 Fig. Time-course brightfield images cropped from the videos. The progression of the images shows the morphological changes to the cells staining with TB or PI. (TIF)

S1 Data. Measurement and analysis of morphological changes for TB-stained Jurkat cells. (XLSX)

S2 Data. Measurement and analysis of morphological changes for TB-stained mouse splenocytes. (XLSX)

S3 Data. Measurement and analysis of morphological changes for TB-stained human PBMCs. (XLSX)

S4 Data. Measurement and analysis of the light intensity of TB-stained Jurkat cells in various concentrations of PBS. (XLSX)

S5 Data. Measurement and analysis of the light intensity of TB-stained Jurkat cells in various concentrations of sucrose. (XLSX)

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