## 2.2.3 Disaggregation of spheroids

Five spheroids per day were selected for each disaggregating treatment, either through accutase or trypsin/EDTA. Each spheroid was disaggregated by Accutase or 0.5x trypsin/EDTA (T/E). Then, 100 µl of disaggregating reagents was added to each spheroid for disaggregation. Then, a sufficient volume of PBS (2.9 ml) was added to make up the cell suspension up to 3 ml of each spheroid and pipetted well to form a uniform suspension. Then, 20 µl from these suspensions was mounted on a hemocytometer, and the number of cells in each spheroid was counted.

## 2.2.5 Calculating the number of cells in a spheroid from its diameter (through mathematical calculation)

To estimate the number of cells in a spheroid from its diameter, spheroids were washed with phosphate-buffered saline (PBS) and transferred from a 96-well culture plate to an 8-well plate along with one drop of PBS. Then, one side of the spheroid was mechanically dissociated with the tip of the pipette inside the 8-well plate, as shown in Figure 1(b). Then, we imaged the spheroids with confocal microscopy in DIC (differential interference contrast) mode. The mean diameter of these disaggregated cells is essential for the interconversion of spheroid parameters. These parameters include the number of cells in the spheroid, diameter of the spheroid, volume of the spheroid, cellular layer in a spheroid and estimation of dead cell and viable cell regions.

For this purpose, the diameter of 20 disaggregated cells, as mentioned in the encircled area of Figure 2, was measured through confocal software, and the mean diameter was calculated. The calculated mean diameter (d) of a single cell was approx.: 15 µm. The volume of a single cell (v) was also calculated by the formula v = 4/3\({\pi }\)r3 (where r is radius of a single cell), which was approximately equal to 1800 µm3.

The conversion of spheroid parameters was calculated by the following formulas:

From diameter to spheroid volume and from volume to number of cells (without haemocytometry)

Radius was calculated from the diameter of the spheroid (R=D/2), where R is the radius and D is the diameter.

To calculate volume of a spheroid (V); V= 4/3\({\pi }\)R3

To calculate number of cells from volume of spheroid

Number of cells = Volume of spheroid/volume of a single cell

From number of cells to spheroid diameter (using haemocytometry):

From the number of cells, the volume of spheroid is calculated by the formula:

V= single cell volume x number of cells in a spheroid

To estimate the diameter of the spheroid from its volume, the following formula was used:

D = (6 V/π)1/3

Similar equations were applied to estimate the number of cells, volume and diameter in dead cell and viable cell regions.

The diameter and volume of a single cell is essential for the interconversion of spheroid parameters. Therefore, it was calculated after disaggregation of cells from a spheroid. It should be noted that the HT-29 cell monolayer, when attached to the glass surface, shows a higher diameter (18 µm) than the diameter obtained from disaggregated spheroid cells (15 µm). The reason for this variation in diameter is that HT-29 cells attached to the glass surface may attain a flat shape, while cells after disaggregation from spheroids are rounded in shape. Therefore, the diameter of freshly disaggregated cells was considered for calculation.

## 2.2.7 Interconversion of spheroids parameters

For interconversion of spheroid parameters, the diameter and volume of a single cell is mandatory. Therefore, the HT-29 single-cell diameter was measured, which was approx. 15 µm. Then, the volume of a single cell was calculated from the diameter, which was approximately equal to 1800 µm3. The diameter of spheroids was measured through confocal microscopy, while the total cell number and viability status of each spheroid were determined through haemocytometry. Afterwards, through interconversion of spheroid parameters, the diameter, volume per cell, number of cells in dead and viable cell regions of each spheroid and radii (radii of spheroids, shell of live and core of dead cell regions) were calculated.