Enumeration of human embryonic stem cell (hESC) numbers through single cell digestion can be time consuming especially in high-throughput or multi-factorial analysis containing 50+ samples. We have developed a reproducible, cost-effective method of counting hESCs in clumps circumventing the need to manually dissociate each sample to single cells. The method is based on the DNA binding capacity of propidium iodide (PI) and subsequent fluorescent signal detection. Standard curves generated for cell numbers versus PI fluorescence as single cells or clumps showed an almost identical relationship in the lines of best fit. The reproducibility of the assay was first demonstrated by seeding hESC clumps at specific cell densities ranging 0.05–2 × 105 cells/well and then secondly by using the assay to count cell numbers after different growth conditions. Validation tests showed that consistent seeding densities are important in maintaining undifferentiated hESC culture and that the assay can be used to estimate relative cell numbers and growth curves with high accuracy.
Human embryonic stem cells (hESCs) have the potential to differentiate into many cell types, making them ideal candidates for therapeutic applications. The earliest methods of hESC culture were labor intensive, time consuming, and did not lend themselves to scale-up. The evolution of hESC culture techniques has seen a move from manual passage of hESC colony clumps to enzymatic digestion and routine plating of clumps or single cells (1,2,3,4). In order to produce the number of cells required for downstream differentiation experiments and subsequent therapeutic applications (on the order of 109 cells), culture methods are being developed that permit rapid cell expansion in an almost completely automated process (5,6). These automated cell culture systems generally use split ratios rather than absolute cell number in sub-culturing protocols. However, the cell density of seeding, as well as the clump size, is critically important for maintaining both undifferentiated growth and controlled differentiation of hESCs, with both sparsely-and too-densely–seeded hESCs having a propensity for slow growth or increased differentiation (7,8,9). Subculturing of cells as clumps then generates the issue of how to accurately determine seeding density based on the overall cell number. Treating a small sample of clumps with a dissociating agent such as trypsin or cell dissociation buffer followed by cell counting on a hemocytometer is feasible for a small number of samples. However, in higher-throughput analysis of hESCs, upwards of 50 individual culture conditions are examined, making manual dissociation and subsequent cell counting both time-consuming and labor-intensive. Here, we demonstrate a method of counting total cell numbers in a 96-well plate format based on the DNA binding capacity of propidium iodide (PI) that avoids dissociating cell clumps. PI intercalates almost indiscriminately between DNA strands and once bound to DNA, its fluorescence is increased 20–30-fold. This method is reliable and cheap, allows for a rapid estimation of total cell numbers per well for many samples at once, and removes operator variability in cell counting. This cell-counting method will be useful for large scale automated culture of multiple samples (5,6,10) where specific, expensive imaging technology (11) is not easily applicable.Materials and Methods Tissue culture
All reagents were from Invitrogen (Carlsbad, CA, USA) unless otherwise stated. hESC lines MEL1 and MEL2 (Chemicon, Millipore, Billerica, MA, USA) were cultured on mouse embryonic fibroblasts (MEFs, 14,000/cm2) in DMEM-F12 with L-glut supplemented by 20% knockout serum replacement (KOSR), 1 × non-essential amino acids and 90 µM β-mercaptoethanol. Cells were passaged in clumps using 200 U/mL collagenase IV. Differentiation controls were used in all experiments consisting of MEL1 or MEL2 grown in the media described above without feeders or maintenance factors.Preparation of single cells for standard curve
hESCs were harvested as clumps as described above and washed twice with PBS (without Ca2+ or Mg2+) before addition of TrypLE Express to dissociate cells. Cells were gently aspirated to dissociate. Cell counts were performed using a standard hemocytometer and cells resuspended in PBS at a concentration of 2 × 105 cells/150 µL. Cells were transferred to a black-walled 96-well plate (Greiner Bio-One, Monroe, NC, USA) from 0–2 × 105 cells/well and final volumes adjusted to 150 µL/well.Preparation of cell clumps for standard curve
hESC colonies were washed twice in warm PBS (without Ca2+ or Mg2+) before the addition of dispase (1 mg/mL; Sigma-Aldrich, St. Louis, MO, USA). Cells were incubated for 7 min and colonies broken into clumps using gentle pipetting. Suspensions were gently inverted several times to evenly distribute clumps immediately before dispensing 2 equal volumes from the center of the tube into separate tubes. One sample was treated with TrypLE Express to dissociate clumps into single cells and a cell count performed that represented cell numbers both in the single cell and clump suspensions. Equal clump size was determined using microscopy. Next, both the single cell and clump suspensions were centrifuged at 200× g for 3 min. Both samples were resuspended in PBS at 2 × 105 cells/150 µL. Both samples, having been derived from the same original suspension, were assumed to have the same total cell numbers. Cells were transferred (as described in section “Preparation of single cells for standard curve”) to black-walled, 96-well plates in cell numbers ranging 0–2 × 105 cells/well and final volumes adjusted to 150 µL/well.