15. Several methods can be used for cell counting in MC cultures.

The simplest method is to take sample from the culture, spin

down, aspirate the medium from the sample, wash the pellet

with DPBS(
), aspirate, and add TrypeLE™-Express. Allow

cells to incubate in TrypeLE™-Express until they dissociate

from the MC surface (generally 10 min at room temperature),

then pipette to obtain a single-cell suspension. Cells can then

count with a hemocytometer using Trypan blue. Microcarriers

generally do not get under the cover slip, so they will not

interfere with the count.

16. Aggregate sizes should be increasing over the course of 6-day

cell expansion (see Fig. 6), ideally reaching 0.3–0.42 mm² in

size after 6 days [3]. Aggregates will not be uniform in size,

measure a few aggregates of different sizes to properly evaluate

these cultures. Do note that cell aggregate sizes serve as a

guideline, and sizes may be larger if they are not due to over-

confluency of cells but rather more MCs encapsulated into an

aggregate. In lieu, cell counts on day 6 of cell expansion should

be tenfold or more than the original seeded amount [3] (see

Fig. 6). Refer to Fig. 7 that illustrates an example of success-

fully attached and proliferating cell lines on MCs and the

aggregate size present after 6 days of culture. This is in com-

parison to a cell line that failed to attach and proliferate on MCs

even after 6 days of culture.

17. To evaluate successful cardiomyocyte differentiation, aggregate

sizes should be increasing after Day 1 of differentiation (see

Fig. 8). In addition, cell counts should show a steady increase

after day 3 of differentiation (see Fig. 9). CM yields are calcu-

lated by multiplying percentage of cells positive for cTnT with

Fig. 6 (a) Cell counts of a well-expanding hiPSC cell line on Cytodex 1 microcarriers [8]. (b) Aggregate sizes of

a well-expanding hiPSC cell line on Cytodex 1 microcarriers [3]

Integrated Cardiomyocyte Differentiation in Microcarrier Culture

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