the microcarriers. Therefore, periodically check under the

microscope to determine whether the cells have detached.

37. For cell counts performed between the first to tenth day of

culture in the bioreactor, re-suspend the cell pellet in a smaller

volume of media. However, for cells cultured more than

10 days in the bioreactor, re-suspend the cell pellet in a larger

volume of media.

38. The 65 μm pore size of the mesh filter allows the separation of

microcarriers from the cell solution during harvest.

39. Use a peristaltic pump (100–200 mL/min) or gravity to intro-

duce the F3 hPSC passaging solution to the bioreactor vessel

and to remove the cell culture medium from the vessel to the

waste bag.

40. For every 1/3 decrease in volume, lower the agitation speed by

10 rpm. When the impeller is visible, discontinue agitation.

41. The bag of F3 hPSC passaging solution can also be connected

to the harvest line. Use a peristaltic pump (100–200 mL/min)

or gravity to fill the vessel with 1.5 L of F3 hPSC passaging

solution.

42. The cell-microcarriers can be incubated with the F3 hPSC

passaging solution for another 5–10 min to ensure release of

cells from the microcarriers. Longer incubation of the F3 hPSC

passaging solution will not adversely impact the health of the

cells. Take another 5 mL sample to confirm.

43. At this juncture, single cells will pass through the mesh filter to

the media bag and the microcarriers will be deposited to the

other side instead. Therefore, the media bag will contain 3 L of

single cell suspension composed of single cells in 1.5 L of F3

hPSC passaging solution and 1.5 L of L7™TFO2 hPSC

complete medium.

44. The expanded cells can be characterized using qualitative and

quantitative methods to ascertain pluripotency such as immu-

nofluorescence staining and flow cytometry, respectively.

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