Retinal pigment epithelial (RPE) cells are an important component of intravitreal and epiretinal membranes in proliferative vitreoretinopathy (PVR) that contract and cause recurrent retinal detachment. Cryotherapy has been shown to enhance release of viable RPE cells into the vitreous cavity. This study demonstrates that repeat cryotherapy (refreezing) releases viable RPE cells in even larger numbers. Repeated indentation of the sclera after transscleral cryotherapy also releases more viable RPE cells than does cryotherapy alone or scleral indentation preceding cryotherapy. These findings suggest that refreezing should be avoided, and repeated indentation of the sclera should be minimized after cryotherapy.