Brain microvascular endothelial cells (BMECS) can be generated from pluripotent stem cells, and possess membrane barrier functions along with capability for maturation into other developing tissues. This cell type has not been successfully frozen with loss of significant viability and/or BMEC functional properties. For example, BMECs can be used to model barrier function in blood brain barrier, by calculating the trans-endothelial resistance (TEER). However, thawed primary BMECs lose TEER resistance. By optimizing cell preparation, freezing media selection, and the controlled freezing, the Inventors have achieved complete recovery of frozen cells, achieving proper tight junction protein expression and physiologically relevant TEER. The freezing methods and compositions described herein, thereby allow for BMECs to be manufactured, frozen and distributed at scale.