One of the important challenges in developing successful cryopreservation protocols for biological samples relies on avoiding the freezing damage imposed by the intracellular ice formation (IIF). Experimental studies have shown that the formation of a traceable quantity of IIF is lethal. This complex phenomenon is affected by various factors including the cooling rate

water transport

cell membrane integrity

extracellular ice

and cryoprotectant. The main goal of this work is to review the four classical intracellular ice formation theories that have been previously proposed: 1) external ice grows through pores in the cell membrane and catalyzes the IIF

2) external ice causes a conformational change in the cell membrane

transforming it into an effective heterogeneous form and causing IIF

3）the IIF is catalyzed heterogeneously through intracellular structures or homogeneously

and 4) the IIF is a result of plasma membrane damage. The latest experimental observations support the idea that the contact between the external ice and cell membrane leads directly or indirectly to the formation of intracellular ice. Aquaporins are likely a key factor affecting the IIF. The use of an aquaporin inhibitor and a gene knockout to study the IIF is also proposed in this article.