A Novel Nanomaterial-Based Approach for Cryopreservation of Individual Sperm Cells Using Addressable Nanoliter Containers

The research and development of a matrix of Addressable Nanoliter Containers (ANLC) is the focus of this work. The ANLC introduces a novel approach for cryopreserving single sperm cells. A significant increase in sperm cell mortality was observed after cryopreserving nanoliter-scale cell suspensions, attributed to the diffusion of water from the aqueous droplets into the surrounding oil phase. This process elevated the salt concentration within the droplets. A practical solution was devised by saturating the oil with water, significantly reducing the concentration gradient and, consequently, the diffusion. For ANLCs smaller than a few nanoliters, locating individual sperm cells within the containers became highly feasible. Using saturated oil, the survival rate reached 100%. Optical simulations were conducted to evaluate the impact of ANLCs on light scattering, enabling the selection of designs with minimal scattering. The simulations conclusively demonstrated that a cylindrical container with a flat bottom produced the least light scattering. This device was tested under clinical conditions in an in vitro fertilization (IVF) laboratory, revealing its strong potential as a practical tool for housing individual sperm cells. It enables characterization using interferometric indicators and facilitates the selection of sperm cells for IVF.