by History and Techniques
The ability to successfully cryopreserve and store living cells at ultra-low temperatures has enabled major advances in fields ranging from medicine to conservation biology. The roots of modern cell cryopreservation can be traced back to 1949 when Polge, Smith and Parkes discovered that glycerol could prevent ice crystal formation in freezing sperm cells from fowl. However, it was not until the 1970s and 80s that consistent and reliable protocols for freezing mammalian cells were developed.
When cells are frozen, ice crystals begin to form both inside and outside of the cell membrane. This ice formation can disrupt the cell membrane and cause intracellular ice crystal damage. To avoid this, Cell cryopreservation like glycerol are added to the freezing medium. Glycerol works by lowering the freezing point of water inside and outside the cell, limiting ice formation. As the cells are cooled, their water content gradually replaces with glycerol through osmosis. This allows intracellular freezing to be avoided, leaving the cell in a vitrified or glass-like state at ultralow temperatures.
Common freezing protocols involve controlled-rate freezing where the cell suspension is cooled at around 1°C/minute until it reaches -80°C for storage in liquid nitrogen. Improper cooling rates can still cause ice crystal damage. Thawing requires rapidly warming cells to 37°C to reverse the osmotic gradient before assessing viability. Development of new cryoprotectants and controlled freeze-thaw devices have helped achieve higher post-thaw survival rates for many cell types.
Applications in Reproductive Biology and Medicine
One of the first major applications of cell cryopreservation was in the field of reproductive biology. Sperm banking allowed for preservation of male fertility for conditions like cancer treatment as well as artificial insemination in livestock. Ovarian and testicular tissue cryopreservation also enables fertility preservation for pediatric oncology patients.
Today, IVF clinics routinely freeze fertilized embryos and human oocytes for long-term storage. This has significantly helped increase success rates as multi-cycle IVF allows embryos to be transferred when uterine conditions are optimal. Embryo banks also support embryo donation programs for couples struggling with infertility. In some countries, embryonic stem cells derived from frozen embryos aid research into conditions like diabetes and Parkinson’s disease.
In medicine, cryopreservation has revolutionized bone marrow transplantation. Storing hematopoietic stem cells from blood or bone marrow of healthy donors allows them to be a readily available graft source for patients requiring transplantation for conditions like leukemia. Cord blood banking also provides stem cells for regenerative therapies. Skin banks provide cryopreserved skin allografts for treating severe burns and wounds.
Cell Therapies and Tissue Engineering Applications
Emerging fields of cell therapies and tissue engineering are highly reliant on the ability to cryopreserve and bank viable cell sources. Mesenchymal stem cells isolated from sources like bone marrow, adipose tissue or dental pulp can be expanded and frozen in master cell banks for off-the-shelf availability in regenerative medicine. Upon thawing, these stem cells retain differentiation capacity to generate bone, cartilage or other connective tissues.
Producing these customizable tissue constructs often requires combining stem cells with 3D matrices and growth factors. Freezing complete tissue engineered constructs rather than just the cells allows preserving the tissue-like characteristics achieved. For example, heart valve conduits or skin substitutes can be cryopreserved for on-demand delivery. Banking finished tissues circumvents repeated production and validation runs.
Cryopreservation also helps standardize cell-based therapies, many of which now enter clinical trials. Productions of induced pluripotent stem cells, Natural Killer cells, T-cells for immunotherapy or genetically modified cellular therapeutics first involves developing microbial-safe master cell banks through controlled rate freezing. Only vials that maintain post-thaw viability and identity undergo further expansion and manufacturing of clinical-grade cell batches. This enhances quality assurance.
Challenges and Future Perspectives
While major progress has been made, further work is still required to optimize cryopreservation of certain primary cell types and complex tissues. For example, difficulties in preserving pancreatic islets have slowed development of cell therapies for diabetes. Three-dimensional viability and functionality assays that better mimic in vivo conditions are also needed to comprehensively assess post-thaw cell quality.
With continued research, we may see novel biomaterials and cryoprotectants designed specifically for different cell sources. Controlled freeze-dry techniques may facilitate long-term ambient temperature storage, reducing liquid nitrogen dependency. Standardization of automated closed freezing/thawing devices, electronic cell banking information systems will support industrial-scale production of cellular products and global sharing of donor samples for research and therapies. Altogether, advances in cryopreservation ensure cells and tissues remain a viable resource, further fueling the next wave of biological and medical innovations.
