There is another side to the Hawaii Endangered Bird Conservation Program that happens at the San Diego Zoo’s Beckman Center for Conservation Research. Our Genetics Division has preserved the genetic material of many Hawaiian birds in the Frozen Zoo®, a large collection of frozen samples, including thousands of cell lines. Most of the cell lines in the Frozen Zoo are grown using a piece of skin tissue from a small biopsy, which can be taken during an animal’s regular veterinary exam. This is not as feasible for birds due to their fragile skin and small size, so our chance to obtain tissue from Hawaiian birds comes after they have died. Usually we receive an eyeball or a section of trachea. Then that piece of tissue is diced up into tiny pieces and put into an enzyme that digests the connective tissue, freeing up the individual cells.
Andrea places prepared flasks in an incubator.
Once the diced tissue has been “digested” by the enzyme for a few hours, we put the remaining material in a tissue culture flask with the appropriate cell culture medium, which is a liquid containing the nutrients cells need to survive, and place them in a heated incubator. We will “feed” the cells every few days by emptying the flask and putting new medium in. If all goes as planned, the cells will attach to the bottom of the flask and will proceed to divide until they take up all the space. When there are enough cells, we will apply another enzyme, called trypsin, that breaks up the bonds holding the cells to the flask so they float freely, allowing us to move the cells into new, larger flasks for continued growth. Eventually the population of cells has doubled several times. It typically takes around a month for this to happen; tissue from younger animals tends to grow more rapidly than from aged animals.
Preserved cells in the Frozen Zoo
Then it’s time to put them in the Frozen Zoo. Ordinarily, cells die when frozen because the water they contain forms sharp ice crystals, tearing the fragile cell membranes apart. To inhibit crystal formation, we add a chemical called a cryoprotectant (in this case, dimethyl sulfoxide) to the cells. The cells are then placed into several tiny one-milliliter vials and put in a computerized cryogenic freezer that lowers the temperature at a carefully controlled rate. A little over an hour later the cells have reached 80 degrees below zero, and they’re ready to be put into boxes for storage in the Frozen Zoo, where liquid nitrogen keeps them frozen at an even colder temperature: 196 degrees below zero. At any time, they can be pulled out of the liquid nitrogen, thawed, and put back into cell culture medium and they will continue growing! Cells can be kept frozen for many years in this suspended but living state; nobody knows exactly how long because the technology has only been around for a few decades. The Frozen Zoo thus provides a self-renewing source of DNA for researchers studying the genetic makeup of particular species.
Currently there are cell lines from over 45 Hawaiian birds in the Frozen Zoo, including nene, ‘alala, Maui parrotbill, puaiohi, palila, and Hawaii creeper. Sadly, in 2005 the po’ouli became the first extinct species represented in the Frozen Zoo. It will doubtless not be the last, but thanks to all the people working hard on this project, there is reason to hope that no more Hawaiian birds will end up on that tragic list.
Andrea Johnson is a research technician for the San Diego Zoo.