METHOD ON LONG-TERM STRUCTURAL PRESERVATION OF HEMOCYTE UTILIZING CELLULAR LYOPHILIZATION TECHNIQUE

摘要

This invention was made on the method of preserving cells structurally for a short or long time using lyophilization technique. The technique to preserve lyophilized cells of human for a long time has its limitation, but our research institution has found a way to preserve lyophilized cells structurally and safely for a short and long time. The model of cells to preserve is human hemocytes. Explanations will be given on the method of storing in two categories: short-term storage and long-term storage. To preserve hemocytes safely for a short time, hemocytes is incubated in 800mM trehalose buffer solution at 37°C for 15 hours. The names and concentration of cell protection material are 5 OE10^-4M L-ascorbic acid, 5 OE10^-5M a-lipoic acid, 5 OE 10^-5M Melatonin, 3 OE10^-4M N-acetyl-L-cysteine, 10^-5M d-tocopherol, 5 OE10^-5M insulin, and 10^-5M prostaglandin E1. Long-term storage starts with lyophilization of incubated hemocytes, which will be added with glycerol later. Hemocytes will be incubated for 15 hours and will be added with lyophilization buffer until the hematocrit reaches to 5%. Then, the hemocytes will be cooled at a rate of 1? per minute until they get to -40?, in which temperature the hemocytes will be incubated for 30 minutes. Primary drying will be performed at -30? for 7 hours at a vacuum of 30mTorr. During secondary drying which will be performed for 6 hours, shelf temperature will be increased from -30? to 20? at a rate of 0.8? per minute using a vacuum of 50mTorr. After then, samples will be put into a specially manufactured container. The air in the container will be removed and the container will be filled with anhydrous glycerol(=99.5%) for a long-term storage. Though a lot of studies are required to perfectly recover the function, this method ensures structural stabilization which enables semi-permanent storage. This technique to store lyophilized cells semi-permanently can be utilized in many ways in our daily lives. For example, for remembering deceased people in funeral or for commemoration between lovers. Despite various purposes in our lives, this technique has never been developed. Thus, we apply for a patent on the method and utilization of this technique.