The Complete Guide to the Cryopreservation of Hybridoma Cells

Candy Swift
Aug 8, 2017 · 3 min read

In the process of establishing hybridoma cells, sometimes one fusion can produce too many “positive” holes to deal with these cells in time for further work, so that we need to freeze some of them. On the other hand, in order to prevent accidents that may occur in the laboratory from bringing disasters to hybridoma cells, such as power cuts, contamination, failure of the temperature or CO2 controller, etc., the common solution is to freeze a portion of the cells as seeds as early as possible. After the establishment of hybridoma cells, it is much more needed to freeze a large number of these cells for future use.

The principle of cell cryopreservation is that cells can be preserved for a long time by soaking them in the culture medium with serum and dimethyl sulfoxide in it, lowering the temperature at a certain slow rate (1℃/min between 0℃ — 20℃; 1℃/ 2 mins between -20℃- -40 ℃), and storing them in liquid nitrogen of -196℃ or liquid nitrogen vapor. Listed below is the one of the cell cryopreservation methods, with which the survival rate of the cells can reach over 80% after several years of experiment in other types of cells.

A. Materials

a. A suction tube with cap (made of aluminum or tin). Put 1–2 layers of asbestos paper or asbestos cloth on its inner wall.

b. HT medium containing 10–20% of serum and 5–10% of DMSO. Decline the temperature to 0℃ or so with ice bath (used as cryoprotectant).

c. Sterilized ampoules or vials with cap.

d. A refrigerator whose lowest temperature can reach -70℃.

e. Liquid nitrogen and a liquid nitrogen tank.

f. Hybridoma cells that are in the middle of logarithmic growth, and are healthy and strong in activity.

B. Methods

a. Centrifuge the hybridoma cells (or other cells), and resuspend them in precooled cryopreserved solution at a concentration of about 106–107 cells/ ml. Put 1ml of the solution into each ampoule, and place in ice bath. Label the ampoules with cells name, date of cryopreservation, batch number, etc.

b. Seal the ampoules and remain them in ice bath.

c. Put the ampoules inside a small cloth bag with binding rope. Label the cloth bag with cells name, date of cryopreservation, etc, and immediately put the bag into the suction tube, and store in refrigerator with the temperature of -70℃.

d. After 2–4 hours or overnight, take out the suction tube and remove the cloth bag to the liquid nitrogen tank. Label on its rope with marks or codes. Finally, make detailed notes about this liquid nitrogen cryopreservation on a notebook.

e. Liquid nitrogen should be added into the liquid nitrogen tank regularly (It is better to have a specific personnel manage this). Ware protective gloves and glasses while adding liquid nitrogen or getting access to cells to avoid being frostbited by the liquid nitrogen.

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