Factors Affecting the Stability of Cryogenically Preserved Human Granulocytes.
Abstract
Human granulocytes free of other cell types were obtained by counterflow centrifugation, cryogenically preserved, and studied for stability and function after thawing. Isolation of granulocytes by counterflow centrifugation was optimal at reduced temperatures (4 -10C) in phosphate buffered saline (or Ca++ free buffers) at pH 7.1. Granulocytes could not be cryogenically preserved in plasma or serum. Heating or prefreezing of serum was ineffective, but dialysis or addition of EDTA overcame the destructive effect of serum. Neither treatment was an improvement over the standard freeze procedure using buffered albumin and cryoprotective components. Beta-mercaptoethanol added to the freezing medium caused the production of a single homogeneous population of osmotically inert, non-viable, ethidium reactive granulocytes. This suggests that osmoregulation by granulocyte membranes is a critical requirement for cryopreservation. Preservation efficiency is species dependent, increasing in the order of human, baboon, guinea pig, and dog. Dog granulocytes can be stored for at least 8 months in liquid nitrogen with small loss of cells and functionality. The present efficiency of preservation of human granulocytes for 3-4 weeks of liquid nitrogen storage is 90-100% morphological and 40% functional recovery. Attempts to increase stability of thawed granulocytes with other additions to our current procedure have so far proved fruitless. These have consisted of inosine, adenine, pyruvate, gluconate, vitamin C, beta-mercaptoethanol, p-phenyl-methyl-sulfonylfluoride, and mannitol.
Document Details
- Document Type
- Technical Report
- Publication Date
- Dec 18, 1979
- Accession Number
- ADA078906
Entities
People
- A. B. Callahan
- C. R. Valeri
- F. L. Luscinskas
- Fabian J. Lionetti
- S. M. Hunt