Factors affecting the stability of cryogenically preserved granulocytes

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 (4–10 °C) in phosphate-buffered saline (or Ca²⁺-free buffers) at pH 7.1. A stabilizing protein, or HES was required. Routinely, 1.2% human or bovine serum albumin was used. Hyperosmolar (310 m0sm) buffers and post isolation handling in ice water baths was optimal for cryogenic preservation. Addition of DMSO at 22 °C produced transient shrinkage initially which depended on the rate of addition, concentration, and temperature. Within 10–15 min granulocytes returned to volume, but continued to swell, equilibrating for 1 hr at 20% larger volume. Ethidium uptake gradually increased. After 24 hr, extreme swelling, lysis, and ethidium uptake was observed at the highest concentration (10%) of DMSO. DMSO-induced swelling was prevented with HES.Granulocytes (30 × 10⁶ ? 50 × 10⁶) were frozen in 2.0-ml volumes in plastic tubes. The combination of 5% DMSO, 6% HES, 4% albumin, 0.056 M glucose in NormosolR at pH 7.1 produced the best yields. Granulocytes were first cooled to 4 °C, then to ?80 °C (approx rate 4 °C per min) in a mechanical freezer and finally stored in liquid nitrogen. Storage varied from days to months. Granulocytes were thawed at 42 °C by manually twirling the freezing tubes and they were subsequently maintained in ice water. They were diluted 3:1 dropwise with a room temperature solution of 7% HES, 1.2% albumin, and 0.026 M glucose in Normosol. Particle ingestion tests were conducted by incubation at room temperature for forty minutes with yeast or zymosan opsonized with autologous serum. Particles ingested were counted by microfluorimetry after two washings at 150g.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. β-mercaptoethanol added to the freezing medium caused the production of a single homogeneous population of osmotically inert, nonviable, 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, β-mercaptoethanol, para-phenylmethyl-sulfonylfluoride, and mannitol.