Stephen M. Hunt

Cryopreservation of human red cells in liquid nitrogen with hydroxyethyl starch

Summary Erythrocytes were frozen in plastic bags in liquid nitrogen with 14% (w/v) hydroxyethylstarch (HES). Both a small volume (25.0 ml) and a full unit system (405 ml) were employed. Small volume freezings were investigated to optimize container configuration, conditions, composition of media, and postthawed stability of cells. Based on this experience a method for full unit freezing of packed erythrocytes was devised. The results of the 10 most recent experiments with 25.0 ml freezing gave postthawed cell recoveries of 99.2 ± 0.3%. Stability for 1 hr in 0.15 M NaCl at 22.0°C was 85.9 ± 4.3%. The cells gained 17 mequiv of Na + and lost 21 mequiv of K + . The ATP decreased 23% while 2,3 DPG was unchanged. In the large volume method using all the packed red cells in a unit after removal of platelet rich plasma and buffy coat, a single bag was sandwiched between large perforated aluminum plates. The results of eight recent freezings and short-term storage in liquid nitrogen vapor gave cell recoveries of 97.2 ± 1.1%, and stabilities in saline of 75.7 ± 1.8%. Relatively small losses of ATP and 2,3 PDG were observed. The cells gained 33 mequiv of Na + and lost 31 mequiv of K + . Assuming acceptable 24-hr posttransfusion survival can be achieved, the feasibility of freezing full units of red cells in a one-step procedure is now demonstrated.

Isolation of Human Blood Phagocytes by Counterflow Centrifugation Elutriation

The principle of cell separation by counterflow centrifugation was first enunciated by Lindahl (1948) who derived an equation defining the position of particles in a centrifugal field opposed by fluid flowing in the centripetal direction. He designed a “counterstreaming” centrifuge which concentrated yeast particles in planes of equilibrium dependent upon the radius and density of the yeast and the viscosity and density of the medium (Lindahl, 1956). In an early study with the “counterstreaming” centrifuge eosinophilic leukocytes from horse blood were concentrated and the final cell suspension contained 20–30% (Lindahl and Lindahl, 1955). A significant advance was made by McEwen et al. (1968) who designed rotors for use with a standard preparative centrifuge. Their studies were largely responsible for the production of the simple equipment which makes counterflow cell separation generally available. Polystyrene micro spheres, yeasts, plant, and blood cells were resolved into subpopulations with diameters which ranged from 0–20 µm. In the first attempts to isolate leukocytes, whole blood with a ratio of 700 red cells to 1 leukocyte was concentrated to a ratio of 4:1. In a sample of whole blood in which 73% of the white cells consisted of granulocytes, counterflow centrifugation recovered 94% of them in the separation chambers. With a prototype rotor designed later, McEwen et al. (1971) achieved 99% recovery of leukocytes from malaria-infected monkey blood.

Factors affecting the stability of cryogenically preserved 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 (4–10 °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.

Control of cell size in the cryopreservation of mouse marrow

Abstract Electronic size analysis of mouse marrow cells enables the rapid assessment of quantity and quality of the cells. Three major distributions for mouse marrow were observed corresponding to erythrocytes, lymphocytes plus normoblasts and myelocytes. Marrow was most stable in balanced salt or tissue culture media at 2 °C. When stored in isotonic NaCl at 4 °C for 24 hr, the myelocyte population swelled and lysed. Glycerol (1.55 m ) greatly shrunk the lymphocytic and myelocytic populations and required an hour to restore initial volume, while DMSO up to 15% was rapidly equilibrated without volume change. Machine analysis was applied to optimize conditions for freezing mouse marrow. Preliminary experiments revealed good recovery of cells after thawing and good survival of transfused cells.