Observations on Cryopreservation of Lymphocytes in Chronic Lymphocytic Leukaemia and Normal Human Lymphocytes

Abstract

A set of conditions is defined for freeze‐storage of lymphocytes circulating in chronic lymphocytic leukaemia (CLL‐lymphocytes) and in healthy individuals (normal lymphocytes). Isolated cells are frozen in culture medium containing 15 % serum and 8.3 % (v/v) dimethyl sulphoxide (DMSO) at a post‐freezing cooling rate in the range 1.8‐–3.4° per min to −40° or below followed by immersion in liquid nitrogen, and thawed rapidly (ca. 400° per min) at 37°. This procedure induced consistently low mortalities (2–7%) in normal and CLL populations, uninfluenced by freeze‐storage lasting up to 57 weeks, and was accompanied by large‐scale destruction (67–100%, average 95 %) of residual isolated red cells. The lymphocytes classified as surviving cells by cytologic analysis could display partial loss of initial capacity for respiration but capacity for survival in short‐term culture was retained. Brief reference is made to unchanged ultrastructural appearance of surviving cells. No pronounced differences were observed in the capacity of normal populations to transform under the influence of phytohaemagglutinin (PHA) as a result of freezing and thawing, but tests for altered proliferative capacity of transformed cells were hampered by culture conditions proving sub‐optimal. Ultrasensitivity of CLL‐lymphocytes in vitro to cytocidal action of colchicine — a property currently utilized for measurement of abnormal cell content of CLL‐lymphocyte populations — was closely reproduced following freezing and thawing in experiments with 9 patients.

Heterogeneous X‐ray survival characteristics of lymphocytes in prolymphocytic leukaemia: Mathematical analysis distinguishing delayed cell death and true radioresistance

“…The lymphocytes contained in the red-cell depleted upper layer were harvested by centrifugation (200 x g,,). The subsequent processing entailed in their cryopreservation and reconstitution is inimical to the residual, accompanying red cells and results in their removal (19).…”

Killing and Characterizing Action of Colchicine in vitro on Lymphocytes of Chronic Lymphocytic Leukaemia

“…Lymphocyte respiration was measured at p H 7.5 and 37" with a Cartesian diver apparatus exactly as described recently (Thomson & O'Connor 1971).…”

The Use of Directly Frozen Cells in LDA Assay

“…In this assay, the use of frozen cells as targets or effectors makes it possible to study the immune reactivity of patients over a long period of time. Two main methods of cryopreservation have been described using a controlled programmed freezer (Powles et al 1973, Guttermann et al 1973, Thomson & O'Connor 1971, Eijsvoogel et al 1973 or freezing directly in an ultra-low temperature freezer (Wood et al 1972, Stopford et al 1972.…”