A simple box method for freezing lymphocytes

Journal oflrnmunologicalMethods, 65 (1983) 265 268

265

Elsevier JIM02894

Letters to the Editor London, 5 August 1983 Re.: A simple box method for freezing lymphocytes Dear Editor, The suitability of frozen cells for HLA studies depends very much on the viability of the cells. Programmed automated machines, e.g., Cryoson BV4 and TR5 produce nearly 100% viability of cells after freezing. These machines are in common use in many laboratories but they require proper use and maintenance. However, the collection of cells away from laboratory conditions has posed great problems due to inadequate facilities. We attempted to devise a simple, inexpensive and reliable method suitable for cryopreserving lymphocytes in less than normal conditions and in remote locations. Our procedure was based on earlier methods, one of which includes the utilisation of - 80°C refrigerators (Fuller et al., 1979; Wood et al., 1979). This requires a freezing period of 14-18 h and a constant supply of electricity. A second technique related to our method, is the use of a freezing plug and liquid nitrogen as coolant as described by Joysey (1979). This method could prove unsuitable for freezing a large number of samples since the distribution of temperature along the freezing plug would be uneven and the capacity of the plug is limited. Using liquid nitrogen as a cooling source, we have devised a method for freezing lymphocytes which is independent of electrical power and is easily transportable to any location. The freezing unit consisted of two polystyrene boxes, an outer larger box of

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Fig. 1. Freezing unit. A: outer polystyrene box; B: inner polystyrene box; C: Beckman tubes containing lymphocytes; D: liquid nitrogen or dry ice. 0022-1759/83/$03.00 © 1983 Elsevier Science Publishers B.V.

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TABLE I VIABILITY A N D H L A T Y P I N G OF L Y M P H O C Y T E S C R Y O P R E S E R V E D BY T W O D I F F E R E N T METHODS Cell

JS MS ED WV RO TG AJ PF GB JB WS VA SL LD

% Viability of:

Antigen assignments of cells in box and in machine

Fresh cells

Cells in box method

Cells in machine

HLA, -A, -B, -C

HEA-DR

100 96 100 98 96 98 100 96 100 100 100 100 100 95

98 94 98 80 90 90 98 80 95 95 98 95 95 85

NT 95 98 80 88 80 99 80 98 NT NT 98 96 80

2, 28/51, 7/7.2 24, 32/39, 27/1, 7 29, 24/62, 8/3, 2, 28/51, 44/7, 2 Th, 33/57, 53/7, 4 28, 30/62, 18/3, 5 29, 30/8, 13/7, 6 28, 29/44, 6 0 / 3 24, 32/35, 61/4, 2 2, 3/62, 44/3~ 5 3, - / 6 2 , 18/3 2, 3/35, 44/1, 4 1, 2/8, 60/7, 3 26, 33/14.2.35A/8

NT 2, 5 3, 7 1, 5 5 x 8, 8 5, 3, 7 7, 5, 8, 6 5, 7 1, 6 1, 2 NT 2, 7

M B

M B

M B

M B

M B

M B

M B

M B

CC Dwl Dw?

JA Dw3D4

PD Dw2 Dw7,

EW Dw3Dw?

MS Dw2Dw5

BO Dw3 Dw6

DM Dw7 Dw8

RO Dw3 DB3

712 670

864 938

948 687

430 715

4864 655

454 1123

663 1505

1067 686

Autologous cpm

6016 (79) 6308 (68)

5743 (51) 15865 (101)

18234 (87) 13612 (84)

25423 (.105) 14596 (65)

22282 (88) 15065 (69)

12991 (80) 14046 (78)

12362 (86) 16808 (99)

3415~(27) b 1005 (11)

GBR Dwl

8240(101) 12662 (101)

9898 (81) 18837 (89)

22325 (99) 21 118 (97)

9062 (35) 6382 (21)

26928 (98) 28601 (97)

4850 (28) 5384 (22)

14460 (93) 18528 (81)

8950 (65) 9334 (74)

DHT Dw2

Homozygous HLA-D stimulators

(8) (7)

3740 (52) 3799 (30)

3621 (34) 14978 (70)

1646 1630

8911 (39) 30678(101)

16747 (69) 2924 (10)

17515 (113) 23964 (98)

2664 (19) 8851 (38)

8323 (68) 12255 (96)

SCO Dw3

a Median counts per minute (cpm) of quadruplicate cultures. b Double normalized values (DNV) as calculated by the method of Ryder et al. (1975).

(M) (B)

Responder panel machine box

4073 (59) 6355 (77)

8693 (85) 14136 (102)

17814 (94) 10736 (75)

22078 (101 16004 (81)

21113 (92) 16388 (85)

8966 (61) 15288 (96)

2422 (19) 4767 (32)

11232 (97) 5668 (68)

JLO Dw4

HLA-D TYPING OF LYMPHOCYTES CRYOPRESERVED BY TWO D I F F E R E N T METHODS

TABLE II

5 146 (82) 3916 (43)

5 762 (62) 9463 (62)

14319 (82) 15798 (100)

2805 (14) 7343 (34)

22710 (108) 20989 (99)

9 822 (73) 13510 (78)

6633 (55) 13093 (79)

6104 (57) 3439 (38)

DHI Dw5

3427 (43) 6608 (56)

11974(101) 19709 (99)

5 172 (23) 6667 (32)

18674 (73) 15732 (55)

19225 (72) 9533 (34)

10904 (64) 11575 (51)

14906 (98) 21 708 (108)

13207 (98) 6110 (51)

KSH Dw6

5991 (81) 6969 (84)

2921 (27) 2666 (19)

8091 (40) 6899 (48)

13364 (57) 9798 (49)

17093 (69) 10613 (55)

1785 (11) 3 873 (24)

12648 (91) 16085 (107

12904 (104) 2664 (32)

HMI Dw7

--M

268 dimension 34 cm x 29 cm x 24 cm with walls 3 cm in thickness and an inner box which measured 21 cm × 19 cm x 7 cm with walls 1 cm thick (Fig. 1). Both boxes had firmly fitting lids. Lymphocytes for freezing were prepared from defibrinated blood by separation on a Ficoll-Isopaque gradient, washed and re-suspended in 80% pooled AB serum in RPMI 1640 (Flow Laboratories). The cryopreservative was made up of 20% dimethyl sulphoxide (DMSO) in RPMI 1640. Aliquots (0.25 ml) of the mixture of cell suspension and cryopreservative were dispensed into sterile polypropylene Beckman tubes. These were quickly transferred to the smaller freezing compartment and positioned in the larger box which contained liquid nitrogen up to the 1 / 4 mark. The cryopreservation of the cells was effected by cooling down to freezing temperatures by liquid nitrogen vapour. The freezing was allowed to continue for at least 35 min before the tubes of cells were transferred to liquid nitrogen for long term storage. With the aid of a temperature probe (Electroplan Ltd.) an estimate of the drop in temperature with time was achieved. The cooling curve obtained (Fig. 2) was similar to those seen in other methods (Navington and Greaves, 1962; Pegg, 1965). A comparison of the viability of cells stored by this method and with the Cryoson TR5 automated freezing machine is shown in Table I. We have shown that lymphocytes preserved by this method may be typed for H L A - D in mixed lymphocyte culture (MLC) (see Table II). This method of freezing was successfully used in Nigeria for the collection of lymphocytes for HLA typing and was often used in conditions where other forms of cryopreservation would have been impossible. It now also forms a reliable back-up system for our automated freezing machine.

Department of Immunology London Hospital Medical College Turner Street London E1 U.K.

Eka Williams Ralph Okoye Bill Oilier Hilliard Festenstein

References Fuller, T.C., 1979, in: NIAID Manual of Tissue Typing Techniques, ed. J.G. Ray (NIH Publication No. 80-545, Bethesda, MD) p. 225. Joysey, V.C., 1979, in: NIAID Manual of Tissue TypingTechniques, ed. J.G. Ray (NIH Publication No. 80-545, Bethesda, MD) p, 233. Navington, J. and R.I.N. Greaves, 1962, Nature (London) 194, 993. Pegg, P.J., 1965, Br. J. Haemat. II, 586. Ryder, L.P., M. Thomson, P. Platz and A. Svejgaard, 1976, in: HistocompatibilityTesting 1975, ed. F. Kissmeyer-Nielsen (Munksgaard, Copenhagen)p. 557. Wood, N., H. Bashir, J. Greally, D.B. Amos and E.J. Yunis, 1979, in: NIAID Manual of Tissue Typing Techniques, ed. J.G. Ray (NIH Publication No. 80-548, Bethesda, MD) p. 240.