Hum. Genet. 48, 125--126 (1979) © by Springer-Verlag 1979
Improved Technique for Human Leukocyte Cultures Julie R. K o r e n b e r g Department of Medical Genetics, University of Wisconsin, Madison, Wisconsin 53706, USA
Summary. A n i m p r o v e d technique for cultivation of peripheral h u m a n lymphocytes is presented. The settling of the b l o o d cells is replaced by a wash in Ficoll to remove the p o l y m o r p h o n u c l e o c y t e s .
A l t h o u g h culturing of peripheral leukocytes is the preferred technique for o b t a i n ing h u m a n c h r o m o s o m e preparations, the p r o p o r t i o n of cells in metaphase a n d the quality of c h r o m o s o m e s varies considerably between laboratories a n d even between cultures. Even worse, cultures occasionally fail to grow at all. This c o m m u n i c a t i o n presents a n i m p r o v e d lymphocyte culture technique in which one a d d i t i o n a l centrifugation a n d a wash with the use of a Ficoll gradient replace the step of settling the b l o o d in the o r d i n a r y lymphocyte culture technique. The results have been consistently better t h a n with the usual method. A syringe (or a Vacutainer) is used to collect 10 ml whole blood by venipuncture. The concentration of sodium heparin should be 250 units per milliliter of blood. Different techniques involving the use of Ficoll gradients for separation of lymphocytes (cf. B6yum, 1968) were tried successfully. However, the simplest and most practical procedure is the following. The blood is transferred to a 50-ml plastic disposable centrifuge tube (Coming) containing 10 ml Hank's balanced salt solution, and gently mixed with a 10-ml pipette. Fifteen milliliters of Ficoll Hypaque (Pharmacia Fine Chemicals, 800 Centennial Ave., Piscataway, New Jersey, USA) at room temperature is slowly layered below the blood mixture with a syringe fitted with a No. 17 gauge, 11/2in. standard needle or with a spinal needle that is carefully placed through the blood mixture and rested on the bottom of the tube. The tube is then capped and centrifuged at 500 g for 40 min at 5°-18°C. The lymphocytes are located in a more or less sharp band between the Ficoll and the upper phase, a lower temperature yielding a sharper band. The lymphocyte band is transferred by means of a syringe and a needle, with a minimum of the Ficoll layer included, into a 50-ml centrifuge tube, thoroughly mixed with 3-5 vol. Hank's solution, and centrifuged at about 350 g for 5 min. The supernatant is discarded and the pellet suspended in 10ml Hank's solution and recentrifuged at 150g for 5min, after which it is suspended in 1 ml culture medium. At this point the cells may be counted in a hemacytometer to determine the efficiency of separation (usually > 90% lymphocytes) and the lymphocyte yield (approx. 1061ymphocytes/ml whole blood). The cell suspension is then diluted with 20ml medium RPMI 1640 (Gibco) containing 15% calf serum, 0.35 ml phytohemagglutinin (Gibco), and the usual amounts of penicillin and streptomycin, the final concentration of cells being about 0.5 x 106/ml. The cell suspension is cultured in loosely capped 15-ml polycarbonate tubes, with 5 ml per tube, in an atmosphere containing 5% CO2.
0340-6717/79/0048/0125/$01.00
126
J.R. Korenberg
Good metaphase preparations have been obtained with all the following methods. Ceils are harvested after 72 or 96 h in culture with colcemid (0.06 gg/ml) present for the last 1-3 h. When the cultures are harvested at 96 h, metaphase yields can be increased by centrifuging the cultures and resuspending the packed cells in fresh medium after 48 or 72 h. Alternatively, the colcemid may be added 15-20 h before fixation, if wished. Although this, as might be expected, results in a number of cells with overcontracted chromosomes, it also appears to increase the frequency of metaphases with extended chromosomes. Although it is advisable to culture the lymphocytes as soon as possible after venipuncture, the Ficoll method has been successful on blood or plasma samples sent through the mail. The yields of metaphases from Ficoll-separated lymphocytes have as a rule been increased twofold to 6%-20% of the cells; in control cultures of lymphocytes settled in the usual way from whole blood the yield is 3%-10% cells in metaphase. The Ficoll technique has resulted in no failures, and the quality of the chromosome preparations has been as good as or better than that obtained with the ordinary method. The improvement of chromosome preparations obtained with the Ficoll technique probably depends on the removal of polymorphonucleocytes from the culture, since it has been observed that their presence decreases the incorporation of 3H-thymidine by lymphocytes in mixed leukocyte cultures (Bach et al., 1971).
Acknowledgements. Supported by grant GM 22881 from the National Institutes of Health (Washington, DC). This is paper No. 2300 from the Genetics Laboratory, University of Wisconsin.
References Bach, M. L., Bach, F. H., Widmer, M., Oranen, H., Wolberg, W. H.: Lymphocyte reactivity in vitro. Transplantation 12, 283--286 (1971) B6yum, A.: Isolation of leucocytes from human blood. A two-phase system for removal of red cells with methylcellulose as erythrocyte-aggregating agent. Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 9--29 (1968)
Received October 25, 1978
Improved Technique for Human Leukocyte Cultures Julie R. K o r e n b e r g Department of Medical Genetics, University of Wisconsin, Madison, Wisconsin 53706, USA
Summary. A n i m p r o v e d technique for cultivation of peripheral h u m a n lymphocytes is presented. The settling of the b l o o d cells is replaced by a wash in Ficoll to remove the p o l y m o r p h o n u c l e o c y t e s .
A l t h o u g h culturing of peripheral leukocytes is the preferred technique for o b t a i n ing h u m a n c h r o m o s o m e preparations, the p r o p o r t i o n of cells in metaphase a n d the quality of c h r o m o s o m e s varies considerably between laboratories a n d even between cultures. Even worse, cultures occasionally fail to grow at all. This c o m m u n i c a t i o n presents a n i m p r o v e d lymphocyte culture technique in which one a d d i t i o n a l centrifugation a n d a wash with the use of a Ficoll gradient replace the step of settling the b l o o d in the o r d i n a r y lymphocyte culture technique. The results have been consistently better t h a n with the usual method. A syringe (or a Vacutainer) is used to collect 10 ml whole blood by venipuncture. The concentration of sodium heparin should be 250 units per milliliter of blood. Different techniques involving the use of Ficoll gradients for separation of lymphocytes (cf. B6yum, 1968) were tried successfully. However, the simplest and most practical procedure is the following. The blood is transferred to a 50-ml plastic disposable centrifuge tube (Coming) containing 10 ml Hank's balanced salt solution, and gently mixed with a 10-ml pipette. Fifteen milliliters of Ficoll Hypaque (Pharmacia Fine Chemicals, 800 Centennial Ave., Piscataway, New Jersey, USA) at room temperature is slowly layered below the blood mixture with a syringe fitted with a No. 17 gauge, 11/2in. standard needle or with a spinal needle that is carefully placed through the blood mixture and rested on the bottom of the tube. The tube is then capped and centrifuged at 500 g for 40 min at 5°-18°C. The lymphocytes are located in a more or less sharp band between the Ficoll and the upper phase, a lower temperature yielding a sharper band. The lymphocyte band is transferred by means of a syringe and a needle, with a minimum of the Ficoll layer included, into a 50-ml centrifuge tube, thoroughly mixed with 3-5 vol. Hank's solution, and centrifuged at about 350 g for 5 min. The supernatant is discarded and the pellet suspended in 10ml Hank's solution and recentrifuged at 150g for 5min, after which it is suspended in 1 ml culture medium. At this point the cells may be counted in a hemacytometer to determine the efficiency of separation (usually > 90% lymphocytes) and the lymphocyte yield (approx. 1061ymphocytes/ml whole blood). The cell suspension is then diluted with 20ml medium RPMI 1640 (Gibco) containing 15% calf serum, 0.35 ml phytohemagglutinin (Gibco), and the usual amounts of penicillin and streptomycin, the final concentration of cells being about 0.5 x 106/ml. The cell suspension is cultured in loosely capped 15-ml polycarbonate tubes, with 5 ml per tube, in an atmosphere containing 5% CO2.
0340-6717/79/0048/0125/$01.00
126
J.R. Korenberg
Good metaphase preparations have been obtained with all the following methods. Ceils are harvested after 72 or 96 h in culture with colcemid (0.06 gg/ml) present for the last 1-3 h. When the cultures are harvested at 96 h, metaphase yields can be increased by centrifuging the cultures and resuspending the packed cells in fresh medium after 48 or 72 h. Alternatively, the colcemid may be added 15-20 h before fixation, if wished. Although this, as might be expected, results in a number of cells with overcontracted chromosomes, it also appears to increase the frequency of metaphases with extended chromosomes. Although it is advisable to culture the lymphocytes as soon as possible after venipuncture, the Ficoll method has been successful on blood or plasma samples sent through the mail. The yields of metaphases from Ficoll-separated lymphocytes have as a rule been increased twofold to 6%-20% of the cells; in control cultures of lymphocytes settled in the usual way from whole blood the yield is 3%-10% cells in metaphase. The Ficoll technique has resulted in no failures, and the quality of the chromosome preparations has been as good as or better than that obtained with the ordinary method. The improvement of chromosome preparations obtained with the Ficoll technique probably depends on the removal of polymorphonucleocytes from the culture, since it has been observed that their presence decreases the incorporation of 3H-thymidine by lymphocytes in mixed leukocyte cultures (Bach et al., 1971).
Acknowledgements. Supported by grant GM 22881 from the National Institutes of Health (Washington, DC). This is paper No. 2300 from the Genetics Laboratory, University of Wisconsin.
References Bach, M. L., Bach, F. H., Widmer, M., Oranen, H., Wolberg, W. H.: Lymphocyte reactivity in vitro. Transplantation 12, 283--286 (1971) B6yum, A.: Isolation of leucocytes from human blood. A two-phase system for removal of red cells with methylcellulose as erythrocyte-aggregating agent. Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 9--29 (1968)
Received October 25, 1978
