The culture and karyotype of rat lymphocytes stimulated with phytohemagglutinin.код для вставкиСкачать
The Culture and Karyotype of R a t Lymphocytes Stimulated with Phyt ohemagglutinin ' WILLIAM 0. RIEKE AND M. ROY SCHWARZ Department of Biological Stmcture, University of Washington School of Medicine, Seattle, Washington ABSTRACT A simple method leading to growth and mitosis in over 95% of shortterm cultures of lymphocytes from the blood, lymph or thymus of the rat is described. The method which is modified from Moorhead's original technique ('60) employs standard tissue culture medium (Eagles MEM or TC no. 199), 20% fresh rat serum, washed lymphocytes, penicillin, and phytohemagglutinin-P (0.01 cm3/cm3 of culture of a 1:5 dilution of stock solution). Details of culture technique and factors contributing to growth failures are discussed. The chromosomes of male and female Lewis rats were studied in metaphase spreads of cultured cells. The karyotypes of these rats are presented and found to be in agreement with those recently reported by Hungerford and Nowell ( ' 6 3 ) . Since 1960, when Nowell reported that phytohemagglutinin (PHA) initiates mitosis in cultures of human leucocytes, many studies (Elves and Wilkinson, '62; Cooper, Barkhan and Hale, '63; Galton and Holt, '63; Humason and Sanders, '63; Aspegren and Rorsman, '64) based on culture techniques described by Moorhead, Nowell, Mellman, Battips and Hungerford ('60) have confirmed his report and shown that it is the lymphocytes which respond in various laboratory animals. The value of lymphocyte culture techniques makes it important that they be suitable for lymphocytes from one of the commonest of all laboratory animals, the rat, Rattus norvegicus. Unfortunately, attempts to culture rat blood lymphocytes have often ended either in failure or in only partial success. Schrek and Rabinowitz ('63) noted that while normal human lymphocytes stimulated with PHA enlarged to form typical PHAblast cells, rat lymphocytes did not respond. Similarly, Ling and Husband ('64) showed that while staphylococcal protein induced a PHA-like transformation in human blood lymphocytes, rat (and guinea pig) lymphocytes were unreactive. In contrast, Humason and Sanders ('63) reported that leucocytes from several species, including the rat, were successfully grown with PHA, but gave no details specific to the culture of rat cells nor indicated the percentage of successful cultures. Most recently, Dowd, Dunn and ANAT. REC., 150: 383-390. Moloney ('64) have carefully described the culture of lymphocytes from rat blood in a medium requiring fresh glutamine and newborn agamma calf serum. With their method sufficient mitotic activity for adequate chromosome preparations was obtained in 70% of the cultures. The present work describes modifications of Moorheads technique ('60) which have allowed the development of a simple and almost invariably successful method for the culture of rat lymphocytes from blood, thoracic duct or the thymus. Lymphocytes from both sexes in one randomly bred and two inbred strains of rats have been cultured, and the karyotype of one of the inbred strains (Lewis) as obtained from the cultured cells is presented. MATERIALS AND METHODS 1. Animals. More than 60 rats and 300 cultures have thus far been studied. The majority of the cultures were established with cells from inbred male Lewis animals weighing 150-250 gm. However, Lewis females, male and female Chocolate ( E N ) and Sprague-Dawley strain rats ranging from 75 to 475 gm were also used. 2. Obtaining cells for culture. Lymphocytes from the blood, thoracic duct lymph or thymus were secured from rats anesthetized with pentobarbital. Blood leucocytes 1This work was supported by U.S.P.H.S. Grant GM 0639. 383 384 WILLIAM 0. RIEKE AND M. ROY SCHWARZ were obtained by cardiac puncture employing a syringe containing heparin (100 units/cm3 of blood) and phytohemagglutinin (0.01 cm3/cmyof blood of 1:5 dilution of the stock PHA). After the blood was mixed with the PHA and heparin, it was allowed to stand in an ice bath for 20-30 minutes and then was centrifuged at 300500 rpm in a clinical centrifuge in a cold room (4" C). The centrifuging was interrupted at 1-2 minute intervals in order to determine the critical stage at which there was separation of leucocyte-rich plasma from erythrocytes before the formation of a buffy coat. The plasma with its white cells was then removed, mixed with an equal volume of Hanks balanced salt solution and centrifuged at 600-800 rpm until all cells were sedimented. The cell pellet was twice washed by resuspension and centrifugation from 5 cm3of Hanks solution. Finally, the cells were suspended in 1-2 cm3 of nutrient medium (Eagles MEM or TC no. 199), and were counted in a standard hemocytometer. The volume of the nutrient medium was then adjusted so that the final concentration of leucocytes in culture would be 1-5 X loficells/cm'. Lymph from the thoracic duct was obtained by cannulating the duct either in the neck (Reinhardt and Li, '45) or the abdomen (Bollman, Cain and Grindlay, '48); and during the 1-14 hour collection periods the lymph was heparinized and kept in an ice bath. Lymphocytes were separated by centrifugation; were washed twice in Hanks solution as described above; and finally were suspended in nutrient medium. The final concentrations of cells in the cultures were varied from 0.4-15 X lofilymphocytes/cm3. Lymphocytes were obtained from the thymus by cutting thymic lobes into several pieces and gently stirring the pieces in Hanks solution containing 10% serum. In most cases the thymic pieces were further agitated by using a syringe to aspirate and eject a portion of the solution in which they were immersed. The pieces of thymus were then removed and the cells which had been released were washed as described above. Thymus cells were cultured at final concentrations ranging from 5-20 X 10" cells/cm'. 3. Culture and nutrient substances. The nutrient medium most frequently employed was Eagles Minimum Essential Medium with Hanks balanced salt solution. However, tissue culture medium no. 199 with NaHC03 also was successfully used. The contents of one bottle of Bacto Phytohemagglutinin-P (Difco Laboratories, Inc., Detroit, Michigan) were mixed with 5 cm3 of Bacto Phytohemagglutinin buffer, pH 7.2-7.3 to form a stock solution. The stock solution was most often used in a final concentration of 0.01 cm'/ cm3 of culture medium, but concentrations of 0.002 cm3/cm3 and 0.02 cm3/cm3 were also tested. Sufficient Penicillin G to give a concentration of 100 units/cm3 of culture medium was added to all cultures. Rat serum in a final concentration of 20% was the only additive used to enrich the nutrient medium. Serum was routinely used within 1-2 hours after being prepared from cardiac blood, but serum which had been refrigerated overnight or deep-frozen (-20" C ) for 1-2 weeks was also tested. Most commonly, isologous serum was used. However, one experiment involved the sera and cells from rats of three different strains in order to compare the growth-supporting characteristics of homologous and isologous sera. 4. Culture vessels and conditions. Initially, sterile, polystyrene tubes or flasks (Falcon Plastics, Los Angeles, California) were used to culture the cells. These were discontinued, however, in favor of standard 10, 25 or 50 cm3 Erlenmyer (Pyrex) flasks which had been washed in Microsolv detergent (Microbiologcal Associates, Bethesda, Maryland), thoroughly rinsed (15 times) in tap and distilled water, and oven dried. Tissue culture medium amounting to 50-60% of the total volume of the flasks was added and the flasks were tightly stoppered. Room air remained as the gas phase above the cultures which were then incubated in an upright position at 37" C for 2-6 days. Cultures were harvested either by hypotonic treatment (Moorhead et al., '60) after the addition of colchicine (0.1 ~gm/cm')),or by smearing after washing the cells in Hanks solution and resuspending them in 0.01-0.02 cm3 CULTURE O F RAT LYMPHOCYTES 385 when the cultures were sacrificed. In contrast, control cultures of blood, lymph or thymus lymphocytes cultured without PHA showed enlargement, division and labeling in only 0.1% of cells or less. Cultures of blood, lymph or thymus lymphocytes were similar with respect to the rate of appearance and morphology of enlarged cells. The sequence of development and morphology of these "blast" cells have been clearly described for human lymphocytes in culture ( Carstairs, '62; Cooper, Barkhan and Hale, '63; Tanaka, Epstein, Brecher and Stohlman, '63) and are very similar to the present findings. In contrast to cultured human lymphocytes, the enlarged cells in cultures of rat lymphocytes only rarely exhibit clearly defined nucleoli in smears (fig. 1). In rat cultures, as in human, however, the number of enlarged cells and mitoses is maximal at two and one-half-three days and declines thereafter. The work of others has established that the small lymphocyte gives origin to the enlarged cell in cultures of human blood cells (MacKinney, Stohlman and Brecher, '62; Marshall and Roberts, '63), and studies in this laboratory indicate the same origin for the enlarged cell in rat cultures (Schwarz and Rieke, '64). The total number of cells regularly declined during culture until the second day and then (except for thymus cultures [Schwarz and Rieke, '641) stabilized. When RESULTS the initial inoculum of cells was large there were only 10-25% Over 95% of the 300 cultures stimu- ( > 5 X 106/cm3)), lated with PHA showed the following evi- as many found at the third day of culture. dences of successful growth when sacri- When (with the exception of thymus ficed at the second or third day of culture: lymphocytes) the initial number was small ( 1 ) significant numbers (up to 60% of (1-2 X 106/cm3), the percentage present a11 cells) of large, primitive-appearing when the culture was sacrificed increased "blast" cells (fig. 1 ) which had not been to 33-50%. While there was cell growth present in the culture inoculum (fig. 2 ) ; in all ranges of cell concentrations tested, and ( 2 ) mitotic figures (fig. 3 ) in suffi- the most consistent growth with highest cient numbers to provide mitotic indexes mitotic indexes was found when cultures of 1% or more per hour of colchicine were begun with leucocyte concentrations treatment. In addition, those cultures in of 0.4-2 X lo6 cells/cm3 for cells from which H3-thymidine was added terminally lymph; 1-2 X lo6 cells/cm3 from blood; evidenced DNA synthesis in many nuclei and 10-20 X lo6 cells/cm3 from thymus. Prior to the initiation of the above techand chromosome spreads (figs. 4, 5). Sixty-five to 70% of the enlarged cells niques a number of cultures failed. The were labeled by a 30-minute exposure to only factor which by itself led to failure isotope, and these labeled cells amounted in every instance was an increase in pH to as much as 30-40% of all cells present caused by a loss of carbon dioxide in those of serum. Smears were air dried and fixed four minutes in absolute methanol. 5. Labeling and radioautographic techniques. In order to determine DNA synthesis and cell growth, H3-thymidine (Yz-1 vc/ml, specific activity 6.7 c/mM, New England Nuclear Corp., Boston, Massachusetts) was added to approximately one-fourth of the cultures during the terminal one-half-three hours of incubation. In some cases colchicine was added with the thymidine. The cultures were well washed when sacrificed and then used either for chromosome preparations (hypotonic treatment technique) or for smears as described above. Smears were made on slides previously subbed with a gelatin base and chromosome spreads were prepared on clean, unsubbed slides. The smears were processed for radioautography by painting on liquid emulsion (Eastman Kodak NTBz or NTB,) in a manner previously described (Everett, Rieke, Reinhardt and Yoffey, ' 6 0 ) , and the unsubbed slides with the chromosome spreads were coated by being dipped in the emulsion. Film exposures of 1-14 days were allowed. After developing the radioautographs, the dipped slides were dried and the emulsion was wiped from their back sides. They, together with the smear preparations, were then stained with MacNeal's tetrachrome (MacNeal, '22). 386 WILLIAM 0. RIEKE AND M. ROY S C H W A R Z cultures in which the stoppers on the flasks had become loosened. Other factors which in combination were sometimes determining included: ( 1 ) The serum. The use of serum within 1-2 hours after it was prepared was most important. Serum refrigerated overnight supported only decreased or variable growth, and serum deep frozen for 1-2 weeks often allowed no growth. Serum from any age, sex or strain rat was used successfully as long as it was fresh. ( 2 ) The culture vessels. While both round and flat bottom plastic vessels allowed cell growth when all other conditions were favorable, the number of surviving cells and mitotic indexes were usually less when plastic was used than when glass vessels were employed. ( 3 ) Washing the cells. While at first it was believed that the use of washed cells was critical in securing growth, later experiments showed that lymphocytes from the blood at least could be grown, unwashed, in the plasma in which they were isolated (see also Dowd, Dunn and Moloney, '64). The percentage of successful cultures and mitotic indexes were higher, however, when washed cells and serum were used. ( 4 ) Phytohemagglutinin. Although some cells were stimulated with PHA in a concentration of 0.002 cm3/cm3 of culture medium, a larger and more uniform response was observed in cultures with PHA concentrations of 0.01 cm3/cm'. Twice the latter amount of PHA did not produce a greater number of enlarged cells and many of those cells which responded were degenerated by the third day of culture. Although PHA solutions which had been stored in a refrigerator for a month or more often retained their potency, stock solutions were routinely changed every two weeks. At least ten metaphase plates from the cultured lymphocytes of each of three male and female Lewis animals were analyzed to prepare the karyotype shown in figure 6. The chromosomes are arranged in accord with the recent suggestion of Hungerford and Nowell ('63) who have obtained particularly clear preparations from bone marrow and lymph node cells treated with colchicine in vivo. DISCUSSION It is believed that the applicability of the present culture technique to lymphocytes from various sites in the body as well as its simplicity and reliability constitute advantages over methods previously described. In essence, this method calls only for the addition of washed lymphocytes to culture medium containing 20% fresh serum, penicillin, and an appropriate concentration of PHA (0.01 cm3/cm3 of culture of a l : 5 dilution of the stock solution). No other additives are requisite. It seems probable that the use of 20% fresh serum obviated the need for the additives which Dowd et al. ('64) found essential (fresh glutamine) or helpful (agamma calf serum and folic acid) in their cultures which contained only 5% rat plasma. The use of room air as the gas phase over cultures not only contributes to the convenience of the technique, but, as shown by Nowell ('60), leads to as much growth as may be obtained with many combinations of oxygen and carbon dioxide. It is interesting, however, that unlike the conditions Nowell described for his standard cultures of human leucocytes, it was not found possible to leave culture bottles loosely capped without a loss of carbon dioxide and a deleterious increase in pH. The problem of the percentage of small lymphocytes which can be stimulated to respond to PHA remains to be solved. The percentage of responding cells cannot be reliably estimated from the percentage of enlarged cells found in culture for there is too little known about the number and kinds of cells which die, about the number, proliferative rates and progeny of cells which divide, and about the number of cells which persist but do not divide. It may be that some small lymphocytes are more responsive to PHA than others. This possibility is strengthened by the evidence from this laboratory (Caffrey, Rieke and Everett, '62) and from the laboratory of Fitzgerald ('64) that there are two populations of small lymphocytes with respect to their rates of formation and circulating life span. The karyotype of the rat has been published several times in the last few years (Fitzgerald, '61 ; Hungerford and Nowell, CULTURE OF RAT LYMPHOCYTES '63; Dowd et al., '64) but, unfortunately, differences in the description of centromere location have led to different systems of classification. Most recently, Hungerford and Nowell ('63), in a study directed mainly at sex chromosome morphology, have classified the autosomes so that the largest chromosomes and those having subterminal centromeres are in separate groups. Their system avoids some of the previous difficulties and is followed here (fig. 6). The present study confirms their report that in Lewis rats X chromosomes have subterminal centromeres, Y chromosomes are telocentric and larger than pair no. 13, and that chromosome pair no. 3 appears to be satellited. The variability between a median and submedian location for the centromere in pair no. 19 is also noted (fig. 6, cf. pair no. 19 in male and female). LITERATURE CiTED Aspegren, N., and H. Rcrsn?-:i 1361 Shortterm culture of lymphocj tes from gu..iea-pigs allergic to tuberculin. Int. Arch. Mcrgy, 24: 119-123. Bollman, J. L., 3 . C. Cain and J. H. Grindlay 1948 Techniques for the rollection of lymph from the liver, small intestiiie and the thoracic duct of the rat. J. Lab. Clm. Med., 33: 13491352. Caffrey, R. W., W. 0. Rieke and N. B. Everett 1962 Radioautographic studies of small lymphocytes i n the thoracic duct of the rat. Acta Haemat., 28: 145-154. Carstairs, C. 1962 The human small lymphocyte: Its possible pluripotential quality. The Lancet, i: 829-832. Cooper, E. H., P. Barkhan and A. J. Hale 1963 Observations on the proliferation of human leucocytes cultured with phytohaemagglutinin. Brit. J. Haemat., 9: 101-111. Dowd, G., K. Dunn and W. C. Moloney 1964 Chromosome studies in normal and leukemic rats. Blood, 23: 564-571. Elves, M. W., and J. E. Wilkinson 1962 The effects of phytohaemagglutinin on the morphology of culture leucocytes. Nature, 194: 1257-1259. Everett, N. B., W. 0. Rieke, W. 0. Reinhardt and J. M. Yoffey 1960 Radioisotopes in the study of blood cell formation with special reference 387 to lymphocytopoiesis. Ciba Foundation Symposium on Haemopoiesis, J. & A. Churchill Ltd., London, 43-66. Fitzgerald, P. H. 1961 Cytological identification of sex in somatic cells of the rat, Rattus norvegicus. Exp. Cell Res., 25: 191-193. 1964 The immunological role and long life-span of small lymphocytes. J. Theoret. Biol., 6: 13-25. Galton, M., and S. F. Holt 1963 Culture of peripheral blood leucocytes of the golden hamster. Proc. SOC. Exp. Biol. and Med., 114: 218-219. Humason, G. L., and P. C. Sanders 1963 Culture and slide preparation of leukocytes from peripheral blood. Stain Techn., 38: 338-340. Hungerford, D. A., and P. C. Nowell 1963 Sex chromosome polymorphism and the normal karyotype in three strains of the laboratory rat. J. Morph., 113: 275-285. Ling, N. R., and E. M. Husband 1964 Specific and non-specific stimulation of peripheral lymphocytes. The Lancet, i: 363-365. MacKinney, A. A,, Jr., F. Stohlman Jr. and G. Brecher 1962 The kinetics of cell proliferation in cultures of human peripheral blood. Blood, 19: 349-358. MacNeal, W. J. 1922 Tetrachrome blood stain; a n economical and satisfactory imitation of Leischmann's stain. J. Amer. Med. Assn., 78: 1122-1 123. Marshall, W. H., and K. B. Roberts 1963 The growth and mitosis of human small lymphocytes after incubation with a phytohaemagglutinin. Quart. J. Exp. Physiol., 48: 146-155. Moorhead, P. S., P. C. Nowell, W. J. Mellman, D. M. Battips and D. A. Hungerford 1960 Chromosome preparations of leucocytes cultured f r t m human peripheral blood. Exp. Cell Res., 20: 613-t'16. Nowell, P. C. 1962 Phytohemagglutinin: An initiator of mitosis in cultures of normal human leukocytes. Cancer Res., 20: 462-466. Reinhardt, W. O., and C. H. Li 1945 Cell count, rate of flow, and protein content of cervical lymph in the rat. Proc. SOC. Exp. Biol., N. Y., 58: 321-323. Schrek, R., and Y. Rabinowitz 1963 Effects of phytohemagglutinin on rat and normal and leukemic human blood cells. Proc. SOC.Exp. Biol. and Med., 113: 191-194. Schwarz, M. R., and W. 0. Rieke 1964 The in vitro effect of phytohemagglutinin on cultures of rat thymus cells. In preparation. Tanaka, Y., L. B. Epstein, G. Brecher and F. Stohlman, Jr. 1963 Transformation of lymphocytes in cultures of human peripheral blood. Blood, 22: 614-629. PLATE 1 EXPLANATION OF FIGURES 1 Enlarged, “blast-like’’ cells shown in a smear preparation of rat thoracic duct lymphocytes cultured two and one-half days with PHA. x 2,000. 2 The appearance of the lymphocytes from the thoracic duct a t the time of initiation of cultures leading to cells such as shown in figure 1. Note that while an occasional large cell is present, it is morphologically dissimilar from those large cells which develop with PHA. X 2,000. 3 Enlarged and mitotic cells from a three-day PHA culture of rat blood lymphocytes treated with colchicine and sacrificed in hypotonic medium. x 1,600. 4 Radioautograph of rat thoracic duct lymphocytes cultured with PHA. Following the addition oo H3-thymidine, labeled nuclei and labeled and unlabeled mitotic figures are seen. x 2000. 5 388 Radioautograph of chromosomes from a rat thoracic duct lymphocyte exposed to H3-thymidine during the terminal three hours of a PHA culture. X 2,000. CULTURE OF RAT LYMPHOCYTES William 0. Rieke and M. Roy Schwarz PLATE 1 389 CULTURE OF RAT LYMPHOCYTES William 0. Rieke and M. Roy Schwarz 6 390 PLATE 2 The karyotype of male and female Lewis rats as obtained from thoracic duct lymphocytes cultured with PHA.