IN VITRO

Volume14, No. 8, 1978 All rightsreserved 9

H U M A N S E M E N AS A S O U R C E O F E P I T H E L I A L

CELLS FOR CULTURE

STEPHANIE GORDON PHILLIPS, DAVID M. PHILLIPS, ELVIN A. KABAT, ANDORLANDO J. MILLER Departments of Human Genetics and Development, Microbiology, Neurology and Obstetrics and Gynecology, Columbia University, Collegeof Physicians and Surgeons, New York, New York 10032; and The Population Council, Rockefeller University, New York, New York 10021 (D. M. P.)

SUMMARY When washed cells from human semen samples were plated out, epithelial cultures were obtained. The human ejaculates used as starting material contained, in addition to spermatazoa, 103 to 107 cells of other types, including granulocytes, macrophages, lymphocytes, spermatocytes and epithelial cells. Although no fractionation of cell types was attempted, semen samples yielded epithelial cultures uncontaminated by fibroblasts. The cultured cells appeared characteristically epithelial with a polygonal shape, interdigitating cell membranes, and desmosomes. ABH blood-group antigenic determinants of the donor were expressed with variable frequency as a surface antigen on these cells. About half the trials gave some cell attachment. Most cultures remained as small, tight colonies, but a few reached confluency in about 5 weeks and could be subcultured successfully. Cell proliferation, as monitored by [3H]thymidine incorporation into nuclear macromolecules, ceased in less than 2 months. K e y words: epithelial cell culture; blood group; semen. INTRODUCTION When fragments of minced mammalian tissue are explanted into nutrient medium, mixtures of cell types grow or migrate out from the explant. These cells frequently can be classified into two general types: "spindle-shaped" cells and "polygonal" cells (1). In order to obtain pure epithelial cell cultures, one must usually manipulate the tissue so as to select epithelial components from a histologically mixed cell population. Replacement of the tissue-mincing step by more sophisticated enzyme digestion of intracellular material to yield single-cell suspensions prior to plating (2) may still result in cultures containing both fibroblastic and epithelial elements {3). Upon repeated subculture, the fibroblastic cells tend to overgrow the epithelial cells in the culture (4, 5). Several methods have been devised for obtaining purely epithelial cultures. These include: (a) initiating cultures from epithelial cells "spilled" into medium after cleanly slicing carcinomatous tissue (2}; (b) picking epithelial colonies from histologically mixed cultures using stainless-steel cloning cylinders (6-8}; (c) selecting for epithelial cells by continuous growth in medium containing

D-valine instead of L-valine (9); (d) starting with a tissue carefully dissected or collected so as to eliminate as many connective tissue elements as possible {5, 10-13~; or (e} starting with a proliferating carcinoma and waiting for it to overgrow senescing normal cells ~14). All of these techniques require tedious handling of cells and/or luck to arrive at pure populations of epithelial cells. In this paper we describe a very simple method for obtaining epithelial cell cultures from human semen. These cultures appear to contain only one cell type. MATERIALSAND METHODS Semen samples from volunteers were collected in sterile cups and each was diluted with 50 ml Hanks' BSS plus 1000 U per I penicillin and 1000 gg per ml streptomycin. All volunteers were scientists and were instructed to maintain sterility if possible. Processing of samples was begun within 2 hr of ejaculation. Semen samples also were obtained anonymously when they were due to be discarded by the diagnostic fertility laboratory. These samples were not handled aseptically until

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they reached us 3 or 4 hr after collection at which time they were diluted with 50 ml Hanks' BSS plus antibiotics. Somatic cells present in the semen sample were washed three times in Hanks' BSS plus antibiotics by centrifuging at 200 x g for 10 min and resuspending the cell pellet in the same salt solution. Semen itself was found to be toxic to cultured cell lines, but repeated washing removed the toxic substances. Most sperm were decanted in the washes since they did not pellet as quickly as the cells during centrifugation. No attempt was made to eliminate the remaining sperm. Cells from each semen sample were plated into five Falcon plastic T30 flasks in various media. Satisfactory epithelial cell growth was obtained in Eagle's M E M (Hanks' baseJ, Ham's F10, R P M I 1640, McCoy's 5a, or medium 199, supplemented in each case with 10% heat-inactivated fetal bovine serum. In most cases, Eagle's M E M was used. Cultures were fed 4 days after initiation of the culture and thereafter either at intervals of 14 days or more frequently when cell growth was extensive. Cultures that grew to confluency lin some cases 6 to 8 weeks after initiationJ could be subcultured successfully after dislodging the cells with a trypsin-EDTA solution (Grand Island Biological Co. J.

Electron microscopy. For scanning-electron microscopy, cells were grown on glass cover slips, fixed in 2.5% collidine-buffered glutaraldehyde, pH 7.4, dehydrated in increasing concentrations of ethanol, and transferred to acetone. Cover slips were dried with liquid CO2 in a Sorvall Critical Point Drying System, then coated with gold, using an Edwards 306 coater, and viewed in an E T E C Autoscan. For transmission microscopy, cells were grown on Falcon plastic petri dishes, fixed in 2.5% collidine-buffered glutaraldehyde, postfixed in 1% OsO,, dehydrated through alcohol, and embedded in Epon. Washed cells in the pellets obtained from semen samples were fixed in collidine-buffered glutaraldehyde, postfixed in 1% OsO4, and processed as above. Radioautography. Cultures used for radioautography contained about 4 x l0 s cells in 2 ml M E M with 10% fetal bovine serum in Falcon plastic petri dishes (35-mmJ. Cultures were fed 72 hr before adding [3H]thymidine in fresh medium (final concentration of 0.5/~Ci per ml, Sp. Act. 0.23 Ci per mMJ. After 20 hr incubation at 37 ~ C, cultures were fixed in acetic acid: ethanol 3:1 for 1 hr, washed in 70% ethanol, and air dried. The bottoms of the petri dishes were cut out and mounted on glass slides before dipping in Kodak

TABLE 1 SUCCESSR ATE IN O B T A I N I N G C ELL C ULTURES FROM S EMEN Donor

V1 V2

Blood Type of Donor

O A2

Sample No.

12-30 1-8 6-10 7-20 8-6 10-14 10-15 10-20 11-1 1-5 1-27 1-15

S AMPLES FROM VOLUNTEER D O N O R S No. Viable Cells per Semen Sample

Growth in Culture after 1 Month a

N.D. b ++ N.D. +++ N.D. -4-++ N.D. +++ N.D. -4-+ 1.3 x 106 + 2.6 x 103 -42.8 x 106 Contaminated 2.8x 106 +-43.4 x 106 Contaminated 4.5x l0 s +++ V3 A, N.D. -4V4 O 1-20 N.D. Contaminated V5 O 2-10 N.D. -3-15 N.D. -V6 B 5-5 N.D. +++ V7 A, 8-10 N.D. -8-17 N.D. -V8 B 12-7 9 x 106 ++ V9 B 1-11 N.D. + V10 A, 4-2 6.2 x 106 + VII A~ 10-7 N.D. ++ V12 A~ 10-21 3.1 x 106 +++ a No attachment ~--); single cells attached (-4-j; coloniesformed (+ + j; reached confluency(+ + + ~. b N.D. ----not determined.

EPITHELIAL CULTURES FROM SEMEN

FIG. 1-6. Examples of some of the cell types commonly seen in semen samples.

641

FIG. 3. Lymphocyte. x8000.

FtG. 1. Neutrophil. x6000.

FIGS. 4 ~x8000 ) and 5 ~x9000). Possibly macrophage or fibroblast.

FtG 2. Spermatocyte. x3500.

FIG. 6. Unknown cell type. x6000.

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PHILLIPS ET AL.

N T B - 2 emulsion. T h e radioautographs were developed 1 week later at 18 ~ C in K o d a k D-19 developer and stained 7 min with 0.025% Azure B, pH 4.0, at 4 ~ C. T h e number of labeled nuclei in 1000 cells chosen at random was determined for each petri dish. T h e values for two dishes were averaged and the percent of labeled nuclei calculated. Assay for blood group A B H substances. T h e presence of blood-group A B H antigenic determinants on cell surfaces was assayed by mixed agglutination. A suspension of cells was obtained by trypsinization of monolayer cultures with two rinses of t r y p s i n - E D T A (GIBCO). T h e cells were washed three times by suspension in phosphatebuffered saline (PBS~ followed by centrifugation and were finally suspended in 0.1 ml PBS. GroupA determinants were detected using 25/~1 h u m a n anti-A serum SM, titer 512 (15, 16), B-determinants using 25 /~1 h u m a n anti-B serum 310D4, titer 256 (17, 18), and H-determinants using 100 /M of an extract of Ulex europeus seeds, titer 16 (19). After 30 min at room temperature, the cells were washed four times by centrifugation and resuspended in PBS. 0.1 ml of a 0.1% suspension of washed h u m a n red blood cells of the appropriate group was added to the cell pellet, and the suspension was mixed and centrifuged. The pellet was resuspended gently and a drop placed on a slide. T w o fragments of cover slip were placed near the drop and a cover slip supported between

them to form a bridge with the cells floating f r e e l y beneath. It was possible to determine whether red cells in a presumptive rosette were firmly adherent by gently tapping the slide. Rosetted (more than three red cells attached) and nonrosetted cells were counted under phase optics at a x400 magnification.

Determination of blood group substances in semen. The A B H blood group of each volunteer semen donor was determined by agglutination of his red blood cells with commercial anti-A and anti-B sera, crude Ulex europeus extract (Hspecific} (19), and crude Dolichos biflorus extract (A,-specific) (20). Patient information was not available to us about the anonymous donors of semen from the fertility clinic. In the case of secretors, the blood group of the donor could be determined by inhibition in Takatsy microtiter plates of hemagglutination of red ceils of the appropriate group by four hemagglutinating units (HU) of the above mentioned alloagglutinins or lectins (21). RESULTS

Cells in semen. The 29 semen samples for which cell counts were done contained 2 x 103 to 9 x 10 ~ viable (trypan-blue-excluding) nonsperm cells. The number varied widely even in different samples from a single donor (Table 1). The limited information available suggested no correlation between time since previous ejaculation and number of nonsperm cells present in semen.

TABLE 2 SUCCESS RATE IN OBTAINING CELL CULTURES FROM SEMEN SAMPLES FROM THE FERTILITY LABORATORY

Donor

Blood Type of Donor

Sample No.

No. Viable Cells per Semen Sample

Growth in Culture after 1 Month a

F1 B 1-31 1.4x 105 +++ F2 A 2-7 2 x 103 -F3 A 2-8 1 x 105 + + F4 AB 2-9 1 x 106 ++ F5 ? 2-16 2 x 104 -F6 AB 2-17-1 1.2 x l0 s -F7 A 2-17-2 2.5 x 10" -F8 A 2-24-1 1.5 x 104 -F9 ? 2-24-2 1.8 x 104 -F10 ? 3-11 2 x 106 -Fll 0 3-31 2 x 10~ + F12 0 4-13-1 2.3 x 105 ++ F13 0 4-13-2 5.5 x 104 -F14 B 4-14-1 5.5x 106 +++ F15 A 4-14-2 1 x 10" -F16 A 4-20-1 1 x 10" -F17 ? 4-20-2 1.8 x 105 -F18 O 4-27 8 x l0 s + F19 B 5-12 2.5x 10~ -F20 A 5-18 2 x 10~ -a No attachment (--); single cells attached ( + ); colonies formed ( + + ); reached confluency ( + -4-+ ).

EPITHELIAL CULTURES FROM SEMEN

643

FI6.7. Light micrograph of colony of cells cultured from semen of volunteer 2 (type A2}at the second passage. Cells are tightly juxtaposed and polygonal, x1200. Examination of the pelleted cells by electron microscopy revealed several cell types based on morphological criteria. Some cells could be classified as neutrophils, lymphocytes, spermatids, or spermatocytes; others were epitheloid. Many cells were difficult to classify t Fig. 1-6L Growth of cells. Three of the 21 semen samples from volunteers gave rise to contaminated cultures lTable 1); none of the 20 samples from the fertility laboratory did so lTable 2). Attachment of cells to the culture vessel was noted after 1 to 2 days. This occurred in a significantly higher proportion of samples from healthy volunteers ~16/20) than in samples from the fertility laboratory ~7/20). Growth of cells into colonies tfirst noted about 2 weeks after platiugj occurred in 55% Ill/20~ of the volunteer samples but in only 25% ~5/20} of the fertility laboratory samples. A corresponding difference was noted in the number of cultures that grew to confluency ~6/20 and 2/20, respectivelyj after about 5 weeks and could be subcultured. Although these numbers are small, they point to a possible lower percentage of successful cultures from the fertility laboratory, perhaps reflecting delay in processing semen samples or even a relationship to the infertility of most of these donors. The age of volunteer donors

ranged from 29 to 60. All were in good health; 8 of 12 had sired children, lThese data are not presented in tabular form to preserve confidentiality of volunteers.) There was no obvious correlation between age or proven fertility of donor and success in obtaining cultures. Cultures derived from semen appeared to be epithelial. Cells in colonies adhered closely to one another, and the colonies had a closed edge with thinner cells often making up a border ~Fig. 7). In the electron microscope, the cells resembled squamous epithelium. Numerous interdigitations were observed between adjacent cells lFig. 8) and desmosomes were common (Fig. 9). In three successful cultures examined in the scanning-electron microscope, no evidence of contaminating mycoplasma was found le.g. Fig. 13~ although scanning E M is a sensitive method of mycoplasma detection ~22, 23L Two cultures tested for D N A synthesis by radioautography also showed no evidence of mycoplasma ~e.g. Fig. 11 t. Thus a sampling of successful cultures did not suggest that mycoplasma contamination was a problem. Mycoplasma are common in the male tract; it is not known to what extent their presence may have affected cell growth in unsuccessful cultures.

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PHILLIPS ET AL.

Blood group antigenic determinants on the cell surface. Semen is known to be rich in soluble blood group substances (24, 25). Eighteen of the

24 semen samples tested contained material that inhibited agglutination of group-O red cells by 4HU Ulex europeus extract at dilutions ranging

FIG. 8. Transmission-electron micrograph of cell cultured from semen at the first passage. Note characteristicallyepithelial polygonal shape and interdigitatingcell membranes, x7000. FIG. 9. Higher magnification view of membranes of three juxtaposed cultured cells from semen showing desmosomes, x35,000.

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EPITHELIAL CULTURES FROM SEMEN TABLE 3 INHIBITION OF HEMAGGLUTINATION BY SOLUBLE A,

n

AND n SUBSTANCES IN SEMEN

Hemagglutination Inhibition Titer a Against: Sample No.

Anti-A

Dolichos biflorus

Anti-B

Ulex

Presumed Phenotype of Donor

12-7 (known B) 0 0 80 10,800 B, Se 1-31 0 0 320 5,120 B, Se 2-7 106 2 0 4,530 A, Se 2-8 107 5 0 145 A, Se 2-9 80 2 155 620 AB, Se 2-16 0 0 0 0 se 2-17-1 b 495 0 110 >7,070 AB, Se 2-17-2 151 2 0 4,660 A, Se 2-24-1 36 18 0 9,230 A, Se 2-24-2 0 0 0 0 se 3-11 2 4 2 0 se 6-10 (known A2) 170 0 0 15,300 A, Se 3-31 0 0 0 64 O, Se 4-2 (known A,, se) 0 0 0 0 se 4-13-1 0 0 0 >4,100 O, Se 4-13-2 0 0 0 597 O, Se 4-14-1 0 0 38 152 B, Se 4-14-2 20 0 0 >2,500 A, Se 4-20-1 2400 4 0 626 A, Se 4-20-2 0 0 0 0 se 4-27 0 0 0 238 O, Se 5-12 0 0 54 108 B, Se 5-18 >3210 8 0 403 A, Se 10-21 (known A,, se) 0 0 0 0 se a Inhibition of measured semen samples diluted by known volumes was tested against 4HU of human anti-A serum SM with A, red blood cells, 4HU of crude Dolichos biflorus seed extract with A, red blood cells, 4HU human anti-B serum 310 D4 with B red blood cells, and 4 HU crude Ulex europeus seed extract with O red blood ceils. In this assay, 1.6 ~g of a standard A substance, cyst 9 (51, 52), gave the end point in hemagglutination. Cyst 9 at concentrations up to 51.2 ~g per ml did not inhibit 4HU of Dolichos biflorus lectin with A, erythrocytes. b Sample numbers assigned in order by date. from 145 to more than 15,000 (Table 3}. In addiIn the electron microscope, red cells involved in tion, semen samples from fourteen presumed A, rosettes were deformed and appeared bound to B, or AB donors contained substances that inhibthe epithelial cells only at intervals along their ited the agglutination of the corresponding red surfaces (Fig. 14). The percentages of rosettes formed after incubation with anti-A serum and cells at dilutions from 20 to more than 3900 (Table 3}. In the three cases in which the ABH group-A red cells were 28, 19, 5 and 0 in four separate cultures. The frequency of rosettes was not status of the donor was known, the inhibitory subchanged by addition of 250/~g per ml of a purified stances in semen were of the expected type. Six of B substance (Beach OOH insoluble) (27} but was the 21 semen samples were negative for soluble A, reduced to zero by addition of 330 g g / m l of an A B, or H substances when tested by inhibition of substance M S M 10% or 1 mg per ml of A RL 0.52 agglutination. In these cases the donors were probably nonsecretors [in the general population [a blood-group-A specific reduced pentasaccharthe expected frequency of nonsecretors is about ide (28)]. In cells from the same four cultures, 23% (26}]. Support for this conclusion comes anti-B followed by group-B red cells produced no from the finding that saliva from two donors of rosettes; Ulex europeus or Lotus tetragonolobus noninhibiting semen samples had no inhibiting lectin and O red cells produced 0% to 3% rosettes power as expected for nonsecretors. There was no indicating the presence of very little H substance; correlation between the A B H or secretor status of and Dolichos biflorus followed by type A~ red a donor and the number of nonsperm cells in his cells produced no rosettes. These findings suggest ejaculate. that A2 determinants (which are expected to react Some cells from a successful culture from an A2 with A-specific and H-specific reagents} are presdonor formed big, unambiguous rosettes when asent on the surface of some of these cells. sayed by mixed agglutination for the presence of B determinants on cultured cells from a groupsurface blood-group antigens (Figs. 10, 12, 14). B donor were assayed by mixed agglutination 3

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P H I L L I P S E T AL.

FIG. 10. Example of a rosette, as seen in the light microscope, indicating the presence of bloodgroup-A antigenic determinants on a cell cultured from semen of an A2 donor 2 m o n t h s after initiating the culture. Such rosettes could be dissociated by the addition of A substance. FIG. 11. Radioautograph of cells cultured from semen sample n u m b e r 1-31 ~blood group B) pulsed with [3H]thymidine after 4 weeks in cuhure. A series of such radioautographs was scored to yield the data shown in Fig. 12.

months after the initiation of the culture at the 70 second passage. The cells were coated with antiB, washed, split into three tubes, and incubated I 60 I with A, B, or O red cells. No rosettes were found I in the control cells incubated with A or O red I I -_~ 5o blood cells; 31% of the epithelial cells formed ros| ettes in the cells incubated with B erythrocytes. Mixed agglutination using anti-A and AI erythro- _2 4o cytes yielded no rosettes; Ulex and type O erythro- Z cytes gave 1% rosettes, perhaps because of the _qo 3 O presence of some incomplete carbohydrate chains (18). ~ 20 n + In cultures from group O donors there were not enough cells to assay for the presence of surface I0 blood-group determinants by rosetting. However, such cells have been used as the human parental i cell line for human-mouse somatic cell hybrids. 4 6 8 I0 12 14 o The presence of H substance on the hybrid cells Weeks in culture was demonstrated by mixed agglutination with Fit;. 12. Uptake of [~H]thymidine by cells cultured Ulex lectin and O erythrocytes (unpublished data). This suggests that some cells of the original from h u m a n semen as a function of time after initiation of the culture. Decreasing proliferation rate is seen in human cell line synthesized H substance. two independent epithelial cell cultures, one started from Blood-group positive cells occurred with vari- semen sample 1-27 ( x - - x ) and the other from sample able frequency in these morphologically homo1-31 {O . . . . 9 ). [3H]Thymidine was added for 20 hr begenous epithelial cultures. In order to determine fore fixing the cells, and labeled nuclei were scored in whether positive cells gave rise to positive cells, radioautographs. No determinations were made before 4 weeks of culture because too few cells were available. we subcultured a primary culture of A: epithelial C u h u r e 1-31 grew rapidly at first, while culture 1-27 cells, plated the single cells, and waited 2 weeks was slow-growing. Both cultures ceased synthesizing for colonies to form. Mixed agglutination was per- D N A by about 10 weeks.

EPITHELIAL CULTURES FROM SEMEN

647

F 16.13. Scanning-electronmicrograph of colonyof second subculture cellsfrom semen of an As donor 22 weeks after starting the culture. Mixed agglutination with anti-A antiserum and group A red cells was carried out on cells growing on a cover slip. Rosetted cells appear to be randomly distributed in the colony, x200. F 16.14. Transmission-electron micrograph of a rosetted cell from the same culture as shown in Fig. 13. Red ceils are deformed and only adhere to the epithelial cell restricted areas. • formed in situ by pouring off the medium, flooding the monolayer with anti-A serum, washing, and adding 0.1% group A red ceils. Although rosettes were scattered randomly through the colonies t Fig. 13), completely positive colonies were not seen. Thus individual cells within in a single clone behaved autonomously with respect to expression of blood group activity. Growth potential of epithelial cells. None of the cultures derived from semen continued growing for more than about 2 months. After that time, although the cells were viable and reattached to the plastic after subculture, they did not multiply. The decreased growth potential was reflected in a decreasing rate of DNA synthesis, monitored radioautographically ~Fig. 11), with time after initiation of the cultures. Fig. 12 shows the percentage of labeled nuclei in cultures from two semen samples after 20 hr incubation with

[3H]thymidine. The percentage of cells that incorporated [3H]thymidine into acid-insoluble material fell to almost zero at 9 weeks. D ISCUSSION Our results indicate that semen samples can be used to obtain short-term cultures of purely epithelial cells; the other types of cells in semen apparently do not produce colonies under these conditions. The ceils may be identified with certainty as epithelial since epithelial cells are characterized by desmosomes, and cell surface expression of ABH blood-group antigens is limited to red blood cells and epithelial derivatives (29). The ease of initiating cultures from semen and the general accessibility of the starting material without recourse to medical facilities make this a useful system for investigations involving cultures of

648

PHILLIPS ET AL.

normal human epithelial cells. It is apparent from our experience with samples from the fertility laboratory that speed or careful aseptic handling of fresh material are not absolutely essential for obtaining successful cultures. Cultures of normal human epithelial cells have been started from kidney (9), umbilical cord endothelium (30, 31), bladder washings (32), prostate (33, 34), liver (35, 36), esophagus (35), and amniotic fluid i37). The elimination of fibroblast contamination from such epithelial cultures required either physical dissection of epithelial from connective tissue components in the original tissue, differential manipulation or cloning of cells growing out of explants, chemical selection against fibroblasts, or luck. All of these approaches are tedious and successful to rather limited degrees. Use of postweaning milk (38) or of urine (32, 39) as a source of epithelial cells for culture eliminates the need for purification, but postweaning milk is not readily obtainable. We and others i39) have not been successful in attempts to culture cells from urine although good success has been reported (32). It is unclear whether the same type of cell is responsible for the growth of epithelial cell colonies from both urine and semen. In the present study epithelial cultures from adults ceased dividing in less than 2 months. Some human epithelial cell cultures have become permanent cell lines. These were generally derived from malignant tissue, but in vitro transformation of normal human amnion ceils (40) to yield a permanent cell line also has occurred. It is not clear why the epithelial cultures from semen ceased dividing after growing vigorously at first. It may be that a factor necessary to maintain growth and division is missing from the medium, or the cells may be inherently capable of only a limited number of divisions. The epithelial cells in semen-derived cultures express blood-group ABH determinants. The expression of cell surface antigenic determinants is a differentiated function seen only on certain cell types and only at certain times during development. The expression of ABH blood-group antigenic determinants on the cell surface is characteristic of certain epithelial cell types such as blood-vessel endothelium (41); squamous epithelium of the skin, tongue and cervix (29); some cells of the columnar epithelium of stomach and intestine such as parietal cells, pyloric gland epithelium, cells of Brunner's gland and goblet cells i42); transitional epithelium of bladder; and cells lining sinusoids of hepatic parenchyma i43). There are

many other examples of tissues in which the surfaces of some cells are positive for blood-group antigenic determinants while others are negative (41). In addition, cells that lack blood-group determinants at one stage of development may express them at another stage (29, 41). Carcinomas derived from cell types that express blood-group determinants may not express such antigens (43, 44) and vice versa (45, 46). Thus it is not unexpected that the expression of antigenic determinants is variable in cultured cells. Variation in expression of ABH blood-group antigenic determinants has been observed in a number of cultured epithelial lines using mixed agglutination. In amniotic-fluid cultures, Friedhoff and Kuhns i37) have observed a variable proportion (4% to 49%) of A, B or H positive cells, with the frequency of positives unrelated to the length of time in culture. H6gman (47) has found varying degrees of expression of ABH determinants in cultured fetal kidney, liver, spleen, lung, heart and skin, with some cultures from each tissue being completely negative. Cultures of endothelial cells fi'om umbilical cord also have been reported to have A, B or H determinants (30). Cells lacking blood-group antigenic determinants can arise from positive cells, and vice versa, in rabbit epithelial lines (48) and in HeLa cells (49). Cultured epithelial cells from semen provide an additional example of a situation in which there is variation in expression of ABH blood-group antigenic determinants. In an earlier study, we found that human semen contains diploid cells that can fuse spontaneously with cultured mouse cells to yield human-mouse hybrid cell lines (50). More recent studies have shown that the epithelial cells from semen-derived cultures can be hydridized with R A G / R A M cells, from a hypoxanthine-guanine phosphoribosyl transferase-deficient mouse cell line, using inactivated Sendai virus, and some of the hybrid cells express human blood-group determinants (Phillips, Kabat and Miller, unpublished data). It may be possible to map the genes controlling the expression of these epithelial cell surface antigenic determinants, and other membrane components that are similarly expressed, by correlating the loss of expression of these antigens with the loss of human chromosomes from these hybrids. Such work is in progress. REFERENCES 1. Carrel, A., and M. T. Burrows. 1910. Cultivation of adult tissues and organs outside the body. J. Am. Med. Assoc. 55: 1379-1381.

E P I T H E L I A L CULTURES FROM SEMEN 2. Lasfargues, E. Y., and L. Ozzello. 1958. Cultivation of human breast carcinomas. J. Nat. Cancer Inst. 21: 1131-1147. 3. Lasfargues, E . Y . , and D . H . Moore. 1971. A method for the continuous cultivation of mammary epithelium. In Vitro 7: 21-25. 4. Kreider, J. W. 1970. Stimulation of DNA synthesis of rat salivary gland cells in monolayer cultures by isoproterenol. Cancer Res. 30: 980-983. 5. Wigley, C. B., and L. M. Franks. 1976. Salivary epithelial cells in primary culture; characterization of their growth and functional properties. J. Cell Sci. 20: 149-165. 6. Brown, A. M. 1973. In vitro transformation of submandibular gland epithelial cells and fibroblasts of adult rats by methylcholanthrene. Cancer Res. 33: 2779-2789. 7. Coon, H. G. 1968. Clonal culture of differentiated rat liver cell (abstr.). J. Cell Biol. 39: 29. 8. Yasumura, Y. A., H. Tashjian, and G. H. Sato. 1966. Establishment of four functional elonal strains of animal cells in culture. Science 154: 1186-1189. 9. Gilbert, S. F., and B. R. Migeon. 1975. D-Valine as a selective agent for normal human and rodent epithelial cells in culture. Cell 5:11-17. 10. Chang, R. S. 1954. Continuous subcultivation of epithelial-like cells from normal human tissues. Proc. Soc. Exp. Biol. Med. 87: 440-445. 11. Kubns, W. J., Y. Faur, S. Bramson, and F. Friedhoff. 1969. Studies on the variability of ABH blood group antigens on ceils in primary culture. Proc. Soc. Exp. Biol. Med. 131: 67-70. 12. Gallagher, J. G. 1973. Rabbit kidney and skin. In: Paul F. Kruse, Jr., and M. K. Patterson, Jr. (EdsA, Tissue Culture Methods and Applications. Academic Press, New York, pp. 102-105. 13. Ricard, M. A., and R. J. Hay. 1976. Proliferation and agglutinability of primary and transformed human epithelial cells in culture. J. Cell Sci. 21: 553-561. 14. Kondo, T., H. Muragishi, and M. Imaizumi. 1971. A cell line trom a human salivary gland mixed tumor. Cancer 27: 403-410. 15. Lundblad, A., and E. A. Kabat. 1971. Immunochemical studies on blood groups. XXXV. The activity of fucose-containing oligosaccharides isolated from blood groups A, B and H substances by alkaline degradation. Biochemistry 5: 1502-1507. 16. Moreno, C., A. Lundblad, and E. A. Kabat. 1971. Immunochemical studies on blood groups LI, a comparative study of the reaction of A, and A2 blood group glycoproteins with human anti-A,. J. Exp. Mcd. 134: 439-457. 17. Allen, P . Z . , and E, A. Kabat. 1959. Immunochemical studies on blood groups. XXII. Immunochemical studies on the nondialyzable residue from partially hydrolyzed blood group A, B and O~H} substances iP1 fractions). J. Immunol. 82: 340-357. 18. Maisonrouge-McAuliffe, F., and E . A . Kabat. 1976. Immunochemical studies on blood groups; fractionation, heterogeneity, and chemical and immunochemical properties of a blood group substances with B,I, and i activities purified from

19.

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Aided by grants from the National Science Foundation (BMS-72-02219 A04 and P C M 76-81029 to E. A. K.), the Public Health Service (CA 12504 to O. J. M.), and the National F o u n d a t i o n - M a r c h of Dimes. The earlier portions of this study were carried out under a Program Project Grant from the National Institutes of Health (No. 5 P O G M 18153).