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Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres
Journal of Immunological Methods, 119 (1989) 259-268 Elsevier
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JIM 05159
Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres G o r d o n C. Douglas and Barry F. King Department of Human Anatomy, School of Medicine, University of California, Davis, CA 95616, U.S.A. (Received 20 September 1988, revised received 12 December 1988, accepted 13 December 1988)
A procedure has been developed which yields a pure population of villous cytotrophoblast from term human placenta. As a first step, a cell preparation highly enriched for cytotrophoblast (identified by positive cytokeratin staining) was obtained using a modification of the method of Kliman et al. (1986). The remaining contaminating cells (identified by positive vimentin staining) were then removed by treatment with mouse monoclonal antibodies against class I and class II major histocompatibility antigens followed by magnetic microspheres coated with goat anti-mouse IgG. The rationale for this step was based on the fact that villous trophoblast fails to express HLA antigens whereas cells from the villous mesenchyme do express these surface antigens. Rosetted cells were immobilized using a magnet allowing the non-rosetted cells to be easily withdrawn by pipette. When the non-rosetted cells were placed in primary culture, no HLA-positive or vimentin-positive cells could be detected using immunofluorescence microscopy, indicating complete removal of these components by the immunomagnetic separation procedure. The cells were positive for cytokeratin and, after 24 h, showed positive staining for pregnancy-specific/31-glycoprotein (SP1) and human chorionic gonadotropin. Recovery of cytotrophoblast was greater than 92% with only a slight loss of viability Key words: Cytotrophoblast; Placenta; Magnetic microsphere
Introduction
Many procedures have been described for the isolation of cytotrophoblast cells from human placenta (Hall et al., 1977; Lobo et al., 1985; Kliman et al., 1986; Daniels-McQueen et al., 1987; Loke and Burland, 1988; see reviews by Stromberg, 1980; Loke, 1983). However, the nature of the isolation procedures and the cellular heterogeneity of the placenta itself usually dictates that a mixed cell population is obtained, which can, at Correspondence to: G.C. Douglas, Department of Human Anatomy, School of Medicine, University of California, Davis, CA 95616, U.S.A.
best, be described as highly enriched for cytotrophoblast. Contaminating cells may include placental macrophages, fibroblasts, endothelial cells and blood elements. Where estimates of cytotrophoblast purity are reported, these vary from 40 to 95%, depending on the isolation procedure used. Several laboratories have produced monoclonal antibodies reactive against trophoblast cell surface antigens (Lipinski et al., 1981; Butterworth and Loke, 1985; Contractor and Sooranna, 1986; Loke et al., 1986) and these have proved useful in identifying trophoblast in mixed cell populations. Contractor and Sooranna (1988) used a trophoblast-specific antibody and a panning technique in an attempt to isolate cytotrophoblast
0022-1759/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
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from a placental cell suspension, but still obtained a significant number of fibroblasts and macrophages. Kawata et al. (1984) used a variety of monoclonal antibodies, including a trophoblastspecific antibody, and the fluorescence activated cell sorter, to characterize and separate placental cells. They were able to obtain a purified cell preparation which was 'mostly cytotrophoblast'. However, the specialized equipment and reagents required are not readily available to all laboratories, particularly for use on a routine basis. To our knowledge, no procedure has been described which will yield a pure population of cytotrophoblast, which is simple to perform and which makes use of commonly available reagents. Kliman et al. (1986) described a procedure for cytotrophoblast purification which involved trypsin digestion of term villous tissue followed by discontinuous Percoll gradient centrifugation. Using a modification of this procedure as a first step and adding an entirely new step, we have been able to obtain a cell preparation which consists entirely of cytotrophoblast cells. The additional step, which is described in detail here, takes advantage of the fact that villous trophoblast is one of the few tissues to lack major histocompatibility antigens on its surface (Faulk and Temple, 1976; Sunderland et al., 1981; Bulmer and Johnson, 1985). HLA-positive contaminating cells were removed using commercially available monoclonal antibodies to class I and class II major histocompatibility antigens and immunomagnetic microspheres, leaving pure cytotrophoblast cells.
beads were purchased from Pharmacia Fine Chemicals, Piscataway, NJ. Magnetic microspheres (Dynabeads M-450) coated with goat anti-mouse IgG and a magnetic particle concentrator were obtained through Robbins Scientific, Mountainview, CA.
Antibodies Mouse monoclonal antibodies were obtained as follows: anti-cytokeratin 18 and anti-vimentin (clone v-9) were from ICN Immunobiologicals, Lisle, IL. Anti-HLA-ABC (IgG2a, monomorphic) and a n t i - H L A - D R (IgG2b, monomorphic) were from Chemicon International, Los Angeles, CA. Polyclonal goat anti-mouse IgG was obtained as a F(ab')2 fragment conjugated with fluorescein isothiocyanate (FITC) from Accurate Chemical and Scientific Corp., Westburg, NY.
Preformed continuous Percoll density gradients Percoll (38 ml) was mixed with a 10 x stock solution of Hanks' balanced salt solution (10 ml) and made to a final volume of 100 ml with deionized water. This gives an isotonic Percoll suspension with a density of 1.055 g / m l . The isotonic Percoll was dispensed into 50 ml polycarbonate centrifuge tubes (35 ml Percoll/tube). A continuous gradient was then formed by centrifuging the tubes at 30000 x g in an angle rotor (Sorvall, SS-34) for 15 min. The shape of the gradient was routinely checked using density marker beads (Pharmacia Fine Chemicals, Piscataway, N J).
Preparation of trophoblast cells" Materials and methods
Materials Hanks' balanced salt solution (without C a 2~ and Mg 2+) was obtained from Gibco Laboratories, Grand Island, New York. Waymouth's MB 752/1 medium, H a m ' s F12 medium and an antibiotic/antimycotic mixture were purchased from Sigma Chemical Company, St. Louis, MO. Trypsin (a 2.5% solution containing 125 U / m l ) and DNase I (from bovine pancreas) were from Boehringer Mannheim, Indianapolis, IN. Newborn calf serum was obtained from Irvine Scientific, Santa Ana, CA. Percoll and density gradient marker
The initial cell isolation procedure was based on the method described by Kliman et al. (1986). The main differences are that a continuous rather than a discontinuous density gradient is used in the present study and an additional purification step using magnetic microspheres has been developed. For clarity, the entire procedure is described here. All solutions were autoclaved or sterile filtered before use. Term placentas were obtained at the time of Cesarian section. Occasionally, placentas from spontaneous deliveries were used. About 30 g (wet wt.) of villous tissue was dissected from the placenta, care being taken to avoid obvious connective tissue and decidua. The tissue was washed with 0.9% sodium chloride over a stainless
261 steel mesh and coarsely chopped using scissors. The tissue fragments were washed again over the wire mesh and then placed in a conical flask with 150 ml Hanks' balanced salt solution, containing 25 m M Hepes (N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid), p H 7.4, trypsin (0.25%, 12.5 U / m l ) and D N a s e (0.2 m g / m l ) . The mixture was incubated in a shaking water bath for 30 min at 3 7 ° C . About 100 ml of the mixture was then decanted into a measuring cylinder and allowed to stand for 1 min after which 80 ml were carefully pipetted out (avoiding large tissue fragments) and layered over 5 ml fetal bovine serum in 50 ml plastic centrifuge tubes. The tubes were centrifuged at 2200 rpm (1000 x g ) for 5 min after which the supernatant was discarded and the pellet retained. The residue in the cylinder was added back to the tissue fragments remaining in the conical flask and the digestion was repeated twice more with 100 ml and 75 ml, respectively, of fresh t r y p s i n / D N a s e solution. On each occasion the mixture was decanted and centrifuged. The pellets from all three digestions were pooled and made to a final volume of about 25 ml in Waymouth's medium supplemented with fetal bovine serum (17%) and antibiotics. The suspension was poured through four layers of cheesecloth and then through two layers of 0.1 m m polyester mesh before being centrifuged at 1000 × g for 10 min. The pellet was re-suspended in Hanks' balanced salt solution to a final volume of 6 ml. The suspension was divided into two 3 ml fractions each of which was carefully layered on top of a preformed, continuous gradient of isotonic Percoll (35 ml) in a 50 ml polycarbonate tube. The tubes were spun at 400 x g for 15 min after which the material sedementing at densities between 1.053 and 1.060 g / m l was collected. Densities were determined by comparison with an identical gradient run with density marker beads. The collected Percoll fractions were diluted four-fold with culture medium and centrifuged at 1000 x g for 5 min. The supernatant containing the Percoll was discarded and the pellet (cells) was re-suspended in culture medium. Between 3 - 6 x 10 7 cells were obtained from 30 g villous tissue. At this point the cells were either further purified by immunomagnetic separation (see below) or were frozen and stored in liquid N 2 until needed,
whereupon they were thawed and then subjected to the immunomagnetic separation procedure.
Storage of cells under liquid N, The cell pellet was suspended in cold 10% ( v / v ) dimethylsulfoxide in culture medium to give 5 - 1 0 X 10 6 cells/ml. The suspension was aliquoted into 1.8 ml cryogenic vials (Costar) which were then placed in insulated cardboard containers and kept at - 7 0 ° C for at least 1 h. The tubes were then rapidly transferred to liquid nitrogen for long term storage. When required, a tube was removed from the liquid nitrogen and thawed in a water bath at 37 o C. The thawed cells were diluted 1 / 1 0 in culture medium and centrifuged at 400 x g for 5 min. The cell pellet was re-suspended in fresh culture medium to the required cell density.
Irnmunomagnetic cell separation Cells (either freshly isolated or thawed after storage in liquid Nz) were suspended in ice-cold medium containing 17% newborn calf serum to give 4 x 10 6 cells/ml. All subsequent steps were performed in the cold. For simplicity, the following description is based on processing 1 ml of cell suspension. However, the procedure can be scaled up as required. To the suspension (1 ml) was added 20/11 of a monoclonal anti-HLA-ABC antibody and 40 ffl of monoclonal a n t i - H E A - D R antibody. The antibodies were added undiluted. The amounts used were such that when mixed with the cell suspension the resulting dilution would be equivalent to that recommended by the manufacturer for immunofluorescence staining. The mixture was incubated on ice for 45 min with occasional gently shaking and then centrifuged at 350 x g for 5 rain. The supernatant was removed and the cell pellet was re-suspended in Hanks' balanced salt solution (without C a 2+ and Mg 2+) supplemented with 1% newborn calf serum. Centrifugation and resuspension was repeated two more times. After a further centrifugation the pellet was finally re-suspended in medium (sup-~ plemented with 1% newborn calf serum) at a ratio of 1 ml medium : 4 x 106 cells. To this was added 40 ffl of a suspension of magnetic microspheres coated with goat anti-mouse IgG. The amount added was calculated using the manufacturers f~rmulae and by assuming the target cells (i.e., con-
262
taminating cells) accounted for 5% of the total cell population. The mixture was placed on a rotating platform at an angle of about 10 o and at a speed of about 3 rpm for 30 min at 4 ° C and then the tube was clamped to a magnetic concentrator (Dynal Laboratories). After 2 min the supernatant was removed from the tube (which was still clamped to the magnet) and centrifuged at 350 × g for 10 min. The pelletted cells were re-suspended in culture medium and plated as required. The cells attached to the magnetic microspheres were also re-suspended in medium and plated.
Primary culture The routine culture medium consisted of a 1 : 1 mixture of Waymouth's MB 752/1 and Ham's F12 supplemented with fetal bovine serum to a final concentration of 17% (v/v) and Hepes buffer to a final concentration of 25 mM and a p H of 7.4. For immunocytochemistry, the cells were plated into Labtek eight-chamber culture slides at 5 0 0 0 0 - 1 0 0 0 0 0 cells/cm 2 and placed in an a i r / C O 2 (95:5) incubator at 37 ° C.
lmmunocytochemistry Immunocytochemical staining was generally carried out on cells which had been cultured in eight-chamber Labtek slides for about 18 h. Sufficient cells were adherent after 3 h such that staining was occasionally carried out at that time, although greater care had to be taken to avoid cell detachment during processing. Fixation and permeabilization were accomplished by immersing
slides in methanol at - 2 0 ° C for 20 min followed by five rapid dips in acetone at - 2 0 ° C . The slides were then washed in phosphate-buffered saline, pH 7.2, for 5 min. The cells were incubated with the appropriate first antibody for 30 min at 37 o C. All antibodies were diluted in phosphatebuffered saline containing 0.2% ( w / v ) gelatin and 0.02% thimerosal. The dilutions used were as follows: anti-cytokeratin 18 (1/200), anti-vimentin (1/200), anti-HLA-ABC (1/50), a n t i - H L A - D R (1/25). After the first incubation, the cells were washed three times with excess phosphate-buffered saline. FITC-labeled goat anti-mouse IgG (diluted 1/200) was added and the slides incubated for 30 min at 37°C. The washing procedure was repeated and the slides were mounted using an aqueous, glycerol-based mounted medium. The cells were examined using a Leitz Diaplan microscope equipped with epifluorescence optics.
lmmunohistochernistry Samples of chorionic villous tissue were processed for microscopic examination using the protocol described by Sato et al. (1986). Thin, paraffin-embedded sections were deparaffinized and rinsed in phosphate-buffered saline before staining with antibodies, as described above in the immunocytochemistry section. Results
We chose to monitor cell purification by staining cells with monoclonal antibodies against cytokeratin and vimentin. Figs. 1A and 1B show s e c -
Fig. 1. A: micrograph of term placental villi stained for vimentin. Note positive staining of stromal cells and endothelial cells. B: villi stained for cytokeratin. Note intense staining of the trophoblast layer, x 280.
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Fig. 2. Micrographs of cells isolated using continuous density gradient centrifugation and cultured for 18 h. Although a few cells stain positively for vimentin (A), the majority are cytokeratin-positive(B). x 280. tions of term villous tissue stained for vimentin and cytokeratin, respectively, using an indirect immunofluorescence technique. Cytotrophoblast and syncytiotrophoblast were positive for cytokeratin and negative for vimentin, as reported by others (Clark and Damjanov, 1985; Vettenranta et al., 1986; Loke and Butterworth, 1987). No cytokeratin-positive cells were found in the villous mesenchyme although there was clear evidence of vimentin-positive staining, particularly around capillaries. When placental cells were isolated using the procedure described by Kliman et al. (1986), 5-10% of the cells stained positively for vimentin, the remainder being cytokeratin positive. In an a t t e m p t to further reduce the n u m b e r of vimentin-positive cells, we substituted a continu-
ous Percoll density gradient for the discontinuous gradient described in the original method. Kliman et al. (1986) showed that cells banding between 1.048 and 1.062 g / m l Percoll were mostly cytotrophoblast. The continuous gradient used in the present study was designed to expand this region of the gradient and so facilitate greater resolution of the cells contained within it. By fractionating the gradient it was found that cells banding between densities of 1.053 to 1.060 g / m l were 2-5% vimentin-positive, with the remainder being cytokeratin-positive (Figs. 2A and 2B). At lesser and greater densities the proportion of vimentin-positive cells increased markedly. Thus, while the continuous gradient slightly improved cytotrophoblast purity, it did not completely eliminate vimentin-positive cells. However, the use of pre-
@ Fig. 3. Micrographs of ceils isolated using continuous density gradient centrifugation and cultured for 18 h. A few cells stain positively for HLA-ABCantigens (A) and HLA-DR antigens (B). x 280.
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Fig. 4. Micrograph illustrating the appearance of gradient purified cells after being treated in suspension with anti-HLA antibodies followed by magnetic microspheres coated with goat anti-mouse IgG antibody. Arrow points to rosette formed by cell and the microspheres, x 280.
formed c o n t i n u o u s gradients simplified a n d reduced the time taken for reagent p r e p a r a t i o n a n d was used in all s u b s e q u e n t experiments. Between 3 - 6 x 107 cells were recovered from 30 g tissue with a viability greater t h a n 90% as d e t e r m i n e d by t r y p a n blue staining. At this p o i n t in the procedure we have f o u n d that t r o p h o b l a s t cells c a n be stored u n d e r liquid n i t r o g e n u n t i l required a n d then successfully thawed a n d plated. T h e p l a t i n g efficiency of cryopreserved cells was a b o u t 80% whereas for freshly isolated cells the figure was 90%. I n order to achieve further p u r i f i c a t i o n of the cells, a d v a n t a g e was taken of the fact that trophoblast is one of the few tissues to lack histoc o m p a t i b i l i t y antigens on its surface ( F a u l k and Temple, 1976; S u n d e r l a n d et al., 1981; Bulmer
@ Fig. 5. Micrograph of density gradient-purified cells which were subjected to the immunomagnetic separation procedure and then cultured for 18 h. Note the absence of any cells staining positively for HLA-ABC (A), HLA-DR (B) or vimentin (C). The cells were positive for cytokeratin (D). x 280.
265 and Johnson, 1985). In chorionic villi, positive H L A staining is restricted to stromal cells within the villous mesenchyme (Sunderland et al., 1981; Sutton et al., 1983; Sutton et al., 1986). Figs. 3A and 3B show a typical adherent cell population stained (after 18 h in culture) for class I and class II major histocompatibility antigens using a monoclonal anti-HLA-ABC and a monoclonal anti-HLA-DR a n t i b o d y , respectively. T h e frequency of positive cells was similar to that found for vimentin-positive cells (i.e., less than 5% of the population). The morphology of these cells was variable, some being round, some being elongated with dendritic processes and others being large and polygonal. The H L A - D R positive cells probably represent placental macrophages a n d / o r dendritic-like cells (Sutton et al., 1983; Bulmer and Johnson, 1984). Fig. 4 shows cells (obtained by trypsin digestion and continuous Percoll gradient centrifugation) which were treated in suspension with anti-HLA antibodies followed by magnetic microspheres coated with goat anti-mouse I g G antibody. Rosetting of cells by the microspheres was clearly evident. Rosetted cells accounted for about 4% of the total. After immobilization of the rosetted cells using a magnet, the remaining non-rosetted cells were removed and cultured. Immunofluorescence staining after 18 h (Fig. 5) showed that the removal of H L A positive cells was complete in that no H L A - A B C or H L A - D R positive cells could be found (Figs. 5A and 5B). Furthermore, no vimentin-positive cells were detected (Fig. 5C). The cells were, however, positive for cytokeratin (Fig. 5D). When control cells were treated with magnetic microspheres alone, or with non-immune mouse I g G a n d m a g n e t i c microspheres, rosetted cells accounted for less than 1% of the total. When the control non-rosetted cells were removed and plated, HLA-positive and vimentin-positive cells were found (Figs. 6 A - 6 C ) . The overall recovery of cells using the immunomagnetic separation procedure was about 92%. At the start of the procedure, cell viability, as determined by trypan blue staining, was usually about 95%. After immunomagnetic separation, viability was 85%. To date, this procedure has been used on four separate cell preparations (i.e., cells prepared from four different placentas) and iden-
Fig. 6. Micrograph of gradient-purified cells treated with goat anti-mouse IgG-coated magnetic microspheres alone and then cultured for 18 h. HLA-ABC-positive(A), HLA-DR-positive (B) and vimentin-positive(C) cells were observed. × 280. tical results have been obtained each time. At least 10 000 cells h a v e b e e n examined by immunofluorescence microscopy for each run and no vimentin-positive, or H L A - A B C / D R - p o s i t i v e cells have been observed. When maintained in culture, the purified cytokeratin-positive, H L A -
266 negative cells aggregated and formed colonies. After 2-3 days, most, but not all, colonies stained positively for human chorionic gonadotropin (HCG). Single cells (representing less than 10% of the culture) were mostly negative for HCG. More than 90% of the colonies and more than 50% of single cells stained positively for pregnancy specific fla glycoprotein (SP1). It was noticed that some colonies or areas within a colony stained positively, although weakly, for HLA-ABC after 24-36 h in culture (not illustrated). Attempts were made to culture the cells rosetted by the magnetic microspheres. Even after 2 days in culture, many of the cells were still coated with microspheres. Some cells, however, were spreading out from under the particles. Immunofluorescence staining revealed a mixture of cytokeratin-positive and vimentin-positive cells in both experimental and control preparations.
Discussion
The isolation of cytotrophoblast cells from human placenta is confounded by the presence of other cells released from the tissue during the isolation procedure. Possible contaminants include placental macrophages (Hofbauer cells), endothelial cells, and fibroblasts. Contamination with maternal or fetal blood elements is also possible. Before attempting to isolate a pure population of cytotrophoblast it was important to have a simple and reliable means of screening for the presence of desired and undesired cell types. The starting material for the procedure described here is chorionic villous tissue from term placenta. Immunocytochemical analyses of sections of this material carried out by ourselves and by others (Clark and Damjanov, 1985; Vettenranta et al., 1986; Loke and Butterworth, 1987) confirm that the multinucleated syncytiotrophoblast and the mononucleated cytotrophoblast layers are cytokeratin-positive, vimentin-negative and HLAnegative. By contrast, all other identifiable cells in this tissue are cytokeratin-negative, vimentin-positive and HLA-positive. It was therefore decided to attempt to separate cytotrophoblast from contaminating cells by exploiting the difference in cell
surface HLA expression and to monitor purification on the basis of intermediate filament expression and HLA expression. The cells obtained by trypsin digestion, continuous Percoll densitygradient centrifugation and immunomagnetic separation, as described here, were mononucleated, cytokeratin-positive, vimentin-negative and HLAnegative. Thus, by the above criteria, the procedure yields a pure preparation of villous cytotrophoblast cells. It should be noted that endometrial gland cells and extravillous cytotrophoblast from the placental bed are cytokeratin-positive (Loke and Butterworth, 1987). However, in the procedure described here these cells are avoided by selecting only villous tissue as the starting material. When placed in culture, the cells aggregated and formed colonies which, after about 2 days, showed increased staining for H C G and SP~, typical of trophoblast (Thiede and Choate, 1963; Tatarinov et al., 1976; Kliman et al., 1986). The observation that some of the colonies showed weakly positive staining for HLA-ABC after 1-2 days in culture, has also been noted by others (Feinman et al., 1987; Loke and Burland, 1988). The immunomagnetic separation step is efficient and simple to perform and requires relatively inexpensive reagents and equipment. The time taken adds about 2 h to the basic isolation procedure (trypsin digestion and Percoll gradient centrifugation), given a total time of about 6 h in our hands. Alternatively, the cells can be frozen after the density gradient step and immunomagnetic separation performed at a later time after thawing out the cells. The ability to store cells frozen and successfully thaw them when required gives greater control over the timing and planning of experiments. The cost of the immunomagnetic separation step is largely dependent on the availability of the anti-HLA antibodies. Some laboratories may generate their own hybridomas, others may have to purchase antibodies commercially. For many applications, a pure cytotrophoblast preparation may be unnecessary and the procedures described by others (Lobo et al., 1985; Kliman et al., 1986; Daniels-McQueen et al., 1987; Loke and Burland, 1988) may suffice. Cells isolated using the continuous density gradient described in this paper also comprise a highly purl-
267 fied villous cytotrophoblast
population
and may
be satisfactory for many experiments. Furtherm o r e , t h e c o n t i n u o u s g r a d i e n t is s i m p l e r a n d less t e d i o u s to p r e p a r e t h a n t h e d i s c o n t i n u o u s g r a d i e n t . H o w e v e r , i n m a n y i n s t a n c e s it is d e s i r a b l e t o work with a pure preparation of cytotrophoblast and the immunomagnetic separation method des c r i b e d h e r e a p p e a r s t o p r o v i d e this.
Acknowledgements We are grateful to Ellen Tucker for expert technical assistance, to Grete Fry for photographic assistance and to Dr. Robert Scibienski for helpful discussions. We also thank the nursing and medical staff at Sutter Memorial Hospital, Sacramento, CA, for their help in obtaining placentas. This work was supported by NIH Grant HD 11658.
References Bulmer, J.N. and Johnson, P.M. (1984) Macrophage populations in the human placenta and amniochorion. Clin. Exp. Immunol. 57, 393. Bulmer, J.N. and Johnson, P.M. (1985) Antigen expression by trophoblast populations in the human placenta and their possible immunological relevance. Placenta 6, 127. Butterworth, B.H. and Loke, Y.W. (1985) Immunocytochemical identification of cytotrophoblast from other mononuclear cell populations isolated from first trimester human chorionic villi. J. Cell. Sci. 76, 189. Clark, R.K. and Damjanov, I. (1985) Intermediate filaments of human trophoblast and choriocarcinoma cell lines. Virchows Arch. Abt. A Pathol. Anat. Histopathol. 407, 203. Contractor, S.F. and Sooranna, S.R. (1986) Monoclonal antibodies to cytotrophoblast and syncytiotrophoblast of human placenta. J. Dev. Physiol. 8, 277. Contractor, S.F. and Sooranna, S.R. (1988) Human placental cells in culture: a panning technique using a trophoblastspecific monoclonal antibody for cell separation. J. Dev. Physiol. 10, 47. Daniels-McQueen, S., Krichevsky, A. and Boime, I. (1987) Isolation and characterization of human cytotrophoblast ceils. Trophoblast Res. 2, 423. Fanlk, W.P. and Temple, A. (1976) Distribution of r2 microglobulin and HLA in chorionic villi of human placentae. Nature 262, 799.
Feinman, M.A., Kliman, H.J. and Main, E.K. (1987) HLA antigen expression and induction by "t-interferon in cultured human trophoblasts. Am. J. Obstet. Gynecol. 157, 1429. Hall, C.St.G., James, T.E., Goodyer, C., Branchard, C., Guyda, H. and Giroud, C.J.P. (1977) Short term tissue culture of human midterm and term placenta: parameters of hormonogenesis. Steroids 30, 569. Kawata, M., Parnes, J.R. and Herzenberg, L.A. (1984) Transcriptional control of HLA-A,B,C antigen in human placental cytotrophoblast isolated using trophoblast- and HLA-specific monoclonal antibodies and the fluorescenceactivated cell sorter. J. Exp. Med. 160, 633. Kliman, H.J., Nestler, J.E., Sermasi, E., Sanger, J.M. and Strauss III, J.F. (1986) Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118, 1567. Lipinski, M., Parks, D.R., Roose, R.V. and Herzenberg, L.A. (1981) Human trophoblast cell-surface antigens defined by monoclonal antibodies. Proc. Natl. Acad. Sci. U.S.A. 78, 5147. Lobo, J.O., Bellino, F.L. and Bankert, L. (1985) Estrogen synthetase activity in human term placental cells in monolayer culture. Endocrinology 116, 889. Loke, Y.W. (1983) Human trophoblast in culture. In: Y.W. Loke and A. Whyte (Eds.), Biology of Trophoblast. Elsevier, Amsterdam, p. 663. Loke, Y.W. and Burland, K. (1988) Human trophoblast cells cultured in modified medium and supported by extracellular matrix. Placenta 9, 173. Loke, Y.W. and Butterworth, B.H. (1987) Heterogeneity of human trophoblast populations. In: T.J. Gill and T.G. Wegmann (Eds.), Immunoregulation and fetal survival. Oxford University Press, New York, p. 197. Loke, Y.W., Butterworth, B.H., Margetts, J.J. and Burland, K. (1986) Identification of cytotrophoblast colonies in cultures of human placental cells using monoclonal antibodies. Placenta 7, 221. Sato, Y., Mukai, K., Watanabe, S., Gato, M. and Shimosato, Y. (1986) A simplified technique of tissue processing and paraffin embedding with improved preservation of antigens for immunostaining. Am. J. Pathol. 125, 431. Stromberg, K. (1980) The human placenta in cell and organ culture. In: C.C. Harris, B.F. Trump and G.D. Stoner (Eds.), Methods in Cell Biology 21b, Normal Human Tissue in Cell Culture. Academic Press, New York, p. 227. Sunderland, C.A., Naiem, M., Mason, D.Y., Redman, C.W.G. and Stirrat, G.M. (1981) The expression of major histocompatability antigens by human chorionic villi. J. Reprod. Immunol. 3, 323. Sutton, L., Mason, D.Y. and Redman, W.G. (1983) HLA-DR positive cells in the human placenta. Immunology 49, 103. Sutton, L., Gadd, M., Mason, D.Y. and Redman, C.W.G. (1986) Cells bearing class II MHC antigens in the human placenta and amniochorion. Immunology 58, 23. Tatorinov, Y.S., Falaleeva, D.M., Kalashnikov, V.V. and Toloknov, B.O. (1976) Immunofluorescent localization of
268 human pregnancy-specific ill-globulin in placenta and chorioepithelioma. Nature 260, 263. Thiede, H.A., and Choate, J.W. (1963) Chorionic gonadotropin localization in the human placenta by immunofluorescent staining. Obstet. Gynecol. 22, 433.
Vettenranta, K., Von Koskull, H., Heikinheimo, M. and Raivio, K.O. (1986) Cytoskeletal markers and specific protein production in cells cultured from human first and third trimester placentae. In Vitro Cell. Dev. Biol. 22, 100.
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JIM 05159
Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres G o r d o n C. Douglas and Barry F. King Department of Human Anatomy, School of Medicine, University of California, Davis, CA 95616, U.S.A. (Received 20 September 1988, revised received 12 December 1988, accepted 13 December 1988)
A procedure has been developed which yields a pure population of villous cytotrophoblast from term human placenta. As a first step, a cell preparation highly enriched for cytotrophoblast (identified by positive cytokeratin staining) was obtained using a modification of the method of Kliman et al. (1986). The remaining contaminating cells (identified by positive vimentin staining) were then removed by treatment with mouse monoclonal antibodies against class I and class II major histocompatibility antigens followed by magnetic microspheres coated with goat anti-mouse IgG. The rationale for this step was based on the fact that villous trophoblast fails to express HLA antigens whereas cells from the villous mesenchyme do express these surface antigens. Rosetted cells were immobilized using a magnet allowing the non-rosetted cells to be easily withdrawn by pipette. When the non-rosetted cells were placed in primary culture, no HLA-positive or vimentin-positive cells could be detected using immunofluorescence microscopy, indicating complete removal of these components by the immunomagnetic separation procedure. The cells were positive for cytokeratin and, after 24 h, showed positive staining for pregnancy-specific/31-glycoprotein (SP1) and human chorionic gonadotropin. Recovery of cytotrophoblast was greater than 92% with only a slight loss of viability Key words: Cytotrophoblast; Placenta; Magnetic microsphere
Introduction
Many procedures have been described for the isolation of cytotrophoblast cells from human placenta (Hall et al., 1977; Lobo et al., 1985; Kliman et al., 1986; Daniels-McQueen et al., 1987; Loke and Burland, 1988; see reviews by Stromberg, 1980; Loke, 1983). However, the nature of the isolation procedures and the cellular heterogeneity of the placenta itself usually dictates that a mixed cell population is obtained, which can, at Correspondence to: G.C. Douglas, Department of Human Anatomy, School of Medicine, University of California, Davis, CA 95616, U.S.A.
best, be described as highly enriched for cytotrophoblast. Contaminating cells may include placental macrophages, fibroblasts, endothelial cells and blood elements. Where estimates of cytotrophoblast purity are reported, these vary from 40 to 95%, depending on the isolation procedure used. Several laboratories have produced monoclonal antibodies reactive against trophoblast cell surface antigens (Lipinski et al., 1981; Butterworth and Loke, 1985; Contractor and Sooranna, 1986; Loke et al., 1986) and these have proved useful in identifying trophoblast in mixed cell populations. Contractor and Sooranna (1988) used a trophoblast-specific antibody and a panning technique in an attempt to isolate cytotrophoblast
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from a placental cell suspension, but still obtained a significant number of fibroblasts and macrophages. Kawata et al. (1984) used a variety of monoclonal antibodies, including a trophoblastspecific antibody, and the fluorescence activated cell sorter, to characterize and separate placental cells. They were able to obtain a purified cell preparation which was 'mostly cytotrophoblast'. However, the specialized equipment and reagents required are not readily available to all laboratories, particularly for use on a routine basis. To our knowledge, no procedure has been described which will yield a pure population of cytotrophoblast, which is simple to perform and which makes use of commonly available reagents. Kliman et al. (1986) described a procedure for cytotrophoblast purification which involved trypsin digestion of term villous tissue followed by discontinuous Percoll gradient centrifugation. Using a modification of this procedure as a first step and adding an entirely new step, we have been able to obtain a cell preparation which consists entirely of cytotrophoblast cells. The additional step, which is described in detail here, takes advantage of the fact that villous trophoblast is one of the few tissues to lack major histocompatibility antigens on its surface (Faulk and Temple, 1976; Sunderland et al., 1981; Bulmer and Johnson, 1985). HLA-positive contaminating cells were removed using commercially available monoclonal antibodies to class I and class II major histocompatibility antigens and immunomagnetic microspheres, leaving pure cytotrophoblast cells.
beads were purchased from Pharmacia Fine Chemicals, Piscataway, NJ. Magnetic microspheres (Dynabeads M-450) coated with goat anti-mouse IgG and a magnetic particle concentrator were obtained through Robbins Scientific, Mountainview, CA.
Antibodies Mouse monoclonal antibodies were obtained as follows: anti-cytokeratin 18 and anti-vimentin (clone v-9) were from ICN Immunobiologicals, Lisle, IL. Anti-HLA-ABC (IgG2a, monomorphic) and a n t i - H L A - D R (IgG2b, monomorphic) were from Chemicon International, Los Angeles, CA. Polyclonal goat anti-mouse IgG was obtained as a F(ab')2 fragment conjugated with fluorescein isothiocyanate (FITC) from Accurate Chemical and Scientific Corp., Westburg, NY.
Preformed continuous Percoll density gradients Percoll (38 ml) was mixed with a 10 x stock solution of Hanks' balanced salt solution (10 ml) and made to a final volume of 100 ml with deionized water. This gives an isotonic Percoll suspension with a density of 1.055 g / m l . The isotonic Percoll was dispensed into 50 ml polycarbonate centrifuge tubes (35 ml Percoll/tube). A continuous gradient was then formed by centrifuging the tubes at 30000 x g in an angle rotor (Sorvall, SS-34) for 15 min. The shape of the gradient was routinely checked using density marker beads (Pharmacia Fine Chemicals, Piscataway, N J).
Preparation of trophoblast cells" Materials and methods
Materials Hanks' balanced salt solution (without C a 2~ and Mg 2+) was obtained from Gibco Laboratories, Grand Island, New York. Waymouth's MB 752/1 medium, H a m ' s F12 medium and an antibiotic/antimycotic mixture were purchased from Sigma Chemical Company, St. Louis, MO. Trypsin (a 2.5% solution containing 125 U / m l ) and DNase I (from bovine pancreas) were from Boehringer Mannheim, Indianapolis, IN. Newborn calf serum was obtained from Irvine Scientific, Santa Ana, CA. Percoll and density gradient marker
The initial cell isolation procedure was based on the method described by Kliman et al. (1986). The main differences are that a continuous rather than a discontinuous density gradient is used in the present study and an additional purification step using magnetic microspheres has been developed. For clarity, the entire procedure is described here. All solutions were autoclaved or sterile filtered before use. Term placentas were obtained at the time of Cesarian section. Occasionally, placentas from spontaneous deliveries were used. About 30 g (wet wt.) of villous tissue was dissected from the placenta, care being taken to avoid obvious connective tissue and decidua. The tissue was washed with 0.9% sodium chloride over a stainless
261 steel mesh and coarsely chopped using scissors. The tissue fragments were washed again over the wire mesh and then placed in a conical flask with 150 ml Hanks' balanced salt solution, containing 25 m M Hepes (N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid), p H 7.4, trypsin (0.25%, 12.5 U / m l ) and D N a s e (0.2 m g / m l ) . The mixture was incubated in a shaking water bath for 30 min at 3 7 ° C . About 100 ml of the mixture was then decanted into a measuring cylinder and allowed to stand for 1 min after which 80 ml were carefully pipetted out (avoiding large tissue fragments) and layered over 5 ml fetal bovine serum in 50 ml plastic centrifuge tubes. The tubes were centrifuged at 2200 rpm (1000 x g ) for 5 min after which the supernatant was discarded and the pellet retained. The residue in the cylinder was added back to the tissue fragments remaining in the conical flask and the digestion was repeated twice more with 100 ml and 75 ml, respectively, of fresh t r y p s i n / D N a s e solution. On each occasion the mixture was decanted and centrifuged. The pellets from all three digestions were pooled and made to a final volume of about 25 ml in Waymouth's medium supplemented with fetal bovine serum (17%) and antibiotics. The suspension was poured through four layers of cheesecloth and then through two layers of 0.1 m m polyester mesh before being centrifuged at 1000 × g for 10 min. The pellet was re-suspended in Hanks' balanced salt solution to a final volume of 6 ml. The suspension was divided into two 3 ml fractions each of which was carefully layered on top of a preformed, continuous gradient of isotonic Percoll (35 ml) in a 50 ml polycarbonate tube. The tubes were spun at 400 x g for 15 min after which the material sedementing at densities between 1.053 and 1.060 g / m l was collected. Densities were determined by comparison with an identical gradient run with density marker beads. The collected Percoll fractions were diluted four-fold with culture medium and centrifuged at 1000 x g for 5 min. The supernatant containing the Percoll was discarded and the pellet (cells) was re-suspended in culture medium. Between 3 - 6 x 10 7 cells were obtained from 30 g villous tissue. At this point the cells were either further purified by immunomagnetic separation (see below) or were frozen and stored in liquid N 2 until needed,
whereupon they were thawed and then subjected to the immunomagnetic separation procedure.
Storage of cells under liquid N, The cell pellet was suspended in cold 10% ( v / v ) dimethylsulfoxide in culture medium to give 5 - 1 0 X 10 6 cells/ml. The suspension was aliquoted into 1.8 ml cryogenic vials (Costar) which were then placed in insulated cardboard containers and kept at - 7 0 ° C for at least 1 h. The tubes were then rapidly transferred to liquid nitrogen for long term storage. When required, a tube was removed from the liquid nitrogen and thawed in a water bath at 37 o C. The thawed cells were diluted 1 / 1 0 in culture medium and centrifuged at 400 x g for 5 min. The cell pellet was re-suspended in fresh culture medium to the required cell density.
Irnmunomagnetic cell separation Cells (either freshly isolated or thawed after storage in liquid Nz) were suspended in ice-cold medium containing 17% newborn calf serum to give 4 x 10 6 cells/ml. All subsequent steps were performed in the cold. For simplicity, the following description is based on processing 1 ml of cell suspension. However, the procedure can be scaled up as required. To the suspension (1 ml) was added 20/11 of a monoclonal anti-HLA-ABC antibody and 40 ffl of monoclonal a n t i - H E A - D R antibody. The antibodies were added undiluted. The amounts used were such that when mixed with the cell suspension the resulting dilution would be equivalent to that recommended by the manufacturer for immunofluorescence staining. The mixture was incubated on ice for 45 min with occasional gently shaking and then centrifuged at 350 x g for 5 rain. The supernatant was removed and the cell pellet was re-suspended in Hanks' balanced salt solution (without C a 2+ and Mg 2+) supplemented with 1% newborn calf serum. Centrifugation and resuspension was repeated two more times. After a further centrifugation the pellet was finally re-suspended in medium (sup-~ plemented with 1% newborn calf serum) at a ratio of 1 ml medium : 4 x 106 cells. To this was added 40 ffl of a suspension of magnetic microspheres coated with goat anti-mouse IgG. The amount added was calculated using the manufacturers f~rmulae and by assuming the target cells (i.e., con-
262
taminating cells) accounted for 5% of the total cell population. The mixture was placed on a rotating platform at an angle of about 10 o and at a speed of about 3 rpm for 30 min at 4 ° C and then the tube was clamped to a magnetic concentrator (Dynal Laboratories). After 2 min the supernatant was removed from the tube (which was still clamped to the magnet) and centrifuged at 350 × g for 10 min. The pelletted cells were re-suspended in culture medium and plated as required. The cells attached to the magnetic microspheres were also re-suspended in medium and plated.
Primary culture The routine culture medium consisted of a 1 : 1 mixture of Waymouth's MB 752/1 and Ham's F12 supplemented with fetal bovine serum to a final concentration of 17% (v/v) and Hepes buffer to a final concentration of 25 mM and a p H of 7.4. For immunocytochemistry, the cells were plated into Labtek eight-chamber culture slides at 5 0 0 0 0 - 1 0 0 0 0 0 cells/cm 2 and placed in an a i r / C O 2 (95:5) incubator at 37 ° C.
lmmunocytochemistry Immunocytochemical staining was generally carried out on cells which had been cultured in eight-chamber Labtek slides for about 18 h. Sufficient cells were adherent after 3 h such that staining was occasionally carried out at that time, although greater care had to be taken to avoid cell detachment during processing. Fixation and permeabilization were accomplished by immersing
slides in methanol at - 2 0 ° C for 20 min followed by five rapid dips in acetone at - 2 0 ° C . The slides were then washed in phosphate-buffered saline, pH 7.2, for 5 min. The cells were incubated with the appropriate first antibody for 30 min at 37 o C. All antibodies were diluted in phosphatebuffered saline containing 0.2% ( w / v ) gelatin and 0.02% thimerosal. The dilutions used were as follows: anti-cytokeratin 18 (1/200), anti-vimentin (1/200), anti-HLA-ABC (1/50), a n t i - H L A - D R (1/25). After the first incubation, the cells were washed three times with excess phosphate-buffered saline. FITC-labeled goat anti-mouse IgG (diluted 1/200) was added and the slides incubated for 30 min at 37°C. The washing procedure was repeated and the slides were mounted using an aqueous, glycerol-based mounted medium. The cells were examined using a Leitz Diaplan microscope equipped with epifluorescence optics.
lmmunohistochernistry Samples of chorionic villous tissue were processed for microscopic examination using the protocol described by Sato et al. (1986). Thin, paraffin-embedded sections were deparaffinized and rinsed in phosphate-buffered saline before staining with antibodies, as described above in the immunocytochemistry section. Results
We chose to monitor cell purification by staining cells with monoclonal antibodies against cytokeratin and vimentin. Figs. 1A and 1B show s e c -
Fig. 1. A: micrograph of term placental villi stained for vimentin. Note positive staining of stromal cells and endothelial cells. B: villi stained for cytokeratin. Note intense staining of the trophoblast layer, x 280.
263
Fig. 2. Micrographs of cells isolated using continuous density gradient centrifugation and cultured for 18 h. Although a few cells stain positively for vimentin (A), the majority are cytokeratin-positive(B). x 280. tions of term villous tissue stained for vimentin and cytokeratin, respectively, using an indirect immunofluorescence technique. Cytotrophoblast and syncytiotrophoblast were positive for cytokeratin and negative for vimentin, as reported by others (Clark and Damjanov, 1985; Vettenranta et al., 1986; Loke and Butterworth, 1987). No cytokeratin-positive cells were found in the villous mesenchyme although there was clear evidence of vimentin-positive staining, particularly around capillaries. When placental cells were isolated using the procedure described by Kliman et al. (1986), 5-10% of the cells stained positively for vimentin, the remainder being cytokeratin positive. In an a t t e m p t to further reduce the n u m b e r of vimentin-positive cells, we substituted a continu-
ous Percoll density gradient for the discontinuous gradient described in the original method. Kliman et al. (1986) showed that cells banding between 1.048 and 1.062 g / m l Percoll were mostly cytotrophoblast. The continuous gradient used in the present study was designed to expand this region of the gradient and so facilitate greater resolution of the cells contained within it. By fractionating the gradient it was found that cells banding between densities of 1.053 to 1.060 g / m l were 2-5% vimentin-positive, with the remainder being cytokeratin-positive (Figs. 2A and 2B). At lesser and greater densities the proportion of vimentin-positive cells increased markedly. Thus, while the continuous gradient slightly improved cytotrophoblast purity, it did not completely eliminate vimentin-positive cells. However, the use of pre-
@ Fig. 3. Micrographs of ceils isolated using continuous density gradient centrifugation and cultured for 18 h. A few cells stain positively for HLA-ABCantigens (A) and HLA-DR antigens (B). x 280.
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Fig. 4. Micrograph illustrating the appearance of gradient purified cells after being treated in suspension with anti-HLA antibodies followed by magnetic microspheres coated with goat anti-mouse IgG antibody. Arrow points to rosette formed by cell and the microspheres, x 280.
formed c o n t i n u o u s gradients simplified a n d reduced the time taken for reagent p r e p a r a t i o n a n d was used in all s u b s e q u e n t experiments. Between 3 - 6 x 107 cells were recovered from 30 g tissue with a viability greater t h a n 90% as d e t e r m i n e d by t r y p a n blue staining. At this p o i n t in the procedure we have f o u n d that t r o p h o b l a s t cells c a n be stored u n d e r liquid n i t r o g e n u n t i l required a n d then successfully thawed a n d plated. T h e p l a t i n g efficiency of cryopreserved cells was a b o u t 80% whereas for freshly isolated cells the figure was 90%. I n order to achieve further p u r i f i c a t i o n of the cells, a d v a n t a g e was taken of the fact that trophoblast is one of the few tissues to lack histoc o m p a t i b i l i t y antigens on its surface ( F a u l k and Temple, 1976; S u n d e r l a n d et al., 1981; Bulmer
@ Fig. 5. Micrograph of density gradient-purified cells which were subjected to the immunomagnetic separation procedure and then cultured for 18 h. Note the absence of any cells staining positively for HLA-ABC (A), HLA-DR (B) or vimentin (C). The cells were positive for cytokeratin (D). x 280.
265 and Johnson, 1985). In chorionic villi, positive H L A staining is restricted to stromal cells within the villous mesenchyme (Sunderland et al., 1981; Sutton et al., 1983; Sutton et al., 1986). Figs. 3A and 3B show a typical adherent cell population stained (after 18 h in culture) for class I and class II major histocompatibility antigens using a monoclonal anti-HLA-ABC and a monoclonal anti-HLA-DR a n t i b o d y , respectively. T h e frequency of positive cells was similar to that found for vimentin-positive cells (i.e., less than 5% of the population). The morphology of these cells was variable, some being round, some being elongated with dendritic processes and others being large and polygonal. The H L A - D R positive cells probably represent placental macrophages a n d / o r dendritic-like cells (Sutton et al., 1983; Bulmer and Johnson, 1984). Fig. 4 shows cells (obtained by trypsin digestion and continuous Percoll gradient centrifugation) which were treated in suspension with anti-HLA antibodies followed by magnetic microspheres coated with goat anti-mouse I g G antibody. Rosetting of cells by the microspheres was clearly evident. Rosetted cells accounted for about 4% of the total. After immobilization of the rosetted cells using a magnet, the remaining non-rosetted cells were removed and cultured. Immunofluorescence staining after 18 h (Fig. 5) showed that the removal of H L A positive cells was complete in that no H L A - A B C or H L A - D R positive cells could be found (Figs. 5A and 5B). Furthermore, no vimentin-positive cells were detected (Fig. 5C). The cells were, however, positive for cytokeratin (Fig. 5D). When control cells were treated with magnetic microspheres alone, or with non-immune mouse I g G a n d m a g n e t i c microspheres, rosetted cells accounted for less than 1% of the total. When the control non-rosetted cells were removed and plated, HLA-positive and vimentin-positive cells were found (Figs. 6 A - 6 C ) . The overall recovery of cells using the immunomagnetic separation procedure was about 92%. At the start of the procedure, cell viability, as determined by trypan blue staining, was usually about 95%. After immunomagnetic separation, viability was 85%. To date, this procedure has been used on four separate cell preparations (i.e., cells prepared from four different placentas) and iden-
Fig. 6. Micrograph of gradient-purified cells treated with goat anti-mouse IgG-coated magnetic microspheres alone and then cultured for 18 h. HLA-ABC-positive(A), HLA-DR-positive (B) and vimentin-positive(C) cells were observed. × 280. tical results have been obtained each time. At least 10 000 cells h a v e b e e n examined by immunofluorescence microscopy for each run and no vimentin-positive, or H L A - A B C / D R - p o s i t i v e cells have been observed. When maintained in culture, the purified cytokeratin-positive, H L A -
266 negative cells aggregated and formed colonies. After 2-3 days, most, but not all, colonies stained positively for human chorionic gonadotropin (HCG). Single cells (representing less than 10% of the culture) were mostly negative for HCG. More than 90% of the colonies and more than 50% of single cells stained positively for pregnancy specific fla glycoprotein (SP1). It was noticed that some colonies or areas within a colony stained positively, although weakly, for HLA-ABC after 24-36 h in culture (not illustrated). Attempts were made to culture the cells rosetted by the magnetic microspheres. Even after 2 days in culture, many of the cells were still coated with microspheres. Some cells, however, were spreading out from under the particles. Immunofluorescence staining revealed a mixture of cytokeratin-positive and vimentin-positive cells in both experimental and control preparations.
Discussion
The isolation of cytotrophoblast cells from human placenta is confounded by the presence of other cells released from the tissue during the isolation procedure. Possible contaminants include placental macrophages (Hofbauer cells), endothelial cells, and fibroblasts. Contamination with maternal or fetal blood elements is also possible. Before attempting to isolate a pure population of cytotrophoblast it was important to have a simple and reliable means of screening for the presence of desired and undesired cell types. The starting material for the procedure described here is chorionic villous tissue from term placenta. Immunocytochemical analyses of sections of this material carried out by ourselves and by others (Clark and Damjanov, 1985; Vettenranta et al., 1986; Loke and Butterworth, 1987) confirm that the multinucleated syncytiotrophoblast and the mononucleated cytotrophoblast layers are cytokeratin-positive, vimentin-negative and HLAnegative. By contrast, all other identifiable cells in this tissue are cytokeratin-negative, vimentin-positive and HLA-positive. It was therefore decided to attempt to separate cytotrophoblast from contaminating cells by exploiting the difference in cell
surface HLA expression and to monitor purification on the basis of intermediate filament expression and HLA expression. The cells obtained by trypsin digestion, continuous Percoll densitygradient centrifugation and immunomagnetic separation, as described here, were mononucleated, cytokeratin-positive, vimentin-negative and HLAnegative. Thus, by the above criteria, the procedure yields a pure preparation of villous cytotrophoblast cells. It should be noted that endometrial gland cells and extravillous cytotrophoblast from the placental bed are cytokeratin-positive (Loke and Butterworth, 1987). However, in the procedure described here these cells are avoided by selecting only villous tissue as the starting material. When placed in culture, the cells aggregated and formed colonies which, after about 2 days, showed increased staining for H C G and SP~, typical of trophoblast (Thiede and Choate, 1963; Tatarinov et al., 1976; Kliman et al., 1986). The observation that some of the colonies showed weakly positive staining for HLA-ABC after 1-2 days in culture, has also been noted by others (Feinman et al., 1987; Loke and Burland, 1988). The immunomagnetic separation step is efficient and simple to perform and requires relatively inexpensive reagents and equipment. The time taken adds about 2 h to the basic isolation procedure (trypsin digestion and Percoll gradient centrifugation), given a total time of about 6 h in our hands. Alternatively, the cells can be frozen after the density gradient step and immunomagnetic separation performed at a later time after thawing out the cells. The ability to store cells frozen and successfully thaw them when required gives greater control over the timing and planning of experiments. The cost of the immunomagnetic separation step is largely dependent on the availability of the anti-HLA antibodies. Some laboratories may generate their own hybridomas, others may have to purchase antibodies commercially. For many applications, a pure cytotrophoblast preparation may be unnecessary and the procedures described by others (Lobo et al., 1985; Kliman et al., 1986; Daniels-McQueen et al., 1987; Loke and Burland, 1988) may suffice. Cells isolated using the continuous density gradient described in this paper also comprise a highly purl-
267 fied villous cytotrophoblast
population
and may
be satisfactory for many experiments. Furtherm o r e , t h e c o n t i n u o u s g r a d i e n t is s i m p l e r a n d less t e d i o u s to p r e p a r e t h a n t h e d i s c o n t i n u o u s g r a d i e n t . H o w e v e r , i n m a n y i n s t a n c e s it is d e s i r a b l e t o work with a pure preparation of cytotrophoblast and the immunomagnetic separation method des c r i b e d h e r e a p p e a r s t o p r o v i d e this.
Acknowledgements We are grateful to Ellen Tucker for expert technical assistance, to Grete Fry for photographic assistance and to Dr. Robert Scibienski for helpful discussions. We also thank the nursing and medical staff at Sutter Memorial Hospital, Sacramento, CA, for their help in obtaining placentas. This work was supported by NIH Grant HD 11658.
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268 human pregnancy-specific ill-globulin in placenta and chorioepithelioma. Nature 260, 263. Thiede, H.A., and Choate, J.W. (1963) Chorionic gonadotropin localization in the human placenta by immunofluorescent staining. Obstet. Gynecol. 22, 433.
Vettenranta, K., Von Koskull, H., Heikinheimo, M. and Raivio, K.O. (1986) Cytoskeletal markers and specific protein production in cells cultured from human first and third trimester placentae. In Vitro Cell. Dev. Biol. 22, 100.