CULTURE OF RCHO-1 CELLS

 

The Rcho-1 cells were derived from a transplantable rat choriocarcinoma (Rcho) originally established by Teshima et al. (1983).  We received the Rcho transplantable tumor from Michel Vandeputte of Leuven University in Belgium.  Vandeputte's group has a long history of studying yolk and trophoblast tumors, mainly from a cancer biologist perspective.  We collaborated with Vandeputte to examine the expression of placental PRLs by the Rcho transplantable tumor (Faria et al., 1990).  Vandeputte's group has published two papers with the cells:  1) a morphological description of the tumors (Verstuyf et al. 1989) and 2) the establishment of a continuous line from the tumors (Verstuyf et al. 1990).  An important observation from these studies is that the cells are aneuploid.  We did not receive the line from Vandeputte but have established our own "continuous line" (Rcho-1) from the Rcho transplantable tumor (Faria and Soares, 1991).  Monoclonal antibodies directed to RCHO cells specifically recognize proliferative cells located in the ectoplacental cone but not with trophectoderm of the blastocyst (Verstuyf et al., 1992). Several reports have appeared investigating the effect of Rcho-1 cell transplantation on the production of pituitary prolactin (Tomogane et al., 1991, 1993; Arbogast et al., 1992, 1993; Mathiasen et al., 1992).  A few laboratories have utilized the Rcho-1 cell line or the Vandeputte-derived Rcho cell line for studies on the control of trophoblast cell-specific gene expression (Shida et al., 1993; Vuille et al., 1993; Ng et al., 1994; Yamamoto et al., 1994, 1995, 1996; Cross et al., 1995; Dai et al., 1996; Scatena & Adler, 1996; Orwig et al., 1997) and the control of trophoblast cell differentiation (Kamei et al., 1997). 

 

1.            CULTURE MEDIA

 

NCTC-135 culture medium (Sigma) supplemented with:

 

50 μM 2-mercaptoethanol (BIO-RAD)

1 mM sodium pyruvate (Sigma)

100 μg/ml penicillin (Sigma)

100 units/ml streptomycin (Sigma)

20% (heat inactivated) fetal bovine serum (JRH Biosciences)

 

[This medium is routinely used for growth and general maintenance of the cell line.  We have previously used RPMI-1640 as a base medium but it does not adequately maintain the pH at higher cell densities.  HEPES (10-20 mM) can also be added to the culture medium.  Under longer term differentiation experiments we shift the cells to medium containing the same supplements with 1-10% horse serum substituted for the the 20% FBS (FBS is a requirement for proliferation).  Cells are maintained in horse serum but show very limited proliferation.  Consequently, differentiation in horse serum enriches for the trophoblast giant cell population.  Please note that lots of FBS and horse serum should be screened.  We have had difficulty in maintaining proper proliferation or differentiation with some lots of serum.]   

 

2.            MAINTENANCE OF THE RCHO-1 CELL LINE

 

The Rcho-1 cell line contains a mixture of stem and differentiated cells. These features of the Rcho-1 cell line make the line valuable for studying the process of trophoblast cell differentiation; however, they are a nuisance for routine cell culture maintenance. Manipulating various aspects of the culture procedure can influence the cellular composition of the cell line. Cell composition can influence growth rates and features of differentiation. The stem cell population can be enriched by growing the cells at low densities and passaging following brief trypsinization. Maintaining the cells at higher densities leads to spontaneous differentiation (giant cell formation).  The differentiated cells are more adherent than the stem cells and require more vigorous dissociation methods such as scraping with a rubber policeman. Consequently, the method of passaging can affect the cellular composition of the cell line. Consistency in cell culture practices is extremely important in working with the Rcho-1 cell line. Variations in culture densities, passaging methods, and splitting ratios significantly influence the phenotype of the cell line.

We routinely maintain the cells at subconfluent conditions in either RPMI-1640 or NCTC-135 culture media supplemented with 10-20% fetal bovine serum. The cells grow more vigorously in RPMI-1640 culture medium but at the cost of poor pH regulation. Cellular proliferation is dependent upon the presence of some unidentified factors present in fetal bovine serum.  The cells grow much better under conditions of high humidity.  Differentiation is induced by growing the cells to confluence in fetal bovine serum supplemented culture medium and then replacing the serum supplementation with horse serum (1-10%). High cell density and the absence of growth stimulation (removal of fetal bovine serum) facilitate trophoblast giant cell formation. The nutritive needs of differentiating Rcho-1 cells appear to be less than the needs of proliferating Rcho-1 cells; however, differentiating Rcho-1 cells minimally require some factors present in horse serum and cannot tolerate the absence of serum for more than 48 hours.

Differentiation is a continuum and will proceed over at least a two to three week period in culture. Differentiation will also occur in the presence of fetal bovine serum; however, under these conditions the cultures are comprised of a greater proportion of stem cells, and thus the cultures are more heterogeneous. In the presence of horse serum, the stem cell population is greatly arrested. The horse serum-arrested stem cell population can be revived by reintroduction of fetal bovine serum. Development of serum free culture conditions facilitating either proliferation or differentiation of the Rcho-1 cells will be valuable for future studies on the control of trophoblast cell differentiation.  Differentiation can be assessed by monitoring the expression of members of the prolactin gene family or the biosynthesis of steroid hormones (Soares et al., 1991, 1993, 1995, 1996).

  

3.            PASSAGING THE RCHO-1 CELLS IN VITRO

 

The cells are routinely maintained in 75 cm² flasks.  The cells are initially plated at 1-2 million cells per flask and fed at two day intervals.  The cells will not form a "homogeneous" monolayer.  You should observe clusters of small cells surrounding islands of larger more adherent cells (including giant cells).  The cells are usually ready to be passaged after 3-4 days.

If the cells are not passaged then they begin to use the medium very rapidly as demonstrated by an increase in its acidity (especially when using RPMI culture medium).  The cells are harvested by brief exposure (30-60 sec) to a trypsin-EDTA solution (0.25% trypsin and 0.02% EDTA in Hanks balanced salt solution).  Medium containing serum is added to inhibit the trypsin and then the flask is scraped to remove all of the cells.  The use trypsin-EDTA without scraping results in the gradual selection of a different population of cells.  Giant cells are not satisfactorily removed with the enzyme-chelator treatment.  This procedure can be used to select for an enriched population of PL-I producing cells.  The use of scraping without enzyme-chelator treatment is also an ineffective method for passaging.  Scraping the dishes results in the generation of large clumps that do not readily grow when seeded into new flasks.

After harvesting, the cells are washed with fresh medium and replated at a splitting ratio of 1:6.  It is possible to split the cells at even a higher ratio but again we are concerned about the gradual selection of different cell types. 

 

 4.            PASSAGING THE RCHO-1 CELLS IN VIVO

 

Thus far, we have only transplanted the Rcho-1 cells beneath the kidney capsule.  Experiments are in progress to transplant the cells to other locations.  One group has successfully transplanted the cells to the cerebral ventricles (Arbogast et al., 1992).

Rcho-1 cells (1-5 million) grown in vitro are harvested as described above and transferred beneath the kidney capsule of 4 week old rats (we have used Lewis and Holtzman strains) in a volume of 25-50 microliters using a 27 gauge needle and 1 ml syringe.  The cells grow rapidly and need to be harvested within two weeks, preferably 10-12 days.  If the transplants are not harvested then they become necrotic and are useless.  A good check for a successful transplant is the presence of stimulated mammary glands.

 

 

5.            STORING THE RCHO-1 CELLS

 

We routinely freeze aliqots of cells containing 1-4 million cells/ml in our standard growth medium containing 10% dimethyl sulfoxide.  After gradual acclimation to -70 C over 1-4 weeks the cells are stored indefinitely in liquid nitrogen.

 

 

6.            TRANSFECTION OF THE RCHO-1 CELLS

 

Linzer and colleagues have successfully utilized Lipofectin or Lipofectamine (GIBCO/BRL) in their experiments of the mouse placental lactogen-I gene promoter (Shida et al., 1993; Ng et al., 1994; Cross et al., 1995).  We have also recently used these reagents in experiments studying the P450scc, P450c17, PLP-Cv, and d/tPRP promoters in Rcho-1 trophoblast cells (Yamamoto et al., 1994, 1995, 1996; Dai et al., 1996; Orwig et al., 1997).

  

 

7.            CLONAL LINES DERIVED FROM RCHO-1 CELLS

 

Various clonal lines have been derived from the parent Rcho-1 cell line (Faria and Soares, 1991).  Each of the lines is maintained as described for the parent Rcho-1 cell line.

 

 

8.            REFERENCES

 

Arbogast LA, Soares MJ, Robertson MC, Voogt JL 1993 A trophoblast-specific factor increases tyrosine hydroxylase activity in fetal hypothalamic cell cultures.  Endocrinology 133: 111-120.

 

Arbogast LA, Soares MJ, Tomogane H, Voogt JL 1992 A trophoblast-specific factor(s) suppress circulating prolactin levels and increases tyrosine hydroxylase activity in tuberoinfundibular dopaminergic neurons.  Endocrinology 131: 105-113.

 

Cross JC, Flannery ML, Blanar MA, Steingrimsson E, Jenkins NA, Copeland NG, Rutter WJ, Werb Z 1995 Hxt encodes a basic helix-loop-helix transcription factor that regulates trophoblast cell development.  Development 121: 2513-2523.

 

Dai G, Imagawa W, Liu B, Szpirer C, Levan G, Kwok SCM, Soares MJ 1996 Rcho-1 trophoblast cell placental lactogens: complementary deoxyribonucleic acids, heterologous expression, and biological activities.  Endocrinology 137: 5020-5027.

 

Dai G, Liu B, Szpirer C, Levan G, Kwok SCM, Soares MJ 1996 Prolactin-like protein-C variant:  complementary DNA, unique six exon gene structure, and trophoblast cell-specific expression.  Endocrinology 137, 5009-5019.

 

Duckworth ML, Robertson MC, Schroedter IC, Szpirer C, Friesen HG 1993 Molecular genetics and biology of the rat placental prolactin family.  In:  MJ Soares, S Handwerger, F Talamantes, editors, Trophoblast Cells:  Pathways for Maternal-Embryonic Communication, Springer-Verlag, New York, pp. 169-190.

 

Faria TN, Deb S, Kwok SCM, Vandeputte M, Talamantes F, Soares MJ 1990 Transplantable rat choriocarcinoma cells express placental lactogen:  identification of placental lactogen-I immunoreactive protein and messenger ribonucleic acid.  Endocrinology  127: 3131-3137. 

 

Faria TN, Soares MJ 1991 Trophoblast cell differentiation:  establishment, characterization, and modulation of a rat trophoblast cell line expressing members of the placental prolactin family.  Endocrinology 129: 2895-2906.

 

Grummer R, Hellmann P, Traub O, Soares MJ, Winterhager E 1996 Regulation of connexin 31 gene expression upon retinoic acid treatment in rat choriocarcinoma cells.  Exp Cell Res 227: 23-32

 

Hamlin GP, Lu X-J, Roby KF, Soares MJ 1994 Recapitulation of the pathway for trophoblast giant cell differentiation in vitro:  stage-specific expression of members of the prolactin gene family.  Endocrinology 134: 2390-2396.

 

Hamlin GP, Soares 1995 Regulation of DNA synthesis in proliferating and differentiating trophoblast cells:  involvement of transferrin, transforming growth factor-β, and tyrosine kinases.  Endocrinology 136: 322-331.

 

Kamei T, Hamlin GP, Chapman BM, Burkhardt AL, Bolen JB, Soares MJ 1997 Signaling pathways controlling trophoblast cell differentiation: src family protein tyrosine kinases.  Biol Reprod, in press

 

Mathiasen JR, Tomogane H, Voogt JL 1992 Serotonin-induced decrease in hypothalamic tyrosine hydroxylase activity and corresponding increase in prolactin release are abolished at midpregnancy and by transplants of rat choriocarcinoma cells.  Endocrinology 131: 2527-2532

 

Ng, YK, Engel, JD, Linzer DIH 1994 GATA factor activity is required for the trophoblast-specific transcriptional regulation of the mouse placental lactogen I gene.  Development 120: 3257-3266

 

Orwig KE, Dai G, Rasmussen CA, Soares MJ 1997 Decidual/trophoblast prolactin-related protein:  characterization of gene structure and cell-specific expression.  Endocrinology 138, 2491-2500

 

Scatena CD, Adler S 1996 Trans-acting factors dictate the species-specific placental expression of corticotropin-releasing factor genes in choriocarcinoma cell lines.  Endocrinology 137:3000-3008

 

Shida M, Ng YK, Linzer DIH 1993 Transcriptional regulation of the placental lactogen genes in mouse trophoblast giant cells.  In:  MJ Soares, S Handwerger, F Talamantes, editors, Trophoblast Cells:  Pathways for Maternal-Embryonic Communication, Springer-Verlag, New York, pp. 2433-252.

 

Shida M, Ng YK, Soares MJ, Linzer DIH 1993 Trophoblast-specific transcription rom the mouse placental lactogen I gene promoter.  Molecular Endocrinology 7:  181-188

 

Soares MJ, Chapman BM, Kamei T, Yamamoto T (1995) Control of trophoblast cell differentiation:  lessons from the genetics of early pregnancy loss and trophoblast neoplasia.  Development, Growth, & Differentiation 37: 355-364.

 

Soares MJ, Chapman BM, Rasmussen CA, Dai G, Kamei T, Orwig KE 1996 Differentiation of trophoblast endocrine cells.  Placenta 17: 277-289

 

Soares MJ, Faria TN, Hamlin GP, Lu X-J, Deb S (1993) Trophoblast cell differentiation:  expression of the placental prolactin family.  In:  MJ Soares, S Handwerger, F Talamantes, editors, Trophoblast Cells:  Pathways for Maternal-Embryonic Communication, Springer-Verlag, New York, pp. 43-65.

 

Soares MJ, Faria TN, Roby KF, Deb S 1991 Pregnancy and the prolactin family of hormones:  coordination of anterior pituitary, uterine, and placental expression.  Endocrine Reviews 12: 402-423

 

Teshima S, Shimosato Y, Koide T, Kuroki M, Kikuchi Y, Aizawa M 1983 Transplantable choriocarcinoma of rats induced by fetectomy and its biological activities.  Gann 74: 205-212

 

Tomogane H, Arbogast LA, Soares MJ, Robertson MC, Voogt JL 1993 A factor(s) from a rat trophoblast cell line inhibits prolactin secretion in vitro and in vivo.  Biology of Reproduction 48: 325-332

 

Tomogane H, Mistry AM, Voogt JL 1991 Late pregnancy and rat choriocarcinoma cells inhibit nocturnal prolactin surges and serotonin-induced prolactin release.  Endocrinology 130: 23-28.

 

Verstuyf A, Fonteyn E., Sobis H, Vandeputte M. 1992 A rat cytotrophoblast antigen defined by a monoclonal antibody.  Am J Reprod Immunol 28, 6-11.

 

Verstuyf A, Sobis H, Goebels J, Fonteyn E, Cassiman JJ, Vandeputte M 1990 Establishment and characterization of a continuous in vitro line from a rat choriocarcinoma.  Int J Cancer 45: 752-756

 

Verstuyf A, Sobis H, Vandeputte M 1989 Morphological and immunological characteristics of a rat choriocarcinoma.  Int J Cancer 44: 879-884

 

Vuille J-C, Cattini PA, Bock ME, Verstuyf A, Schroedter IC, Duckworth ML, Friesen HG 1993 Rat prolactin-like protein A partial gene and promoter structure:  promoter activity in placental and pituitary cells.  Mol Cell Endocrinol 96: 91-98

 

Yamamoto T, Chapman BM, Clemens J, Richards JS, Soares MJ 1995 Analysis of cytochrome P450 side-chain cleavage gene promoter activation during trophoblast cell differentiation.  Mol Cell Endocrinol 113: 183-194.