Culture of HRP/LRP/R8 Trophoendodermal Cell Lines

             In  rodents, specifically the mouse and rat, there is a stem cell population that arises after midgestation with the capability of differentiating into cells with characterstics resembling trophoblast and yolk sac phenotypes (Damjanov et al., 1985; Soares et al., 1987; Vandeputte and Sobis, 1988).   We refer to these progenitor cells as trophoendodermal stem cells.  This population of cells can be harvested from the midgestation chorioallantoic placenta and is readily adaptable to propagation both in vitro and in vivo  (Log et al., 1981; Beeson et al., 1984; Soares et al., 1987).  In general, among a variety of features, the cells are typified by their abundant synthesis of basement membrane components, especially laminin (Soares et al., 1987; 1988).   

Several years ago, we established a number of cell lines from the normal rat midgestation chorioallantoic placenta with characteristics similar to trophoendodermal stem cells (see Soares et al., 1987; Hunt et al., 1989, for a description and characterization of the cell line).  The cell lines are very easy to maintain and very hardy [please make sure not to contaminate other cell lines with these HRP/LRP/R8 cell line].  This cell line does not appear to express any members of the placental prolactin gene family.  Consequently, we do not believe the cells are capable of differentiating along the trophoblast giant cell or spongiotrophoblast lineages. The cells are very responsive to activators of the cAMP/protein kinase A pathway (Soares et al., 1989) and very recently we have found the cells are capable of expressing α-CG promoter/reporter constructs (Yamamoto and Soares, unpublished).  This may be related to the recent observations of β-CG transgene expression in a midgestation parietal endoderm-like population of cells (Strauss et al. 1994).  Additionally, the HRP-1 cell line has recently been shown to be a useful model for studying transport, permeability, and metabolic properties of trophoblast cells (Das et al., 1997; Shi et al., 1997).

 

1.            CULTURE MEDIA

RPMI 1640 culture medium (Sigma) supplemented with:

50 micromolar 2-mercaptoethanol (BIO-RAD)

1 millimolar sodium pyruvate (Sigma)

100 micograms/ml penicillin (Sigma)

100 units/ml streptomycin (Sigma)

5-20% (heat inactivated) fetal bovine serum (JRH Biosciences)

 

(This medium is routinely used for growth and general maintenance of the cell line.  If the density of the cultures is to high then the RPMI medium will not adequately maintain the pH.  Under longer term differentiation experiments we shift the cells to NCTC-135 (Sigma) medium containing the same supplements.  We have not rigorously tested the effects of different concentrations of fetal bovine serum.  A concentration of 20% fetal bovine serum probably exceeds the minimum requirement.)   

  

2.            PASSAGING THE HRP/LRP/R8 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 form a monolayer.  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.  The cells are harvested by either scraping or brief exposure (30-60 sec) to a trypsin-EDTA solution (0.25% trypsin and 0.02% EDTA in Hanks balanced salt solution).  When trypsin is used, medium containing serum is added to inhibit the trypsin. 

After harvesting, the cells are washed with fresh medium and replated at a splitting ratio of 1:10.  It is possible to split the cells at even a higher ratio but we are concerned about the gradual selection of different cell types. 

  

3.            STORING THE HRP/LRP/R8 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.

  

4.            REFERENCES

 

Beeson, J.H., Daynes, R.A., Weinert, A.M., and Gahring, L.C. (1984) Masses arising from injection of cultured cells into normal animals:  direct evidence for placental origin of masses of two histologic types.  J. Natl. Cancer Inst. 73, 705-709

 

Damjanov, I., Damjanov, A., and Andrews, P.W. (1985) Trophectodermal carcinoma:  mouse teratocarcinoma-derived tumour stem cells differentiating into trophoblastic and yolk sac elements.  J. Embryol. exp. Morph. 86, 125-141

 

Das, U.G., Sadiq, H.F., Soares, M.J., Hay, W.W., and Devaskar, S.U. (1997) Time dependent physiological regulation of rodent and ovine placental glucose transporter (Glut 1) protein.  Submitted to:  American J Physiology

 

De M, Hunt JS, Soares MJ 1988 Stimulation of rat placental cell DNA synthesis by transferrin.  Biol Reprod 38: 1123-1128

 

De SK, Larsen DB, Soares MJ (1995) Trophoendodermal stem cell-derived extracellular matrices:  absence of detectable entactin and presence of multiple laminin species.  Placenta 16: 701-718

 

Hunt JS, Deb S, Faria TN, Wheaton D, Soares MJ 1989 Isolation of phenotypically distinct trophoblast cell lines from normal rat chorioallantoic placentas Placenta 10: 161-177.