A serum-free system for culturing human placental trophoblasts

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In Vitro Cell. Dev. Biol. 26:865-870, September1990 9 1990Tissue Culture Association 0883-8364/90 $01.50+0.00

A SERUM-FREE

SYSTEM FOR CULTURING

HUMAN PLACENTAL

TROPHOBLASTS

CHARLOTTE L. BRANCHAUD', CYNTHIA G. GOODYER, HARVEY J. GUYDA, ANDYVES LEFEBVRE McGiH University-Montreal Children's Hospital Research Institute and Uuiversitg de MontrgabH$pital Notre Dame

~Received 12 February 1990; accepted 3 May 19901

SUMMARY

We have compared hormone production by early gestation and term human placental trophoblasts cultured in Ham's F10 medium containing 10% fetal bovine serum with that by cells cultured in serum-free HB102 medium. Mean daily production of progesterone on Days 3 to 7 was approximately 25% less by both early gestation and term cells cultured in HB102 as compared to Ham's F10, but production was maintained at a stable level for at least 7 d longer than the cells in Ham's. Estradiol production from 10-6 M dehydroepiandrosterone by both early gestation and term cells was comparable in both media. Human placental lactogen production on Days 3 to 7 was 40% less by cells cultured in HB102. Human chorionic gonadotropin ~hCG) output by early gestation cells was also 50% less in HB102 but term cells in HB102 produced twice as much hCG as those in Ham's F10. 3B-Hydroxysteroid dehydrogenase ~3BI-ISD) activity in early gestation and term cells and llB-hydroxysteroid dehydrogenase (llBHSD) activity of early gestation cultures was comparable in the two media, l l B H S D activity was decreased in the term cultures, and this decrease was more marked in Ham's than in HB102. Sulfatase and aromatase activities in the early gestation cultures were comparable in both media; sulfatase activity was comparable and aromatase activity only 20% less in the term ceils cultured in HB102. These results indicate that serum-free HB102 supports differentiated function of human trophoblast cells and is useful for studies of placental activity for as long as 14 d in culture.

Key words: placental cultures; progesterone; estradiol;

chorionic gonadotropin;

placental

lactogen;

serum-free medium.

INTRODUCTION

and bovine serum albumin (700 /ag/ml}; HB102 contains human low density lipoprotein (1 to 2 tag/ml~ in addition. These media also maintain JEG3 cells, a choriocarcinoma cell line, without added serum tLai, W. H., personal communicationL We now report that normal placental cells may also be maintained in HB medium, and compare the functional activity of both early gestation and term placental cells cultured in Ham's F10 vs. HB102.

We have previously described a method for culturing human midgestation and term placental cells il,TL The culture medium used at that time was Ham's F10 supplemented with 10% fetal bovine serum IFBSL The sensitivity of placental cells to FBS has since become a matter of concern. Morphology of cells from the same placenta was found to differ vastly when exposed to various lots of FBS (unpublished observationsL Steroid and peptide hormone production also varied both quantitatively and in terms of the time in culture over which they were maintained. In an attempt to overcome these variable responses, we sought a serum-free system for maintaining human placental cells in culture. I-IB101 and HB102 media IHana Media Inc., Alameda, CA) were developed to support the growth of certain myeloma, hybridoma, and lymphoblastoid cell lines (10,14). HB101 is a modified R P M I medium containing human transferrin (10 gg/ml), bovine insulin (15 ~g/ml),

MATERIALS AND METHODS

Placental cultures. Early gestation placentae were obtained at the time of therapeutic abortion by dilation and curettage (Dept. of Obstetrics and Gynecology, Hopital Notre Dame} with approval from the local Institutional Ethics Committees. Term placentae were obtained after caesarean section at 38 to 40 wk from the Royal Victoria Hospital. Tissue was transported in ice cold Ca**- and Mg§247 Earles balanced salt solution and processed as soon as possible. Monolayer cultures were established as described previously (1L Briefly, trophoblast cells were dispersed with 0.25% trypsin and 5 /ag/ml DNAase; the cells were harvested every 10 min for approximately 90 min. The final pooled pellet was lysed

' To whom correspondence should be addressed at Montreal Children's Hospital, 2300 Tupper Street, Rm. C-1237, Montreal, Quebec H3H 1P3, Canada.

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with ammonium chloride phosphate buffer to remove the large number of red cells. The lysed pellet was resuspended in Ham's F10 IGIBCO, Burlington, Ont., Canada) supplemented with 10% FBS (Hyclone, Logan, UT) and antibiotics [5 /ag/ml of amphotericin B (Fungizone, Squibb, Montreal, Que., Canadal, 200 !IU/ml of penicillin G (Crystapen, Glaxo, Montreal, Que., Canada J, and 40 #g/ml of gentamicin (Garamicin, Schering, Point Claire, Que., Canada,] and the cells plated confluenfly in 60 X 15-ram plastic petri dishes (Lux, Miles Canada Inc., Rexdale, Ont.). After 24 h, once the cells had attached, the plating medium was removed and, after one rinse, replaced with either serum-free HB102 medium or Ham's F10 containing 10% FBS. The media were collected and replaced daily thereafter. Hormone determinations. Media were kept frozen until processed for determination of their hormone content. Progesterone and estradiol were measured by radioimmunoassay (RIA~ as described previously (1~. Human chorionic gonadotropin (hCG} and human placental lactogen (hPL} levels were determined by RIA using N I A D D K reagents (7}. Endogenous levels of progesterone, estrone, and estradiol in Ham's + 10% FBS medium were
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