Isolation of Cells from the Feto‐Maternal Interface

Victoria Male1, Lucy Gardner2, Ashley Moffett2

1 Imperial College, London, United Kingdom, 2 Department of Pathology, University of Cambridge, Cambridge, United Kingdom
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.40
DOI:  10.1002/0471142735.im0740s97
Online Posting Date:  April, 2012
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Abstract

The mucosal lining of the human uterus is host to a specialized population of leukocytes, which, during pregnancy, interact with invading placental cells (trophoblast) of fetal origin. Of particular interest are uterine natural killer cells, which account for around 70% of the leukocytes at this site during the first trimester of pregnancy, and seem to be specially adapted to recognize invading trophoblast cells. In order to understand the interactions between mucosal immune cells and trophoblast, and those among the immune cells themselves, it is useful to be able to isolate and culture these cells. Here, we describe protocols for the isolation of leukocytes, stromal cells, and trophoblast cells from the feto‐maternal interface. Curr. Protoc. Immunol. 97:7.40.1‐7.40.11. © 2012 by John Wiley & Sons, Inc.

Keywords: pregnancy; trophoblast; decidua; endometrium; natural killer cells; stromal cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Decidual and Placental Tissue Pieces
  • Basic Protocol 2: Enzymatic Isolation of Decidual Cells
  • Alternate Protocol 1: Mechanical Disaggregation of Decidual Cells
  • Basic Protocol 3: Isolation of Trophoblast Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Decidual and Placental Tissue Pieces

  Materials
  • Ham's F12 medium (see recipe)
  • RPMI 1640 medium (see recipe)
  • Class II microbiological safety cabinet
  • Large plastic tray
  • Paper towels
  • 150‐mm diameter petri dishes
  • Large metal sieve (100 µm) with waste collection tray, autoclaved to sterilize (Endecotts)
  • Gauze (butter muslin cut to 200‐mm squares and then autoclaved to sterilize)
  • Sterile scissors
  • Sterile forceps
  • 250‐ml bottles, sterile
  • Magnetic stirrer and sterile magnetic stirrer bars

Basic Protocol 2: Enzymatic Isolation of Decidual Cells

  Materials
  • Washed decidual tissue pieces ( protocol 1)
  • RPMI 1640 medium (see recipe), supplemented with 10% fetal bovine serum, 2 µM L‐glutamine, and antibiotics (100 U/ml penicillin and streptomycin, 6.25 µg/ml amphoteracin, 50 µg/ml gentamicin)
  • Collagenase Type V solution (see recipe)
  • Lymphoprep brought to room temperature before beginning the protocol (Nycomed; http://www.nycomed.com)
  • Appropriate cell culture medium or phosphate‐buffered saline (PBS)
  • Sterile disposable scalpels
  • gentleMACS C tubes (Miltenyi Biotec)
  • gentleMACS dissociator (Miltenyi Biotec)
  • 37°C incubator with shaking platform: atmospheric oxygen and carbon dioxide levels
  • 100‐ and 40‐µm cell strainers
  • Centrifuge
  • 50‐ml tubes
NOTE: The fetal bovine serum (FBS) added to the RPMI 1640 medium is heat inactivated at 56°C for 60 min and cooled quickly in cold water. Aliquots of the serum can be stored at –20°C and, after thawing, for up to 1 month at 4°C.

Alternate Protocol 1: Mechanical Disaggregation of Decidual Cells

  • Large metal sieve (75 µm) with collection tray, autoclaved to sterilize (Endecotts; for this protocol, the diameter of the mesh must be 70 µm)
  • Large rubber bung (such as would be used to stop a large piece of laboratory glassware), sterilized

Basic Protocol 3: Isolation of Trophoblast Cells

  Materials
  • Fibronectin (BD Biosciences)
  • Ham's F12 medium (see recipe), supplemented with 20% newborn calf serum (NCS; heat inactivated and stored as for FBS)
  • Trypsin solution (see recipe)
  • Washed pieces of placental villi
  • Lymphoprep (Nycomed)
  • 35‐mm diameter petri dishes, sterile
  • 150‐mm diameter petri dishes, sterile
  • Sterile disposable scalpels
  • Sterile forceps
  • 100‐ml bottles, sterile
  • Magnetic stirrer and sterile magnetic stirrer bars
  • 37°C heat block
  • Centrifuge
  • Plastic funnel
  • Gauze (butter muslin cut to fit in the funnel and then autoclaved to sterilize)
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Figures

Videos

Literature Cited

Literature Cited
   Apps, R., Gardner, L., and Moffett, A. 2008. A critical look at HLA‐G. Trends Immunol. 29:313‐321.
   Apps, R., Sharkey, A., Gardner, L., Male, V., Trotter, M., Miller, N., North, R., Founds, S., and Moffett, A. 2011. Genome‐wide expression profile of first trimester villous and extravillous human trophoblast cells. Placenta 32:33‐43.
   Boyd, J.D., and Hamilton, W.J. 1970. The Human Placenta. W. Heffner and Sons, Cambridge, UK.
   Gardner, L. and Moffett, A. 2003. Dendritic cells in the human decidua. Biol. Reprod. 69:1438‐1446.
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   Hiby, S.E., Walker, J.J., O'shaughnessy, K.M., Redman, C.W., Carrington, M., Trowsdale, J., and Moffett, A. 2004. Combinations of maternal KIR and fetal HLA‐C genes influence the risk of preeclampsia and reproductive success. J. Exp. Med. 200:957‐965.
   Hiby, S.E., Apps, R., Sharkey, A.M., Farrell, L.E., Gardner, L., Mulder, A., Claas, F.H., Walker, J.J., Redman, C.C., Morgan, L., Tower, C., Regan, L., Moore, G.E., Carrington, M., and Moffett, A. 2010. Maternal activating KIRS protect against human reproductive failure mediated by fetal HLA‐C2. J. Clin. Invest. 120:4102‐4110.
   Kam, E.P., Gardner, L., Loke, Y.W., and King, A. 1999. The role of trophoblast in the physiological change of the decidual spiral arteries. Hum. Reprod. 14:2131‐2138.
   King, A. and Loke, Y.W. 1990. Human trophoblast and JEG choriocarcinoma cells are sensitive to lysis by IL‐2‐stimulated decidual NK cells. Cell Immunol. 129:435‐448.
   King, A., Balendran, N., Wooding, P., Carter, N.P., and Loke, Y.W. 1991. CD3‐ leukocytes present in the human uterus during early placentation: Phenotypic and morphologic characterization of the CD56++ population. Dev. Immunol. 1:169‐190.
   Koopman, L.A., Kopcow, H.D., Rybalov, B., Boyson, J.E., Orange, J.S., Schatz, F., Masch, R., Lockwood, C.J., Schachter, A.D., Park, P.J., and Strominger, J.L. 2003. Human decidual natural killer cells are a unique NK cell subset with immunomodulatory potential. J. Exp. Med. 198:1201‐1212.
   Medawar, P.B. 1953. Some immunological and endocrinological problems raised by the evolution of viviparity in vertebrates. Symp. Soc. Exp. Biol. 7:320‐338.
   Mjösberg, J., Berg, G., Jenmalm, M.C., and Ernerudh, J. 2010. FOXP3+ regulatory T cells and T helper 1, T helper 2, and T helper 17 cells in human early pregnancy decidua. Biol. Reprod. 82:698‐705.
   Moffett, A. and Loke, C. 2006. Immunology of placentation in eutherian mammals. Nat. Rev. Immunol. 6:584‐594.
   Pijnenborg, R., Vercruysse, L., and Hanssens, M. 2005. The uterine spiral arteries in human pregnancy: Facts and controversies. Placenta 27:939‐958.
   Sharkey, A.M., Gardner, L., Hiby, S., Farrell, L., Apps, R., Masters, L., Goodridge, J., Lathbury, L., Stewart, C.A., Verma, S., and Moffett, A. 2008. Killer Ig‐like receptor expression in uterine NK cells is biased toward recognition of HLA‐C and alters with gestational age. J. Immunol. 181:39‐46.
   Verma, S., King, A., and Loke, Y.W. 1997. Expression of killer cell inhibitory receptors on human uterine natural killer cells. Eur. J. Immunol. 27:979‐983.
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