Amniotic Membrane: Separation of Amniotic Mesoderm from Amniotic Epithelium and Isolation of Their Respective Mesenchymal Stromal and Epithelial Cells

Antonella Barbati1, Maria Grazia Mameli2, Angelo Sidoni2, Gian Carlo Di Renzo1

1 Department of Medical‐Surgical Specialties and Public Health, Section of Obstetrics and Gynecology, University of Perugia, Perugia, Italy, 2 Department of Experimental Medicine and Biochemical Sciences, Section of Pathologic Anatomy and Histology, University of Perugia, Italy
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1E.8
DOI:  10.1002/9780470151808.sc01e08s20
Online Posting Date:  March, 2012
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The human amniotic membrane (hAM) or amnion contains two principal types of cells: amniotic epithelial cells (hAECs) and amniotic mesenchymal stromal cells (hAMSCs), located in two distinct regions: the epithelium and the stromal layer. Emerging evidence suggests that both of them retain multipotent/pluripotent characteristics, making the amniotic membrane a promising and very attractive source of cells for regenerative medicine. Therefore, the isolation of hAECs and hAMSCs has recently received great interest; they can be released by differential enzymatic digestion and various procedures have been reported; however, significant contamination of hAMCs with hAECs and vice versa frequently occurs. This unit describes an efficient and rapid method to separate, mechanically, amniotic mesoderm from amniotic epithelium in order to obtain, after subsequent enzymatic digestions, purified population of hAMCs and hAECs. In this way, the cells can be cultured or investigated for other aims avoiding additional procedures related to their purification. Curr. Protoc. Stem Cell Biol. 20:1E.8.1‐1E.8.15. © 2012 by John Wiley & Sons, Inc.

Keywords: amniotic membrane; amniotic epithelium; amniotic mesoderm; epithelial/mesenchymal stem cells

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Table of Contents

  • Introduction
  • Basic Protocol 1: Amnion Collection and Separation of Amniotic Mesoderm from Amniotic Epithelium
  • Basic Protocol 2: Release of Amniotic Epithelial Stem Cells
  • Basic Protocol 3: Release of Amniotic Mesenchymal Stem Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Amnion Collection and Separation of Amniotic Mesoderm from Amniotic Epithelium

  • Term placenta, freshly delivered (see Recruitment of Placentas)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Lonza, cat. no. BE17‐517Q), sterile
  • Washing solution (see recipe)
  • Laminar flow cabinet
  • Gloves, sterile
  • Forceps, sterile
  • Scalpels, sterile
  • 150‐ml sterile specimen containers (e.g., for urine collection)
  • Electronic balance (optional)
  • 600‐mm glass beakers, sterile
  • 150‐mm petri dishes, sterile (e.g., Greiner Bio‐One)
  • Sterile microscope slides with smoothed edges
  • 50‐ml Falcon tubes

Basic Protocol 2: Release of Amniotic Epithelial Stem Cells

  • Amniotic epithelium (see protocol 1)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Lonza, cat. no. BE17‐517Q), sterile
  • Digestion solution A (see recipe)
  • Hank's balanced salt solution (HBSS)
  • Fetal bovine serum (FBS)
  • Standard culture medium for hAECs (see recipe)
  • 37°C shaking water bath
  • 300‐ml glass conical flasks, sterile
  • Sterilized magnetic stir bar
  • Magnetic stirrer
  • 37°C incubator
  • Sterilized steel strainer (5‐cm diameter)
  • 150‐ml containers, sterile
  • 50‐ml conical polypropylene centrifuge tubes (e.g., Falcon), sterile
  • Centrifuge
  • Rotator
  • 40‐µm nylon cell strainers (Falcon, cat. no. 2350)
  • Additional reagents and equipment for counting cells using trypan blue staining (Phelan, )

Basic Protocol 3: Release of Amniotic Mesenchymal Stem Cells

  • Falcon tube containing the amniotic mesoderm (see protocol 1)
  • Digestion solution B (see recipe)
  • ACK lysing buffer (Lonza, cat. no. 10‐548E), optional
  • Hank's balanced salt solution
  • Standard culture medium for hAMSCs (see recipe)
  • Rotator
  • 37°C incubator
  • Centrifuge
  • 70‐µm cell strainers
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Literature Cited

Literature Cited
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