Isolation of Mesenchymal Stem Cells from Amniotic Fluid and Placenta

Christopher D. Graham1, Dario O. Fauza1

1 Department of Surgery, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1E.2
DOI:  10.1002/9780470151808.sc01e02s35
Online Posting Date:  November, 2015
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Abstract

Diverse progenitor cell populations, including mesenchymal, hematopoietic, trophoblastic, and possibly more primitive stem cells can be isolated from the amniotic fluid and the placenta. At least some of the amniotic and placental cells share a common origin, namely the inner cell mass of the morula. Indeed, most types of progenitor cells that can be isolated from these two sources share many characteristics. This unit will focus solely on the mesenchymal stem cells, the most abundant progenitor cell population found therein and, unlike some of the other stem cell types, present all through gestation. Protocols for isolation, expansion, freezing, and thawing of these cells are presented. Preference is given to the simplest methods available for any given procedure. © 2015 by John Wiley & Sons, Inc.

Keywords: amniotic fluid; placenta; mesenchymal stem cells; fetus; neonate; amniotic stem cells; stem cells; tissue engineering; fetal tissue engineering

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Mesenchymal Stem Cells from Amniotic Fluid
  • Alternate Protocol 1: Isolation of Mesenchymal Stem Cells from Amniotic Fluid Using Cover Slips
  • Basic Protocol 2: Isolation of Mesenchymal Stem Cells From Placenta
  • Alternate Protocol 2: Isolation of Mesenchymal Stem Cells from Placenta Using Cover Slips
  • Support Protocol 1: Expansion of Mesenchymal Stem Cells from Amniotic Fluid or Placenta
  • Support Protocol 2: Freezing and Thawing Mesenchymal Stem Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Mesenchymal Stem Cells from Amniotic Fluid

  Materials
  • Amniotic fluid (obtained via institutionally approved protocol)
  • Mesenchymal‐20 medium (see recipe)
  • Dulbecco's PBS (cation‐free; Invitrogen)
  • 0.025% (w/v) trypsin/0.04% (w/v) EDTA (Invitrogen)
  • 6‐well culture plate, pre‐coated with collagen I (BD Biosciences or self‐made plates, see recipe)
  • Inverting microscope
  • Centrifuge
  • 15‐ and 50‐ml conical tubes (BD Biosciences)
  • 10‐cm tissue culture dishes (BD Biosciences)

Alternate Protocol 1: Isolation of Mesenchymal Stem Cells from Amniotic Fluid Using Cover Slips

  Additional Materials (also see protocol 1)
  • 10‐cm collagen‐coated plate (see recipe)

Basic Protocol 2: Isolation of Mesenchymal Stem Cells From Placenta

  Materials
  • Human placental tissue (obtained via institutionally approved protocol)
  • 10% type II collagenase (Worthington Biochemical)
  • Dispase II (Roche)
  • Calcium chloride (CaCl 2, as a 2.5 mM solution)
  • Mesenchymal‐20 medium (see recipe)
  • Dulbecco's PBS (cation‐free; GIBCO)
  • 0.025% (w/v) trypsin/0.04% (w/v) EDTA (Invitrogen)
  • 6‐well culture plate pre‐coated with collagen type I (BD Biosciences or self‐made plates, see recipe)
  • Inverting microscope
  • Centrifuge
  • 15‐ and 50‐ml conical tube (BD Biosciences)
  • 100‐μm mesh (Fischer Scientific)
  • 10‐cm tissue culture dishes (BD Biosciences)

Alternate Protocol 2: Isolation of Mesenchymal Stem Cells from Placenta Using Cover Slips

  Additional Materials (also see protocol 3)
  • 10‐cm collagen‐coated plate (see recipe)

Support Protocol 1: Expansion of Mesenchymal Stem Cells from Amniotic Fluid or Placenta

  Materials
  • Mesenchymal stem cells (MSCs) in culture at 80% to 90% confluence
  • Mesenchymal‐10 medium (see recipe)
  • Dulbecco's PBS (cation‐free; Invitrogen)
  • 0.025% (w/v) trypsin/0.04% (w/v) EDTA (Invitrogen)
  • Inverting microscope
  • 10‐cm tissue culture dishes (BD Biosciences)

Support Protocol 2: Freezing and Thawing Mesenchymal Stem Cells

  Materials
  • Mesenchymal stem cells (MSCs) in culture, ∼50% confluent
  • Mesenchymal‐10 medium (see recipe)
  • Freezing solution (DMSO [Sigma‐Aldrich] and mesenchymal‐10 medium; see recipe)
  • Dulbecco's PBS (cation‐free; Invitrogen)
  • 0.025% trypsin/0.04% EDTA (Invitrogen)
  • 70% ethanol
  • Inverting microscope
  • Centrifuge
  • 15‐ml conical tube (BD Biosciences)
  • 2.0‐ml cryogenic vials (Fisher Scientific)
  • 10‐cm tissue culture dishes (BD Biosciences)
CAUTION: DMSO is hazardous. Carry out all operations involving DMSO in a fume hood, and wear gloves and protective glasses.
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Figures

Videos

Literature Cited

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