A Practical Guide to Culturing Mouse and Human Bone Marrow Stromal Cells

K. Nemeth1, B. Mayer1, B.J. Sworder1, S.A. Kuznetsov1, E. Mezey1

1 Health and Human Services, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 22F.12
DOI:  10.1002/0471142735.im22f12s102
Online Posting Date:  October, 2013
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Abstract

Bone marrow stromal cells (BMSCs, frequently also called MSCs) represent a cell population within the bone marrow, a subset of which contains multipotent stem cells. Their primary role is to produce and maintain both bone tissue and bone marrow microenvironment necessary for hematopoiesis. The latter is achieved by secreting a wide variety of different cytokines and growth factors, many of which also have a regulatory role in immune processes. BMSCs have recently been introduced into the field of immunobiology after their successful clinical use in GVHD was reported in 2004. Since then, numerous studies confirmed and expanded the knowledge on the immunosuppressive potential of BMSCs in various in vitro and in vivo models. Although the immunomodulatory capacity of BMSCs is well established, there are still many unanswered questions regarding the cytokines, chemokines, receptors, and molecular pathways that play a role in this effect. To study these cells and answer many of the questions, researchers must be able to reliably and reproducibly isolate, culture, and use these cells. Below a practical guide on how to culture and characterize mouse and human BMSCs, which can then be applied in various in vitro and in vivo assays, is provided. Curr. Protoc. Immunol. 102:22F.12.1‐22F.12.13. © 2013 by John Wiley & Sons, Inc.

Keywords: mesenchymal stem cells; bone marrow stromal cells; isolation; culture; immunomagnetic separation; osteogenic and adipogenic differentiation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Primary Culture of Mouse BMSCs
  • Basic Protocol 2: Culture of Human BMSCs from Fresh Bone Marrow Aspirate
  • Support Protocol 1: Characterization of BMSCs by Facs Analysis
  • Support Protocol 2: Osteogeneic and Adipogeneic Differentiation Assays
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Primary Culture of Mouse BMSCs

  Materials
  • Live mice or frozen carcasses
  • 70% ethanol
  • Complete medium (see recipe)
  • Trypan blue (Lonza)
  • Separation buffer (see recipe)
  • Anti‐mouse CD11b and CD45 magnetic beads (Miltenyi)
  • 1× PBS (Cellgro) or HBSS (Gibco)
  • 0.05% Trypsin‐EDTA (Gibco)
  • Freezing medium (see recipe)
  • Isopropyl alcohol
  • Liquid nitrogen
  • 100‐mm culture dish (BD Falcon)
  • Scalpel (blade size 10)
  • Curved scissors (e.g., Iris scissors, size 4‐1/8)
  • Forceps: tissue forceps or dissecting forceps, length 4‐7
  • 12‐ml syringes
  • 16‐, 19‐, and 23‐G needles
  • 50‐ml conical tubes
  • 25‐ and 75‐cm2 vented‐cap culture flasks (BD Falcon)
  • 37°C, 5% CO 2 humidified cell culture incubator
  • Inverted phase‐contrast microscope
  • Hemacytometer (Neubauer)
  • Light microscope
  • MACS LD separation columns (Miltenyi Biotech)
  • MACS separator
  • Pipets
  • 70‐µm mesh size cell strainer (BD Falcon)
  • 2‐ml freezing tubes
  • Freezing tank
  • 37°C water bath

Basic Protocol 2: Culture of Human BMSCs from Fresh Bone Marrow Aspirate

  Materials
  • Bone marrow
  • Heparinized syringes (BD)
  • 3% acetic acid with methylene blue
  • Complete medium (see recipe)
  • HBSS
  • Automated cell counter or manual cell counting chamber
  • Culture flasks

Support Protocol 1: Characterization of BMSCs by Facs Analysis

  Materials
  • Cells
  • FACS buffer (see recipe)
  • Antibodies (BD Biosciences):
    • Anti‐mouse CD44, Sca‐1, CD45, CD31, Ter‐119, appropriate (fluorophore‐matched) isotype controls
    • Anti‐human CD44, CD73, CD90, CD105, CD106, CD166, CD45, CD31, GPA, appropriate isotype controls
  • Wash buffer
  • 5‐ml FACS tubes
  • Centrifuge
  • Flow cytometer (FACSCalibur, BD Biosciences)

Support Protocol 2: Osteogeneic and Adipogeneic Differentiation Assays

  Materials
  • BMSCs
  • Complete medium (see recipe)
  • Dexamethasone (Sigma)
  • L‐Ascorbic acid 2‐phosphate (Sigma)
  • β‐glycerol phosphate (Sigma)
  • PBS
  • 4% paraformaldehyde in 1× PBS
  • Alizarin Red S (Sigma)
  • 2% ethanol
  • Indomethacin (Sigma)
  • Hydrocortisone (Sigma)
  • Isobutylmethylxanthine (IBMX, Sigma)
  • Oil Red (Sigma)
  • 6‐well plates
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Figures

Videos

Literature Cited

Literature Cited
  Friedenstein, A.J., Piatetzky‐Shapiro, I.I., and Petrakova, K.V. 1966. Osteogenesis in transplants of bone marrow cells. J. Embryol. Exp. Morphol. 16:381–390.
  Friedenstein, A.J., Petrakova, K.V., Kurolesova, A.I., and Frolova, G.P. 1968. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation 6:230‐247.
  Le Blanc, K., Rasmusson, I., Sundberg, B., Götherström, C., Hassan, M., Uzunel, M., and Ringdén, O. 2004. Treatment of severe acute graft‐versus‐host disease with third party haploidentical mesenchymal stem cells. Lancet. 363:1439–1441.
  Uccelli, A., Moretta, L., and Pistoia, V. 2008. Mesenchymal stem cells in health and disease. Nat. Rev. Immunol. 8:726–736.
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