Preparation of Anti‐Inflammatory Mesenchymal Stem/Precursor Cells (MSCs) Through Sphere Formation Using Hanging‐Drop Culture Technique

Thomas J. Bartosh1, Joni H. Ylostalo1

1 Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine at Scott & White Hospital, Temple, Texas
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2B.6
DOI:  10.1002/9780470151808.sc02b06s28
Online Posting Date:  February, 2014
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Abstract

Herein, we describe a protocol for preparation of pre‐activated anti‐inflammatory human mesenchymal stem/precursor cells (MSCs) in 3‐D culture without addition of exogenous chemicals or gene‐transfer approaches. MSCs are an easily procurable source of multipotent adult stem cells with therapeutic potential largely attributed to their paracrine regulation of inflammation and immunity. However, the culture conditions to prepare the ideal MSCs for cell therapy remain elusive. Furthermore, the reported lag time for activation in experimental models has prompted investigations on pre‐activating the cells prior to their administration. In this protocol, standard 2‐D culture‐expanded MSCs are activated by aggregation into 3‐D spheres using hanging‐drop cultures. MSC activation is evaluated by real‐time PCR and/or ELISA for anti‐inflammatory factors (TSG‐6, STC‐1, PGE2), and by a functional assay using lipopolysaccharide‐stimulated macrophage cultures. Further, we elucidate methods to prepare MSC‐sphere conditioned medium, intact spheres, and suspension of single cells from spheres for experimental and clinical applications. Curr. Protoc. Stem Cell Biol. 28:2B.06.1‐2B.06.23. © 2014 by John Wiley & Sons, Inc.

Keywords: MSC; sphere; anti‐inflammatory; macrophage; activation; spheroid; LPS

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Hanging‐Drop Culture Technique for the Development of MSC Spheres
  • Support Protocol 1: Recovery of Frozen MSCs and Expansion as 2‐D Adherent Cultures
  • Basic Protocol 2: Harvest of MSC Spheres and Preparation of Single Cells from Spheres
  • Basic Protocol 3: Preparation of MSC‐Sphere Conditioned Medium Using Transfer Cultures
  • Basic Protocol 4: Gene Expression Analysis of Anti‐Inflammatory Markers
  • Basic Protocol 5: Quantification of PGE2 Secreted by MSC Spheres
  • Basic Protocol 6: Functional Measurement of the Anti‐Inflammatory Properties of MSC Spheres Using Macrophage Cultures
  • Alternate Protocol 1: Measurement of the Anti‐Inflammatory Effects of MSC Spheres and Sphere‐Derived Cells Using a Transwell Macrophage System
  • Support Protocol 2: J774 Macrophage Culture in Petri Dishes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Hanging‐Drop Culture Technique for the Development of MSC Spheres

  Materials
  • Culture‐expanded and harvested bone‐marrow MSCs in CCM ( protocol 2)
  • Complete culture medium (CCM; see recipe)
  • Phosphate‐buffered saline (PBS) without calcium chloride and magnesium, pH 7.4 (Life Technologies, cat. no. 10010)
  • 150 × 25–mm cell culture–treated dishes
  • Lab marker
  • Sterile reagent reservoir
  • Multichannel pipettor with sterile 100‐µl tips

Support Protocol 1: Recovery of Frozen MSCs and Expansion as 2‐D Adherent Cultures

  Materials
  • Complete culture medium (CCM; see recipe)
  • Frozen vial containing approximately 106 passage‐1 or ‐2 bone marrow MSCs (Center for the Preparation and Distribution of Adult Stem Cells, Texas A&M Health Science Center, Institute for Regenerative Medicine)
  • Phosphate‐buffered saline (PBS), pH 7.4 (Life Technologies, cat. no. 10010)
  • 0.25% trypsin‐EDTA (e.g., Life Technologies), prewarmed
  • 150 × 25–mm cell culture–treated dishes
  • Liquid nitrogen tank for cell storage
  • Water bath set to 37°C
  • Upright microscope with a 10× objective
  • 50‐ml sterile conical tubes (e.g., BD Falcon)
  • Centrifuge with swinging‐bucket rotor and adaptors for 50‐ml conical tubes
  • Additional reagents and equipment for counting cells with a hemacytometer (e.g., unit 1.13)

Basic Protocol 2: Harvest of MSC Spheres and Preparation of Single Cells from Spheres

  Materials
  • Hanging‐drop culture dish with day‐3 MSC spheres ( protocol 1)
  • Phosphate‐buffered saline (PBS) without calcium chloride and magnesium chloride, pH 7.4 (Life Technologies, cat. no. 10010)
  • 0.25% trypsin‐EDTA (e.g., Invitrogen), prewarmed
  • Complete culture medium (CCM; see recipe)
  • Fetal bovine serum (FBS; optional)
  • Cell lifter
  • 15‐ml and 50‐ml sterile conical tubes (e.g., BD Falcon)
  • Centrifuge with swinging bucket rotor and adaptors for 15‐ml and 50‐ml conical tubes Water bath set to 37°C
  • Upright microscope with a 10× objective
  • 40‐µm cutoff cell strainer
  • Additional reagents and equipment for counting cells with a hemacytometer (e.g., unit 1.13)

Basic Protocol 3: Preparation of MSC‐Sphere Conditioned Medium Using Transfer Cultures

  Materials
  • Adherent monolayer MSCs ( protocol 2) or MSC spheres or single cells from spheres ( protocol 3)
  • Complete culture medium (CCM; see recipe)
  • 15‐ml sterile conical tubes (e.g., BD Falcon)
  • Centrifuge with swinging bucket rotor and adaptors for 15‐ml conical tubes
  • 6‐well cell culture dish, treated
  • 0.22 µm filter (optional)
  • Additional reagents and equipment for counting cells with a hemacytometer (e.g., unit 1.13)

Basic Protocol 4: Gene Expression Analysis of Anti‐Inflammatory Markers

  Materials
  • Adherent monolayer MSCs ( protocol 2)
  • MSC spheres ( protocol 3)
  • RNeasy Mini Kit (Qiagen, cat. no. 74104)
  • RNase‐Free DNase Set (Qiagen, cat. no. 79254)
  • High Capacity cDNA Reverse Transcription Kit (Life Technologies, cat. no. 4368814)
  • GAPDH TaqMan Gene Expression Assay FAM (Life Technologies, cat. no. Hs02758991_g1)
  • PTGS2 (COX‐2) TaqMan Gene Expression Assay FAM (Life Technologies, cat. no. Hs00153133_m1)
  • TNFAIP6 (TSG‐6) TaqMan Gene Expression Assay FAM (Life Technologies, cat. no. Hs01113602_m1)
  • STC1 (STC‐1) TaqMan Gene Expression Assay FAM (Life Technologies, cat. no. Hs00174970_m1)
  • TaqMan Fast Universal PCR Master Mix (2×), No AmpErase UNG (Life Technologies, cat. no. 4352042)
  • QIAshredder (Qiagen, cat. no. 79654)
  • 1.5‐ml RNase‐free sterile microcentrifuge tubes
  • Microvolume spectrophotometer (e.g., NanoDrop)
  • 0.2‐ml RNase and DNase‐free sterile PCR tubes or strips with caps
  • Thermal cycler
  • Real‐time PCR system capable of detecting FAM and ROX reporter dyes (e.g., ABI 7900HT; Life Technologies)

Basic Protocol 5: Quantification of PGE2 Secreted by MSC Spheres

  Materials
  • MSC monolayer and sphere‐conditioned medium ( protocol 4)
  • Prostaglandin E2 Parameter Assay Kit (R&D Systems, cat. no. KGE004B)
  • Horizontal orbital microplate shaker capable for 500 rpm speed
  • Multichannel vacuum aspirator
  • Microplate reader capable of measuring absorbances at 450 nm and 540 nm
  • Computer software that can generate a four parameter logistic curve fit

Basic Protocol 6: Functional Measurement of the Anti‐Inflammatory Properties of MSC Spheres Using Macrophage Cultures

  Materials
  • Macrophage medium (see recipe)
  • MSC‐conditioned medium from adherent monolayer, sphere, and sphere‐derived cell cultures ( protocol 4)
  • Complete culture medium (CCM; see recipe)
  • J774 mouse macrophages ( protocol 9)
  • 0.1 mg/ml lipopolysaccharide (LPS; e.g., Sigma, cat. no. L4130) solution in PBS (Life Technologies, cat. no. 10010)
  • 12‐well cell culture dish, treated
  • 15‐ml and 50‐ml sterile conical tubes (e.g., BD Falcon)
  • Water bath set to 37°C
  • Upright microscope with a 10× objective
  • Mouse TNF‐α Quantikine ELISA kit (R&D Systems, cat. no. MTA00B)
  • Mouse IL‐10 Quantikine ELISA kit (R&D Systems, cat. no. M1000)

Alternate Protocol 1: Measurement of the Anti‐Inflammatory Effects of MSC Spheres and Sphere‐Derived Cells Using a Transwell Macrophage System

  Materials
  • Macrophage medium (see recipe)
  • Adherent monolayer MSCs ( protocol 2)
  • MSC spheres and single cells from spheres ( protocol 3)
  • Complete culture medium (CCM; see recipe)
  • J774 mouse macrophages ( protocol 9)
  • 0.1 mg/ml lipopolysaccharide (LPS; e.g., Sigma, cat. no. L4130) solution in PBS (Life Technologies, cat. no. 10010)
  • Costar 6‐well tissue culture plates with 24‐mm permeable Transwell supports, pore size 0.4 µm (Corning, cat. no. 3412)
  • Sterile forceps
  • Upright microscope with 10× objective
  • 15‐ml and 50‐ml sterile conical tubes
  • Additional reagents and equipment for TNF‐α and IL‐10 ELISAs using commercial kits (see protocol 7)

Support Protocol 2: J774 Macrophage Culture in Petri Dishes

  Materials
  • Frozen vial containing approximately 106 J774 mouse macrophages (J774A.1; ATCC no. TIB‐67)
  • Macrophage medium (see recipe), prewarmed
  • Liquid nitrogen tank for cell storage
  • Water bath set to 37°C
  • 15‐ml and 50‐ml sterile conical tubes
  • Centrifuge with swinging‐bucket rotor and adaptors for 15‐ml and 50‐ml conical tubes
  • 150 mm × 15 mm petri dishes
  • Upright microscope with a 10× objective
  • Additional reagents and equipment for counting cells with a hemacytometer (e.g., unit 1.13)
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Figures

Videos

Literature Cited

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