Human Umbilical Cord Mesenchymal Stromal Cell Isolation, Expansion, Cryopreservation, and Characterization

J. Robert Smith1, Adrienne Cromer1, Mark L. Weiss1

1 Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.18
DOI:  10.1002/cpsc.24
Online Posting Date:  May, 2017
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Abstract

Revised methods to derive, expand, and characterize mesenchymal stromal cells (MSCs) from the umbilical cord are provided. Several considerations are taken for GMP compliance including using a closed system isolation method and eliminating several xenogenic components. With this method cells are isolated using mechanical and enzymatic digestion and then expanded with high viabilities that retain >90% viability after cryopreservation. Lastly, characterization methods have been optimized to identify these cells as MSCs according to the ISCT minimal criteria. This method standardizes the process for isolating, expanding, cryopreserving, and characterizing MSCs from the umbilical cord. © 2017 by John Wiley & Sons, Inc.

Keywords: mesenchymal stromal cells (MSCs); umbilical cord (UC); stem cells; isolation; cryopreservation; culture; characterization; platelet lysate

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Isolation of Cells From Umbilical Cord
  • Basic Protocol 2: Culturing and Passaging UC‐MSCS
  • Basic Protocol 3: Cryopreservation and Thawing of UC‐MSCs
  • Basic Protocol 4: Flow Cytometry
  • Basic Protocol 5: Osteogenic, Adipogenic, and Chondrogenic Differentiation of UC‐MSCs
  • Basic Protocol 6: Staining for MSC Differentiation
  • Basic Protocol 7: Colony Forming Units—Fibroblast (CFU‐F)
  • Reagents And Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Cells From Umbilical Cord

  Materials
  • Human umbilical cord
  • Enzyme solution (see recipe)
  • DPBS‐AA (see recipe)
  • Providone‐Iodine solution (see recipe)
  • Human platelet lysate‐enriched medium (HPLM; see recipe)
  • Red blood cell lysing buffer (Hybri‐Max; Sigma, cat. no. R7757)
  • ViaStain live dead cell stain (Nexcelom, cat. no. C52‐0106‐5ML)
  • Personal protective equipment (PPE) including:
    • Cuffed laboratory coat
    • Closed‐toe shoes
    • Nitrile gloves
    • Safety glasses
    • Surgical mask
    • Splash shield
  • Class II biological safety cabinet
  • Pre‐enzyme materials:
    • C‐Tubes (Miltenyi Biotec, cat. no. 130‐096‐534)
    • 250‐ml sterile specimen cups (Fisher, cat. no. 02‐540‐10)
    • Sterile forceps; 2, 8‐cm straight, blunt end, serrated
    • Sterile no. 3 scalpel handle
    • Sterile no. 10 scalpel blade
    • Sterile 150 × 15–mm petri dishes
    • Sterile 100 × 15–mm petri dishes
    • Plastic ruler
  • 37°C water bath
  • GentleMACS Dissociator (Miltenyi Biotec, cat. no. 130‐093‐235)
  • MACSmix Tube Rotator (Miltenyi Biotec, cat. no. 130‐090‐753)
  • Post‐enzyme materials:
    • 100‐µm sterile cell strainer (Fisher, cat. no. 22‐363‐549)
    • 50‐ml, 60‐µm Steriflip filters (Fisher, cat. no. SCNY00060)
    • 50‐ml sterile centrifuge tubes
    • 1.5‐ml microcentrifuge tubes
    • Counting chambers (Nexcelom, cat. no. CHT4‐SD100‐014)
    • Tissue culture treated plates (see materials list for protocol 2)
  • Centrifuge
  • Biohazardous waste container
  • Waste flask (Erlenmeyer flask containing 10% household bleach)
  • Sharps container
  • Auto2000 Cellometer (Nexcelom Bioscience)
  • 37°C, 5% CO 2 incubator (90% humidity)
NOTE: Procedures are performed inside a class II biosafety cabinet (BSC). Materials are wiped down with 70% ethanol prior to placing them in the BSC. Sterile packs are opened within the BSC. Universal precautions are used to prevent possible transmission of human blood borne pathogens.

Basic Protocol 2: Culturing and Passaging UC‐MSCS

  Materials
  • UC‐MSCs at 70% to 90% confluence (see protocol 1 for obtaining cells)
  • Dulbecco's phosphate‐buffered saline (without Ca or Mg, DPBS; Fisher, cat. no. 14190)
  • 0.25% Trypsin‐EDTA (Fisher, cat. no. 25200)
  • Human platelet lysate‐enriched medium (HPLM; see recipe)
  • ViaStain live dead cell stain (Nexcelom, cat. no. C52‐0106‐5ML)
  • Tissue culture treated plates
    • 12‐well plate (CytoOne, USA Scientific, cat. no. CC7682‐7512)
    • 6‐well plate (CytoOne, USA Scientific, cat. no. CC7682‐7506)
    • 25‐cm2 flask (Corning, Fisher, cat. no. 10‐126‐28)
    • 75‐cm2 flask (Corning, Fisher, cat. no. 10‐126‐37)
    • 225‐cm2 flask (Corning, Fisher, cat. no. 10‐126‐63)
  • 37°C, 5% CO 2 incubator
  • Phase‐contrast inverted microscope
  • 50‐ml sterile centrifuge tubes
  • 15‐ml sterile centrifuge tubes
  • Cell counting equipment and supplies (see protocol 1)

Basic Protocol 3: Cryopreservation and Thawing of UC‐MSCs

  Materials
  • UC‐MSCs after passage (see protocol 2)
  • hES freeze medium (Globalstem, cat. no. GSM‐4200)
  • Ice
  • Human platelet lysate‐enriched medium (HPLM; see recipe)
  • Mr. Frosty freezing container (Fisher, cat. no. 15‐350‐50)
  • Cryogenic vials (2 ml; Fisher, cat. no. 03‐337‐7D)
  • −80°C freezer
  • Liquid nitrogen storage tank
  • 37°C water bath
  • 15‐ml sterile centrifuge tubes
  • Centrifuge
  • Cell counting equipment and supplies (see protocol 1)
NOTE: Prior to freezing, MSCs should be in the log growth phase. The final density of MSCs should not exceed 5 million live cells per ml and final volume should not exceed 1.4 ml for a 2‐ml cryovial.

Basic Protocol 4: Flow Cytometry

  Materials
  • UC‐MSCs grown in 75‐cm2 flasks ( protocol 2)
  • Dulbecco's phosphate‐buffered saline (DPBS; Fisher, cat. no. 14190)
  • Accutase (Fisher, cat. no. NC9464543)
  • Human platelet lysate‐enriched medium (HPLM; see recipe)
  • Flow cytometry buffer (FACS buffer; see recipe)
  • Ice
  • Antibodies (see Table 1.18.3)
  • 4% Paraformaldehyde (see recipe)
  • Human MSC analysis kit (BD Biosciences, cat. no. 562245)
  • 30‐µm cell strainer (Miltenyi, cat. no. 130‐098‐458)
  • 50‐ and 15‐ml sterile centrifuge tubes
  • Centrifuge
  • Cell counting equipment and supplies (see protocol 1)
  • 4‐color flow cytometer (BD Biosciences)
  • 12 × 75–mm clear polystyrene tubes (Evergreen, cat. no. 222‐2036‐050)

Basic Protocol 5: Osteogenic, Adipogenic, and Chondrogenic Differentiation of UC‐MSCs

  Materials
  • UC‐MSCs ( protocol 2)
  • StemPro Adipogenesis differentiation kit (Fisher, cat. no. A1007001)
  • StemPro Osteogenesis differentiation kit (Fisher, cat. no. A1007201)
  • StemPro Chondrogenesis differentiation kit (Fisher, cat. no. A1007101)
  • Antibiotic‐antimycotic solution (Fisher, cat. no. 15240)
  • Human platelet lysate‐enriched medium (HPLM; see recipe)
  • 50‐ml, 0‐22 µm filter (Steriflip, Millipore, cat. no. SCGP00525)
  • 12‐well plate (CytoOne, USA Scientific, cat. no. CC7682‐7512)
  • 37°C, 5% CO 2 incubator
  • 1.5‐ml microcentrifuge tubes
  • Centrifuge
  • Additional reagents and equipment for counting live cells with ViaStain (see protocol 1) and for passaging and culturing cells (see protocol 2)

Basic Protocol 6: Staining for MSC Differentiation

  Materials
  • UC‐MSCs ready to be fixed from protocol 5
  • Dulbecco's phosphate‐buffered saline with calcium and magnesium (DPBS; Sigma, cat no. 21‐030‐CV)
  • 4% paraformaldehyde solution (see recipe)
  • Alizarin Red S solution (see recipe)
  • Distilled water
  • Oil Red O working solution (see recipe)
  • 0.1% Safranin O solution (aq.) (Sigma, cat. no. HT904‐8FOZ)
  • Parafilm

Basic Protocol 7: Colony Forming Units—Fibroblast (CFU‐F)

  Materials
  • UC‐MSCs after passage ( protocol 2)
  • Dulbecco's phosphate‐buffered saline (DPBS; Fisher, cat. no. 14190)
  • Methanol
  • 1% Methylene Blue solution (aq.), filtered (see recipe)
  • Distilled water
  • 6‐well plates
  • Phase‐contrast illumination, inverted microscope Fisher
  • EVOS microscope (Life Technologies/Fisher)
  • Additional reagents and equipment for counting live cells using ViaStain (see protocol 1, steps 26 to 28)
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Figures

Videos

Literature Cited

Literature Cited
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