Controlling the Effective Oxygen Tension Experienced by Cells Using a Dynamic Culture Technique for Hematopoietic Ex Vivo Expansion

Abhilasha Tiwari1, Cynthia S. Wong2, Lakshmi P. Nekkanti1, James A. Deane1, Courtney McDonald1, Jingang Li1, Yen Pham1, Amy E. Sutherland1, Graham Jenkin3, Mark A. Kirkland2

1 The Ritchie Centre, Hudson Institute of Medical Research, Victoria, Clayton, 2 Institute for Frontier Materials, Deakin University, Geelong, 3 The Ritchie Centre, Hudson Institute of Medical Research, Department of Obstetrics & Gynaecology, Southern Clinical School, Monash University, Victoria, Clayton
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2A.11
DOI:  10.1002/cpsc.42
Online Posting Date:  February, 2018
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Clinical hematopoietic stem/progenitor cell (HSPC) transplantation outcomes are strongly correlated with the number of cells infused. Hence, to generate sufficient HSPCs for transplantation, the best culture parameters for expansion are critical. It is generally assumed that the defined oxygen (O2) set for the incubator reflects the pericellular O2 to which cells are being exposed. Studies have shown that low O2 tension maintains an undifferentiated state, but the expansion rate may be constrained because of limited diffusion in a static culture system. A combination of low ambient O2 and dynamic culture conditions has been developed to increase the reconstituting capacity of human HSPCs. In this unit, the protocols for serum‐free expansion of HSPCs at 5% and 20% O2 in static and dynamic nutrient flow mode are described. Finally, the impact of O2 tension on HSPC expansion in vitro by flow cytometry and colony forming assays and in vivo through engraftment using a murine model is assessed. © 2018 by John Wiley & Sons, Inc.

Keywords: bone marrow repopulation assay; dynamic culture system; hematopoietic stem/progenitor cell expansion; pericellular oxygen; umbilical cord blood

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Table of Contents

  • Introduction
  • Basic Protocol 1: Expansion and in vitro Characterization of Umbilical Cord Blood–Derived CD34+ Hematopoietic Stem/Progenitor Cells
  • Basic Protocol 2: Measurement of Dissolved pO2
  • Basic Protocol 3: In Vivo Bone Marrow Repopulation Assay in Mice
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Expansion and in vitro Characterization of Umbilical Cord Blood–Derived CD34+ Hematopoietic Stem/Progenitor Cells

  • RetroNectin (RN) (Clonetech, cat. no. T100B)
  • D‐PBS without Ca2+ and Mg2+ (Invitrogen, cat. no. 14190‐250)
  • HSPC expansion medium (see recipe)
  • Methocult (Stem Cell Technologies, cat. no. H4434)
  • Umbilical cord blood CD34+ cells, frozen
  • Stemspan ACF (Stem Cell Technologies, cat. no. 9855), prewarmed
  • Staining cocktails (see Table 2.11.1):
  • FACS buffer
  • Human CD34‐FITC (BD Biosciences, cat. no. 348053)
  • Human CD34‐PE (BD Biosciences, cat. no. 348057)
  • Human CD38‐FITC (BD Biosciences, cat. no. 555459)
  • Human CD45‐FITC (BD Biosciences, cat. no. 347463)
  • Human CD45‐PE (BD Biosciences, cat. no. 555483)
  • Human CD133‐PE (Miltenyi Biotech, cat. no. 130‐090‐853)
  • Human CD33‐FITC (BD Biosciences, cat. no. 555626)
  • Human IgG secondary antibody‐H&L, PE‐conjugated (Abcam, cat. no. ab7006)
  • Human IgG secondary antibody‐H&L, FITC‐conjugated (Abcam, cat. no. ab6854)
  • 7AAD (BD Biosciences, cat. no. 559925)
  • Staining solution (see recipe)
  • Fixing solution (see recipe)
  • Actinomycin D (Sigma Aldrich, cat. no. A1410‐10 mg) (see recipe)
  • Trypan blue (Sigma Aldrich, cat. no. T8154)
  • 24‐well plates (non‐treated) (Costar‐Corning, cat. no. 3738)
  • 37°C water bath
  • Centrifuge
  • 37°C, 5% CO 2 humidified incubator (HERAcell 150i Tri‐gas incubator, Thermo Fisher, cat. no. 51026280)
  • Digital orbital shaker (OM6, Ratek)
  • Vortexer
  • 12‐well plates
  • 3‐ml syringes
  • 16‐G blunt‐end needles (Stem Cell Technologies, cat. no. 28110)
  • 15‐ and 50‐ml tubes (BD Falcon, cat. no. 352096 and cat. no. 352070, respectively)
  • 5‐ml round‐bottom FACS tubes (Grale Scientific, cat. no. P7512T)
  • FACS CantoII flow cytometer, FACSDiva software (BD Biosciences)
Table 2.1.1   MaterialsFlow Cytometry Staining for HSPC Expansion

Unstained Isotype Control Cocktail 1 Cocktail 2 Cocktail 3 Cocktail 4
Antibody Volume (µl) Antibody Volume (µl) Antibody Volume (µl) Antibody Volume (µl) Antibody Volume (µl) Antibody Volume (µl)
FACS buffer 25 IgG PE 1 CD34 PE 1 CD34 PE 1 CD133 PE 1 CD34 PE 1
Cells 25 IgG FITC 1 CD45 FITC 1 CD38 FITC 1 CD34 FITC 1 CD33 FITC 1
FACS buffer 23 7AAD 1 7AAD 1 7AAD 1 7AAD 1
Cells 25 FACS buffer 22 FACS buffer 22 FACS buffer 22 FACS buffer 22
Cells 25 Cells 25 Cells 25 Cells 25

Basic Protocol 2: Measurement of Dissolved pO2

  • pO 2 ‘Bare‐Fibre’ sensor (Oxford Optronix; NX/BF/O/E)
  • OxyLite Pro (Oxford Optronix)

Basic Protocol 3: In Vivo Bone Marrow Repopulation Assay in Mice

  • NSG (NOD.Cg‐Prkdcscid Il2rgtm1Wjl/SzJ) mice (8‐ to 10‐week‐old males and females)
  • 137Cesium source (Nordion)
  • Bactrim antibiotic: trimethoprim and sulfamethoxazole (Roche, cat. no. AUSTR119404)
  • CD34+ cells, post 7‐day expansion (see protocol 1) resuspended in saline
  • PBS
  • Spleen medium (see recipe)
  • 6% ammonium chloride solution (Sigma Aldrich, cat. no. A0171)
  • FACS buffer
  • Antibodies:
  • Mouse CD45‐FITC (BD Biosciences, cat. no. 553080)
  • Human CD45‐PE (BD Biosciences, cat. no. 555483)
  • Human CD33‐V450 (BD Biosciences, cat. no. 561157)
  • Human CD19‐APC Cy7 (BD Biosciences, cat. no. 557791)
  • FITC IgG1,k Isotype control (BD Pharmingen, cat. no. 551954)
  • PE IgG1,k Isotype control (BD Pharmingen, cat. no. 551436)
  • V450 IgG Isotype control (BD Biosciences, cat. no. 560373)
  • APC Cy‐7 IgG Isotype control (BD Biosciences, cat. no. 557873)
  • Micro‐isolators (animal housing)
  • Mouse restraint or 50‐ml tube with breathing hole
  • 40°C water bath
  • 0.5‐ and 1‐ml ultra‐fine syringes (29 G) (BD Ultra‐Fine™ insulin syringes, BD Biosciences, cat. no. 326105)
  • 26‐ and 29‐G needles (BD Biosciences)
  • Sterile disposable transfer pipets (BD Biosciences, cat. no. 357575)
  • Mice dissection kit (Able Scientific)
  • 70‐µm cell strainer (Fisher Scientific, cat. no. 087712)
  • Refrigerated centrifuge
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Literature Cited

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