Expansion of Human Embryonic Stem Cells on Cellulose Microcarriers

Allen K. Chen1, Xiaoli Chen1, Andre B.H. Choo1, Shaul Reuveny2, Steve K.W. Oh1

1 Stem Cell Group, Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), Centros, Singapore, 2 Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.11
DOI:  10.1002/9780470151808.sc01c11s14
Online Posting Date:  September, 2010
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Abstract

This unit describes the routine maintenance and expansion of undifferentiated human embryonic stem cells (hESC) on cellulose microcarriers. Conventionally, hESCs have been maintained on feeder cells or extracellular matrix–coated two‐dimensional tissue culture plates. The expansion of hESC on a tissue culture platform is limited by the available surface area and the requirement of repetitive subculturing to reach the required cell yield. Here, we show that expansion of hESC can be carried out in a three‐dimensional suspension culture using Matrigel‐coated cellulose microcarriers. hESCs from a tissue culture plate can be seeded directly onto the microcarriers; hESC microcarrier culture is passaged and expanded by mechanical dissociation of the cells without enzyme. Expansion of the culture in a 100‐ml spinner flask is also described. Long‐term culture of hESC on the microcarriers maintains typical pluripotent markers (OCT‐4, Tra‐1‐60, and SSEA‐4) and stable karyotype. Spontaneous differentiations of microcarrier‐maintained hESCs in vitro (embryoid body formation) and in vivo (teratoma formation in SCID mouse) have demonstrated formation of the three germ layers. These protocols can also be applied equally well to human induced pluripotent stem cells. Curr. Protoc. Stem Cell Biol. 14:1C.11.1‐1C.11.14. © 2010 by John Wiley & Sons, Inc.

Keywords: human embryonic; stem cells; hESCs; microcarriers; hESC expansion; induced pluripotent stem cells; iPSC

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Seeding hESCs from Two‐Dimensional Culture onto Matrigel‐Coated Cellulose Microcarriers
  • Support Protocol 1: Preparation of Cellulose Microcarriers
  • Support Protocol 2: Coating Microcarriers with Matrigel
  • Basic Protocol 2: Passaging hESCs from Microcarriers to Microcarriers
  • Basic Protocol 3: Expansion of hESC Using a Spinner Flask
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Seeding hESCs from Two‐Dimensional Culture onto Matrigel‐Coated Cellulose Microcarriers

  Materials
  • hESC (HES‐3 and HES‐2; ES International), expanded colonies cultured on Matrigel‐coated dish
  • Conditioned hESC growth medium (see recipe)
  • Matrigel‐coated cellulose microcarriers ( protocol 3)
  • Stereomicroscope
  • 1‐ml and 200‐µl pipettor and sterile‐filtered tips
  • StemPro EZPassage Disposable Stem Cell Passaging Tool (Invitrogen, cat. no. 23181‐010)
  • Cell scraper, 24 cm (TPP, cat. no. 99002)
  • NucleoCounter (ChemoMetec A/S, NucleoCounter SCC‐100)
  • Orbital shaker (e.g., IKA Mixing Oribital Shaker KS260 Control, cat. no. 2980300)
  • 37°C humidified incubator with 5% CO 2

Support Protocol 1: Preparation of Cellulose Microcarriers

  Materials
  • Cellulose carriers (DE52, DE53, or QA52; Whatman, cat no. WH/CH/CO/4057‐050, WH/CH/CO/4058‐050 or WH/CS/OO/4065‐050)
  • 4 M HCl
  • Dulbecco's phosphate‐buffered saline without calcium chloride and magnesium chloride (DPBS; Invitrogen, cat. no. 14190‐144)
  • Weighing balance
  • 500‐ml glass bottles coated with Sigmacote (Sigma‐Aldrich, cat. no. SL2)
  • 1‐ml pipettor and tips
  • pH meter
  • 50‐ml serological pipet
  • Steam sterilizer

Support Protocol 2: Coating Microcarriers with Matrigel

  Materials
  • Sterile cellulose microcarrier suspension (50 mg/ml) in Dulbecco's phosphate‐buffered saline solution (see protocol 2)
  • Unconditioned hESC medium (see recipe)
  • BD Matrigel Basement Membrane Matrix (Beckon Dickson, cat. no. 354234)
  • Conditioned hESC growth medium (see recipe), cold
  • 1.5‐ml and 50‐ml centrifuge tubes, sterile
  • 5‐ml and 25‐ml serological pipets, sterile
  • 1‐ml sterile filtered tips, pre‐cooled
  • Laboratory platform rockers
  • Ultra‐low adherent 6‐well plate (Corning, cat. no. 3471)
  • 37°C humidified incubator with 5% CO 2

Basic Protocol 2: Passaging hESCs from Microcarriers to Microcarriers

  Materials
  • hESC on Matrigel‐coated microcarriers in one well of an ultra‐low adherent 6‐well plate (see protocol 3)
  • Conditioned hESC growth medium (see recipe)
  • 1‐ml and 200‐µl pipettors with sterile‐filtered tips
  • Sterile 5‐ml serological pipet
  • NucleoCounter (ChemoMetec A/S, NucleoCounter SCC‐100)
  • Ultra‐low adherent 6‐well plate (Corning, cat. no. 3471)
  • BD Matrigel Basement Membrane Matrix (Beckon Dickson, cat. no. 354234)
  • Sterile cellulose microcarrier suspension (50 mg/ml) in phosphate‐buffered saline solution ( protocol 2)
  • Orbital shaker (e.g., IKA Mixing Oribital Shaker KS260 Control, cat. no. 2980300)
  • 37°C humidified incubator with 5% CO 2

Basic Protocol 3: Expansion of hESC Using a Spinner Flask

  Materials
  • hESC (HES‐3 and HES‐2, ES International), confluent and adapted (more than 4 passages) to microcarrier culture ( protocol 1)
  • Conditioned hESC growth medium (see recipe)
  • Ultra‐low‐adherent 6‐well plate (Corning, cat. no. 3471)
  • 100‐ml spinner flasks (Bellco, cat. no. 1965‐00100) coated with Sigmacote (Sigma‐Aldrich, cat. no. SL2)
  • Autoclave
  • 37°C humidified incubator with 5% CO 2
  • Orbital shaker (e.g., IKA Mixing Oribital Shaker KS260 Control, cat. no. 2980300)
  • 1‐ml and 200‐µl pipettors with sterile‐filtered tips
  • NucleoCounter (ChemoMetec A/S, NucleoCounter SCC‐100)
  • Stereomicroscope
  • Magnetic stirrer (e.g., Thermolyne Cellgro Stirrer, Thermal Scientific Barnstead, cat. no. S45600)
  • Sterile 5‐ml serological pipets
  • Sterile 15‐ and 50‐ml centrifuge tubes
  • Additional reagents and equipment for preparing microcarriers coated with Matrigel ( protocol 3)
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Figures

Videos

Literature Cited

Literature Cited
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