Laser‐Assisted Generation of Human Induced Pluripotent Stem Cells

Yang Liu1, Dongmei Wu1, Donglin Lao1, Cory Peterson2, Kristi A. Hohenstein Elliott2, Evan Y. Snyder3

1 Stem Cell Research Center and Core Facility, Sanford‐Burnham Medical Research Institute, La Jolla, California, 2 Cell Engineering Unit, Intrexon Corporation, San Diego, California, 3 Department of Pediatrics, University of California at San Diego, La Jolla, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 4A.7
DOI:  10.1002/9780470151808.sc04a07s31
Online Posting Date:  November, 2014
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Abstract

This unit describes a procedure for generating human induced pluripotent stem cells (hiPSCs) using the Laser‐Enabled Analysis and Processing (LEAP®) system, which combines high‐throughput cell imaging with laser‐mediated cell manipulation. Use of this system should not only improve the quality and uniformity of hiPSCs produced, but ultimately enable a more rapid, efficient, high‐throughput, and automated production process. © 2014 by John Wiley & Sons, Inc.

Keywords: iPSC generation; laser‐assisted; high‐throughput; automated

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Fibroblast Reprogramming Using Episomal Vectors
  • Support Protocol 1: Preparation and Quality Control of MEF Conditioned Medium (MEF‐CM)
  • Basic Protocol 2: Human iPSC Colony Isolation, Purification, and Amplification using the LEAP® System
  • Support Protocol 2: LEAP® Calibration Protocol
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Fibroblast Reprogramming Using Episomal Vectors

  Materials
  • 0.1% (w/v) gelatin (Stemcell Technologies, cat. no. 07903)
  • Human skin fibroblast cells (e.g., unit 1.7)
  • FB medium (fibroblast culture medium with antibiotics; see recipe)
  • FB‐A medium (fibroblast culture medium without antibiotics; see recipe)
  • Phosphate‐buffered saline (PBS) (Corning Cellgro, cat. no. 21‐040‐CV)
  • 0.05% (w/v) trypsin/0.48 mM EDTA with phenol red
  • Episomal vector cocktail (see recipe)
  • Electroporation Buffer R (included in the Neon kit; see below)
  • CF1 mouse embryonic fibroblast (MEF) feeder cells (e.g., iGentBio, cat. no. CL0101)
  • KOSR stem cell medium (see recipe)
  • KOSR MEF conditioned medium ( protocol 2)
  • 10‐cm tissue culture dishes (e.g., Fisher Scientific, cat. no. 0877222)
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Hemacytometer
  • 2‐ml microcentrifuge tubes
  • Neon electroporation system (Life Technologies, cat. no. MPK5000)
  • 6‐well culture plates (e.g., Fisher Scientific, cat. no. 072080)
  • Inverted microscope
  • Stereomicroscope in a laminar flow hood

Support Protocol 1: Preparation and Quality Control of MEF Conditioned Medium (MEF‐CM)

  Materials
  • 0.1% (w/v) gelatin (Stemcell Technologies, cat. no. 07903)
  • Inactivated CF1 MEF feeders (1 × 107 cells; prepared in‐house or purchased from commercial sources, e.g., iGentBio, cat. no. CL0102)
  • FB medium (fibroblast culture medium with antibiotics; see recipe)
  • KOSR stem cell medium (see recipe)
  • Matrigel (BD Biosciences)
  • hESC/hiPSC cells (see appropriate units in this manual)
  • 150‐mm tissue culture dishes
  • 0.2‐μm membrane filters

Basic Protocol 2: Human iPSC Colony Isolation, Purification, and Amplification using the LEAP® System

  Materials
  • hiPSCs growing in culture ( protocol 1)
  • Phosphate‐buffer saline (PBS) without calcium and magnesium (Corning, cat. no. 21‐040‐CV)
  • Phototherm solution (Intrexon Corporation)
  • Bar code stickers (provided by Intrexon for identification of the plate type when the plate is loaded)
  • LEAP® instrument (Intrexon Corporation)

Support Protocol 2: LEAP® Calibration Protocol

  Materials
  • LEAP® instrument (Intrexon Corporation)
  • Calibration mirror (Intrexon Corporation)
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Figures

Videos

Literature Cited

Literature Cited
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