Generation of Xeno‐Free, cGMP‐Compliant Patient‐Specific iPSCs from Skin Biopsy

Luke A. Wiley1, Kristin R. Anfinson1, Cathryn M. Cranston1, Emily E. Kaalberg1, Malia M. Collins1, Robert F. Mullins1, Edwin M. Stone1, Budd A. Tucker1

1 Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, Iowa
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 4A.12
DOI:  10.1002/cpsc.30
Online Posting Date:  August, 2017
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Abstract

This unit describes protocols for the generation of clinical‐grade patient‐specific induced pluripotent stem cell (iPSC)–derived retinal cells from patients with inherited retinal degenerative blindness. Specifically, we describe how, using xeno‐free reagents in an ISO class 5 environment, one can isolate and culture dermal fibroblasts, generate iPSCs, and derive autologous retinal cells via 3‐D differentiation. The universal methods described herein for the isolation of dermal fibroblasts and generation of iPSCs can be employed regardless of disease, tissue, or cell type of interest. © 2017 by John Wiley & Sons, Inc.

Keywords: fibroblasts; retina; induced pluripotent stem cells; xeno‐free; current Good Manufacturing Practice; cGMP; photoreceptor precursor cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation and Processing of Patient Skin Biopsy: Isolation, Culture, and Freezing of Patient‐Specific Fibroblasts
  • Basic Protocol 2: Generation of Patient‐Specific Induced Pluripotent Stem Cells: Reprogramming, Maintenance, and Freezing of iPSCs
  • Basic Protocol 3: Patient‐Specific Retinal Cell Generation Via 3‐Dimensional Differentiation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation and Processing of Patient Skin Biopsy: Isolation, Culture, and Freezing of Patient‐Specific Fibroblasts

  Materials
  • Human subject
  • Lidocaine HCl/epinephrine mix (Hospira Inc., cat. no. 0409‐3182‐01)
  • Biopsy collection medium: combine 499 ml MEM‐α (Gibco/Thermo Fisher Scientific, cat. no. 12571‐063) with 1 ml Primocin (InvivoGen, cat. no. ant‐pm‐2)
  • Skin biopsy rinsing solution: combine 500 ml 1× HBSS no calcium, no magnesium (Gibco/Thermo Fisher Scientific, cat no. 14170‐112) with 1 ml Primocin (InvivoGen, cat. no. ant‐pm‐2)
  • CTS TrypLE Select Enzyme (Gibco/Thermo Fisher Scientific, cat. no. A1285901)
  • Fibroblast freezing medium: heat‐inactivated human serum (Innovative Research, cat. no. IPLA‐SERAB‐HI) containing 20% (v/v) DMSO (Sigma‐Aldrich, cat. no. D2438‐5x10ML)
  • University of Iowa xeno‐free biopsy medium (UIxMedium) with 10% heat‐inactivated human serum (see recipe)
  • Liquid nitrogen
  • Covidien Webcol 70% ethanol swabs (Medline Industries, cat. no. KDL5033)
  • Sterile hypodermic needle (Becton Dickinson, cat. no. 305106)
  • Sterile 1‐ml syringe (Becton Dickinson, cat. no. 309628)
  • Sterile 3‐mm disposable skin punch (Integra Miltex, cat. no. 33‐32)
  • Sterile forceps (Stephens Instruments, cat. no. S5‐1570)
  • Sterile scalpel (Bard‐Parker, cat. no. 371610)
  • Covidien Dermacea sterile 2 × 2 gauze (Medline Industries, cat. no. 441205)
  • Tegaderm (3M Healthcare, cat. no. 1624W)
  • 2‐ml cell cryovials (Simport, cat. no. T311‐2)
  • Clean room marker (Mirconova, cat. no. PEN 40IR)
  • Biohazard bags
  • 100‐mm tissue culture–treated sterile petri dishes (Techno Plastic Products, cat. no. 93040)
  • Sterile forceps (Fine Science Tools; Foster City, cat. no. 11251‐10)
  • 15‐ml and 50‐ml polypropylene screw cap conical tubes (CellTreat Scientific Products, cat. no. 229411, 229418, 229421, and 229428)
  • Costar 6‐well tissue culture‐treated sterile plates (Corning Life Sciences, cat. no. 3516)
  • BioSpherix Xvivo closed incubator/cell culture system (BioSpherix, Ltd.)
  • EVOS digital microscope (Thermo Fisher Scientific, cat. no. AME 3300)
  • Sorvall ST 8 tabletop centrifuge (Thermo Fisher Scientific, cat. no. 75007205)
  • Scepter 2.0 Cell Counter and 60 μM sensors (EMD Millipore, cat. no. PHCC20060 and PHCC60050)
  • CoolCell LX cryo‐freeze container (BioCision, cat. no. BCS‐405)
  • Liquid nitrogen container and associated hardware

Basic Protocol 2: Generation of Patient‐Specific Induced Pluripotent Stem Cells: Reprogramming, Maintenance, and Freezing of iPSCs

  Materials
  • Patient‐derived dermal fibroblasts ( protocol 1)
  • University of Iowa xeno‐free biopsy medium (UIxMedium) with 10% human serum (see recipe)
  • University of Iowa xeno‐free biopsy medium (UIxMedium), serum‐free (see recipe)
  • Viral transduction medium (see recipe)
  • 10 µg/ml recombinant human laminin‐521 (Corning Life Sciences, ca. no. 354222)
  • CTS TrypLE Select Enzyme (Gibco/Thermo Fisher Scientific, cat. no. A1285901)
  • Y‐27632 ROCK inhibitor (EMD Millipore, cat. no. 688000)
  • Essential 8 medium (Gibco/Thermo Fisher Scientific, cat. no. A1517001)
  • Recombinant human fibroblast growth factor 2 (rhFGF2, cGMP grade; Waisman Biomanufacturing, cat. no. rhFGF)
  • Versene (Gibco/Thermo Fisher Scientific, cat. no. 15040‐066)
  • Costar 6‐well tissue culture‐treated plates, sterile (Corning Life Sciences, cat. no. 3516)
  • BioSpherix Xvivo closed incubator/cell culture system (BioSpherix, Ltd.)
  • 100‐mm tissue culture‐treated sterile dishes (Techno Plastic Products, cat. no. 93040)
  • Costar 12‐well tissue culture‐treated sterile plates (Corning Life Sciences, cat. no. 3513)

Basic Protocol 3: Patient‐Specific Retinal Cell Generation Via 3‐Dimensional Differentiation

  Materials
  • Patient‐specific iPSCs ( protocol 2)
  • CTS TrypLE Select Enzyme (Gibco/Thermo Fisher Scientific, cat. no. A1285901)
  • 3‐D differentiation medium (see recipe)
  • Y‐27632 ROCK inhibitor (EMD Millipore, cat. no. 688000)
  • IWR1e (Cayman Chemical, cat. no. 13659)
  • ECM mixture:
    • human type 1 collagen (Advanced BioMatrix, cat. no. 5007‐20ML)
    • human type 3 collagen (Advanced BioMatrix, cat. no. 5021‐10MG)
    • human vitronectin (Advanced BioMatrix, cat. no. 5051‐0.1MG)
    • human fibronectin (Advanced BioMatrix, cat. no. 5050‐1MG)
  • StemMACS CHIR99021 (Miltenyi Biotec Inc., cat. no. 130‐103‐926)
  • SAG (Enzo Life Sciences, cat. no. ALX‐270‐426)
  • Neural retina (NR) culture medium (see recipe)
  • DAPT (EMD Millipore, cat. no. 565770)
  • BioSpherix Xvivo closed incubator/cell culture system (BioSpherix, Ltd.)
  • 50‐ml polypropylene screw cap conical tubes (CellTreat Scientific Products, cat. no. 229421 or 229428)
  • EVOS digital microscope (Thermo Fisher Scientific, cat. no. AME 3300)
  • Sorvall ST 8 Tabletop Centrifuge (Thermo Fisher Scientific, cat. no. 75007205)
  • Scepter 2.0 Cell Counter and 40 μM sensors (EMD Millipore, cat. nos. PHCC20040 and PHCC40050)
  • Corning spheroid 96‐well microplate, ultra‐low attachment (Corning Life Sciences, cat. no. 4520 or CLS7007)
  • ClipTip 30 to 300 µl multichannel pipettor (Thermo Fisher Scientific, cat. no. 4661140)
  • 100‐mm ultra‐low attachment culture dishes (Corning Life Sciences, cat. no. 3262)
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Figures

Videos

Literature Cited

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