Using Organotypic (Raft) Epithelial Tissue Cultures for the Biosynthesis and Isolation of Infectious Human Papillomaviruses

Michelle A. Ozbun1, Nicole A. Patterson1

1 Department of Molecular Genetics and Microbiology, The University of New Mexico School of Medicine, The UNM Cancer Center, Albuquerque, New Mexico
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14B.3
DOI:  10.1002/9780471729259.mc14b03s34
Online Posting Date:  August, 2014
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Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro–wherein virion morphogenesis occurs under cooperative viral and cellular cues–requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte‐dermal equivalent is placed at the air‐liquid interface. The apparent floating nature of the cell‐matrix in this method led to the nickname “raft” cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single‐step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection. Curr. Protoc. Microbiol. 34:14B.3.1‐14B.3.18. © 2014 by John Wiley & Sons, Inc.

Keywords: papillomavirus; organotypic epithelial tissue culture; air‐liquid interface; keratinocyte; virion isolation

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation and Growth of Organotypic Epithelial Tissue Cultures
  • Basic Protocol 2: HPV Virion Isolation from Epithelial Tissues
  • Support Protocol 1: Epithelial Medium (E Medium) Preparation
  • Support Protocol 2: Preparing the Stainless Steel Screen Support Grids
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation and Growth of Organotypic Epithelial Tissue Cultures

  • Human or mouse fibroblast “feeder” cells (e.g., J2 3T3 fibroblasts at early passage); estimate about 3 to 6 rafts per confluent 100 mm dish of fibroblasts, depending upon the confluence
  • Keratinocytes that maintain extrachromosomal HPV genomes (e.g., CIN 612‐9E cells; Meyers et al., ; see unit ); estimate 2 to 6 rafts per nearly confluent 100 mm dish of cells, depending upon cell line
  • Rat Tail Type 1 collagen, 3.8 to 4.0 mg/ml; store at 4°C (BD Biosciences, cat. no. 354236)
  • Reconstitution buffer: Dissolve 11.0 g NaHCO 3 and 23.85 g HEPES in 100 ml 0.162 N NaOH. Bring final volume to 500 ml with 0.162 N NaOH, filter sterilize, and store in aliquots at −20°C.
  • Dulbecco's Modified Eagle's Media (DMEM) powder, high glucose, no pyruvate, no bicarbonate, for 10 liters (Life Technologies‐Gibco®, cat. no. 12100‐061): fully dissolve an amount suitable for 1 liter in 100 ml warm ultrapure water. Filter sterilize and store in 20 to 50 ml aliquots at −20°C.
  • 10 N NaOH
  • E medium containing 5% heat‐inactivated fetal bovine serum (FBS) and 10,000 U/ml nystatin suspension (see protocol 3)
  • 10 μg/ml EGF (BD Biosciences, cat. no. 354001), dissolved in sterile ultrapure water
  • 20 mM 1,2‐dioctanoyl‐sn‐glycerol (C8:0; Calbiochem, cat. no. 317505), dissolved in 100% ethanol
  • 70% ethanol (for dipping forceps and spatulas if needed)
  • Flat‐bottom 6‐well plates with lids, sterile
  • 50‐ml graduated conical polypropylene centrifuge tubes with screw‐cap lids, sterile
  • 5‐ and 10‐ml graduated plastic pipets, sterile and individually wrapped
  • 100‐mm tissue culture plates
  • Stainless steel screen grids 3.063 in. diameter, 40 mesh, 0.01 wire (see protocol 4)
  • 2‐liter Pyrex® beakers
  • 9‐inch forceps, individually wrapped, sterile
  • 9‐inch stainless steel spatula/spoon tool (e.g., Fisher, cat. no. S50789A), individually wrapped, sterile
  • Bunsen burner in cell culture cabinet to flame 70% ethanol‐treated forceps and spatulas, if needed

Basic Protocol 2: HPV Virion Isolation from Epithelial Tissues

  • Epithelial raft tissues (from protocol 1)
  • 5 M NaCl, filter sterile
  • 1 M NaCl, filter sterile
  • 150 mM NaCl, filter sterile
  • 0.5 M EDTA, pH 8.0 (see )
  • 0.1 M Sodium phosphate buffer, pH 8.0 (see )
  • 0.1 M Sodium phosphate buffer pH 7.4 (see )
  • Virus extraction buffer 1, ice cold (1 M NaCl/50 mM Na‐phosphate buffer, pH 8.0): 100 ml of 5 M NaCl; 250 ml of 0.1 M Na‐phosphate buffer, pH 8; 150 ml of dH 2O; filter sterile
  • Virus extraction buffer 2, ice cold (50 mM NaCl/100 mM EDTA/50 mM Na‐phosphate buffer, pH 7.4): 25 ml of 1 M NaCl; 100 ml of 0.5 M EDTA; 250 ml of 0.1 M Na‐phosphate buffer, pH 7.4; 125 ml of dH 2O; filter sterile
  • Vesphene 1:128 dilution
  • 70% ethanol
  • 10% chlorine bleach
  • Absorbent bench paper
  • Autoclavable containers with lids (e.g., Nalgene 6910‐0618 Polypropylene Autoclavable Instrument Tray): one for consumables and waste, one for permanent lab items
  • N100 HEPA respirator, 3M model 8233 or equivalent
  • BeadBeater homogenizer (BioSpec Model 1107900,
  • Glass Beads, 1.0 mm (BioSpec, cat. no. 11079110), autoclaved in a beaker
  • BeadBeater chambers:
    • 350 ml polycarbonate chamber for secondary containment (BioSpec, cat. no. 110792)
    • 50 ml chamber for ∼10 to 75 raft tissues (BioSpec, cat. no. 110803)
    • 15 ml chamber for 1 to 5 raft tissues (BioSpec, cat. no. 110803)
  • Stainless‐steel 15 ml (1 tablespoon) measuring spoon, autoclaved
  • 38‐ml Nalgene™ Oak Ridge high‐speed centrifuge tubes, screw cap, sterile
  • High‐speed centrifuge
  • Ultracentrifuge tubes (Beckman, cat. no. 344058); sterilize 10 min in 70% ethanol, rinse 3 times with sterile ultrapure water
  • SW27 (swing‐bucket) ultracentrifuge rotor
  • Ultracentrifuge
  • 2‐ml glass dounce homogenizers
CAUTION: Parts of this extraction and isolation protocol could generate aerosols or spills. Thus, procedures are carried out in a cell‐culture hood or sealed containers to the extent possible. We recommend the additional precautions of HPV vaccination and PPE (disposable Tyvek lab jackets, gloves, protective eyewear, and a HEPA mask). Spills that are potentially contaminated with virus should be decontaminated immediately by treating with either 1:128 Vesphene® solution or 70% ethanol for 30 min before rinsing with water.

Support Protocol 1: Epithelial Medium (E Medium) Preparation

  • Ultrapure water (e.g., Milli‐Q system from Millipore)
  • Ultrapure water, autoclaved
  • Dulbecco's Modified Eagle's Media (DMEM) powder, high glucose, no pyruvate, no bicarbonate, for 10 liters (Life Technologies‐Gibco®, cat. no. 12100‐061)
  • Hams F‐12 nutrient mix powder, 10 × 1 liter (Life Technologies‐Gibco®, cat. no. 21700‐075)
  • HEPES Ultra Pure (Life Technologies‐Gibco®, cat. no. 11344‐041)
  • 10,000 U/ml Pen‐Strep (Life Technologies‐Gibco®, cat. no. 15140‐022)
  • Adenine, suitable for cell culture (Sigma‐Aldrich, cat. no. A‐2786)
  • Insulin, suitable for cell culture (Sigma‐Aldrich, cat. no. I‐6634)
  • Apo‐transferrin, suitable for cell culture (Sigma‐Aldrich, cat. no. T‐1147)
  • 3,3′,5‐Triiodo‐l‐thyronine (T3), suitable for cell culture (Sigma‐Aldrich, cat. no. T‐6397)
  • Hydrocortisone, for cell culture (Sigma‐Aldrich, cat. no. H‐0888)
  • Cholera toxin (Sigma‐Aldrich, #C‐8052)
  • NaHCO 3, cell culture grade (TC grade; Gibco, cat. no. 118110‐025)
  • 12.1 N (concentrated), 10 N, and 0.1 N HCl
  • 10 N and 0.02 N NaOH
  • 10,000 U/ml nystatin suspension (Sigma‐Aldrich, cat. no. N‐1638)
  • Sterile PBS ( )
  • 20‐liter carboy
  • Graduated beakers
  • Graduated cylinders
  • Stericap PLUS 0.22 μm, sterile bottle‐top filter units (Millipore, cat. no. SCGPCAPRE)
  • Pyrex 1‐liter bottles

Support Protocol 2: Preparing the Stainless Steel Screen Support Grids

  • Stainless steel screen disks 3.063 in. diameter, 40 mesh, 0.01 in. diameter wire (TWP Inc.;
  • Oven‐bake clay kit (e.g., Sculpey® Keepsake® ornament kit with 4 in. diameter casting ring, available at hobby stores)
  • Chromic acid cleaning solution: dissolve 400 g potassium dichromate in 4000 ml distilled water; transfer to 4‐liter brown glass bottle. In a fume hood, slowly add 400 ml conc. sulfuric acid.
  • 5‐ml test tube (12 × 75 mm)
  • 2‐liter Pyrex® beakers
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Literature Cited

Literature Cited
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