Functional Stem Cell Integration into Neural Networks Assessed by Organotypic Slice Cultures

David Forsberg1, Charoensri Thonabulsombat2, Johan Jäderstad1, Linda Maria Jäderstad1, Petri Olivius3, Eric Herlenius1

1 Department of Women's and Children's Health, Karolinska Institutet, and Karolinska University Hospital, Stockholm, 2 Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, 3 Center for Hearing and Communication Research, Karolinska Institutet, and Karolinska University Hospital, Stockholm
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.13
DOI:  10.1002/cpsc.34
Online Posting Date:  August, 2017
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Re‐formation or preservation of functional, electrically active neural networks has been proffered as one of the goals of stem cell–mediated neural therapeutics. A primary issue for a cell therapy approach is the formation of functional contacts between the implanted cells and the host tissue. Therefore, it is of fundamental interest to establish protocols that allow us to delineate a detailed time course of grafted stem cell survival, migration, differentiation, integration, and functional interaction with the host. One option for in vitro studies is to examine the integration of exogenous stem cells into an existing active neural network in ex vivo organotypic cultures. Organotypic cultures leave the structural integrity essentially intact while still allowing the microenvironment to be carefully controlled. This allows detailed studies over time of cellular responses and cell‐cell interactions, which are not readily performed in vivo. This unit describes procedures for using organotypic slice cultures as ex vivo model systems for studying neural stem cell and embryonic stem cell engraftment and communication with CNS host tissue. © 2017 by John Wiley & Sons, Inc.

Keywords: brainstem; engraftment; integration; interaction; neuroprotection; neural stem cells; organotypic culture; roller drum; striatum; Stoppini; transplantation

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Table of Contents

  • Introduction
  • Basic Protocol 1: Organotypic Cultures—Interface Method—Brainstem
  • Basic Protocol 2: Organotypic Cultures—Roller Drum Method—Striatum/Brainstem
  • Basic Protocol 3: Neural and Embryonic Stem Cell Preparation
  • Basic Protocol 4: Viral Transfection
  • Basic Protocol 5: Time‐Lapse Calcium Imaging
  • Basic Protocol 6: DYE Spread to Investigate Direct Intercellular Connections
  • Support Protocol 1: Propidium Iodide Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Organotypic Cultures—Interface Method—Brainstem

  • 0.05 mg/ml poly‐D‐lysine (Sigma, cat. no. P7405)
  • 70% ethanol
  • Dissection medium (see recipe)
  • Culture medium (see recipe)
  • 1 to 8 mice or rat pups embryonic age 18 to postnatal day 14; earlier embryonic as well as adult age can work for both mice and rats
  • Washing/transportation solution (see recipe)
  • Millicell culture plate inserts (Millipore, cat. no. PICM03050)
  • Sterile 6‐well culture plates (Sarstedt, cat. no. 83.3920)
  • Petri dishes (diameter 60 mm; Sarstedt, cat. no. 82.1194.500)
  • Petri dishes (diameter 35 mm; Sarstedt, cat. no. 82.1135.500)
  • Scissors, for decapitation
  • Stereomicroscope (e.g., Zeiss Stemi DV 4, Carl Zeiss)
  • Large forceps, for moving the heads
  • Sharp forceps, for holding tissue
  • Ewald forceps, for removing skull
  • Spatulas
  • Small and larger blunt, rounded spatulas, for dissection and transfer of slices
  • Scalpel and blade, for cutting through the skull
  • Double‐edged stainless steel razor blade (e.g., Paragon, cat. no. 10 B.S.2982/Campden Instruments Ltd., Part N. 752/1/SS)
  • Micro scissors, for cutting nerves
  • McIllwain tissue chopper: e.g., The Mickle Laboratory Engineering, Co. Ltd. ( or Campden Instruments (http://www.campden‐
  • 10‐ml sterile syringes

Basic Protocol 2: Organotypic Cultures—Roller Drum Method—Striatum/Brainstem

  • Xylene
  • Acetone
  • Isopropanol
  • 0.05 mg/ml poly‐D‐lysine (Sigma, cat. no. P7405)
  • Chicken plasma (Sigma, cat. no. P3266)
  • Tissue‐culture‐grade H 2O (Invitrogen)
  • Thrombin from bovine plasma (Sigma, cat. no. T3399‐1KU)
  • Gey's balanced salt solution (GBSS; Sigma)
  • OC medium (see recipe)
  • Sprague‐Dawley rats: postnatal age 2 to 14 or mice embryonic day 18 to postnatal age 14; earlier embryonic as well as adult age may work for both mice and rats.
  • Dissection medium (see recipe)
  • 12 × 24 mm glass coverslips (No 1; Gribi,
  • Hot plate
  • McIlwain tissue chopper (e.g., Campden Instruments)
  • Stereomicroscope (e.g., Carl Zeiss)
  • Sterilized surgical cloths
  • Sterile syringe filter: red FB 30/0 (Merck, cat. no. 265622‐0)
  • 35‐mm and 100‐mm Petri dishes (Corning)
  • 15‐ml conical polypropylene tubes (e.g., Corning Falcon, cat. no. 009‐2095)
  • Scissors, for decapitation
  • Forceps, straight, angled, and blunt (e.g., Allgaier Instrumente GmbH, http://www.allgaier‐
  • Angled scissors
  • Double‐edged stainless steel razor blade (e.g., Paragon, cat. no. 10 B.S.2982/Campden Instruments Ltd., Part N. 752/1/SS)
  • Micropipettor and tips: 1 µl, 100 µl, 1000 µl (e.g., Eppendorf)
  • Rounded spatula (e.g., Allgaier Instrumente GmbH)
  • Roller‐drum device (Tissue Culture Roller‐drum, Model TC‐7; New Brunswick Scientific)

Basic Protocol 3: Neural and Embryonic Stem Cell Preparation

  • 70% ethanol
  • Dulbecco's Modified Eagle Medium (DMEM; Invitrogen)
  • Pyruvate with pyridoxine (Invitrogen)
  • Fetal bovine serum (FBS; Sigma)
  • Horse serum (Invitrogen)
  • L‐glutamine (Invitrogen)
  • Penicillin/streptomycin/fucidin (Invitrogen)
  • NSC C17.2 clone (Snyder et al., )
  • DiI (Sigma)
  • Hanks’ balanced salt solution (HBSS; Sigma)
  • Ca2+ and Mg2+–free phosphate‐buffered saline(PBS; Invitrogen)
  • Animals: the authors have used C57Bl6 and DBA/1LacJ mice and Sprague Dawley or Lewis rats, embryonic day 18 fetal, or newborn (postnatal day 0 to 5) rodents
  • Ketalar and Rompun
  • Dissection medium (see recipe)
  • Culture medium (see recipe)
  • Nerve growth factor (mouse, e.g., Invitrogen)
  • 0.25% trypsin (Invitrogen)
  • HBSS containing 1% (w/v) trypsin
  • Petri dishes (Corning)
  • Bürker chamber for cell counting (e.g., VWR Scientific)
  • Stereomicroscope (e.g., Carl Zeiss)
  • Micro scissors
  • 15‐ml conical polypropylene centrifuge tubes (e.g., Corning Falcon)
  • Fire‐polished Pasteur pipets
  • Forceps, straight, angled and blunt (e.g., Allgaier Instrumente GmbH)
  • Poly‐D‐lysine coated Petri dishes (see recipe)
  • Additional reagents and equipment for trypsinization of cells (Phelan, ) and roller drum method for cultures ( protocol 2)

Basic Protocol 4: Viral Transfection

  • Viral solution
  • Culture medium (see recipe)
  • Stereomicroscope
  • Glass capillary pipets
  • Pipet puller (P‐97, Sutter Instruments)
  • Air‐pressure‐controlled microinjector
  • Micromanipulator

Basic Protocol 5: Time‐Lapse Calcium Imaging

  • NaCl (e.g., Sigma)
  • KCl (e.g., Sigma)
  • KH 2PO 4 (e.g., Sigma)
  • CaCl 2 (e.g., Sigma)
  • MgSO 4 (e.g., Sigma)
  • NaHCO 3 (e.g., Sigma)
  • Glucose (e.g., Sigma)
  • Gas mixture 95% O 2 and 5% CO 2 (e.g., AGA Gas AB)
  • Fluo‐4 AM or Fura‐2 AM (Invitrogen)
  • Dimethylsulfoxide (DMSO; e.g., Sigma)
  • Pluronic acid (F‐127, Invitrogen)
  • For Fluo‐4 recordings: a Leica DM IRBE confocal laser scanning microscope equipped with a 40×/1.25 oil‐immersion objective (Leica Microsystems) or a Zeiss Axioexaminer D1 DM epifluorescence microscope equipped with a 20×/1.0 water immersion objective (Carl Zeiss)
  • For Fura‐2 recordings: a Zeiss Axiovert 135 epifluorescence microscope equipped with a 40×/1.4 oil‐immersion objective (Carl Zeiss)
  • Perfusion chamber (e.g., Warner Instruments)
  • Peristaltic pump (e.g., Ismatec)
  • Tubing (e.g., Warner Instruments)
  • Forceps (e.g., Allgaier Instrumente GmbH)
  • ImageJ software (NIH;

Basic Protocol 6: DYE Spread to Investigate Direct Intercellular Connections

  • DiI (Sigma)
  • Calcein (Sigma)
  • Hank's balanced salt solution (HBSS; Sigma)
  • NSC culture (see protocol 3)
  • Ca2+ Mg2+ phosphate‐buffered saline(PBS; Invitrogen)
  • 4% paraformaldehyde (PFA; see recipe)
  • Fluorescence microscope (e.g., Nikon Eclipse E 800)
  • Additional reagents and equipment for trypsinization of cell cultures (Phelan, )

Support Protocol 1: Propidium Iodide Staining

  • 1 mg/ml propidium iodide (PI; Invitrogen)
  • Culture medium (see recipe)
  • Organotypic cultures ( protocol 1 or 2)
  • 4% paraformaldehyde (see recipe)
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