Isolation and Expansion of Mesenchymal Stem Cells from Human Conjunctival Tissue

Samad Nadri1, Shahin Yazdani2

1 Medical Biotechnology and Medical Nanotechnology Department, Faculty of Medicine, Zanjan University of Medical Science, Zanjan, 2 Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.14
DOI:  10.1002/9780470151808.sc01f14s33
Online Posting Date:  May, 2015
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Here we describe a simple protocol for the isolation and culture of mesenchymal stem cells (MSCs) from conjunctiva stromal tissue, with applications to stem cell biology and regenerative medicine. This protocol is based on an explant culture protocol for the adhesion and migration of MSCs from tissue biopsy in primary culture and expansion of the MSC population by passaging cells on the surface of plastic culture dishes. Conjunctiva mesenchymal stem cells (CJMSCs) are generally isolated from small biopsies of human conjunctival tissue. Epithelial sheets are carefully loosened and removed by enzyme treatment after 18 hr and placed in fresh medium supplemented with growth factors. When the cells migrating from tissue segments in primary cultures become nearly confluent, the culture is treated with trypsin. A purified population of CJMSCs that retain proliferation and differentiation potential for many passages can be obtained 2 weeks after the initiation of culture. © 2015 by John Wiley & Sons, Inc.

Keywords: isolation; conjunctiva; mesenchymal stem cells; human

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • Conjunctival biopsy
  • Dulbecco's Modified Eagle Medium (DMEM) with 2 mM l‐glutamine and without ribonucleosides and ribonucleotides (Life Technologies, cat. no. 12100‐046)
  • 100× penicillin‐streptomycin (Sigma, cat. no. P4333)
  • Amphotericin B (Sigma, cat. no. A2942)
  • Gentamicin (Sigma,
  • Supplemented hormonal epithelial medium (SHEM; see recipe)
  • Dispase II (Sigma, cat. no. D4693)
  • Sorbitol
  • Dulbecco's modified Eagle's medium (DMEM)/F‐12 1:1 mixture (DMEM/F‐12 medium; Life Technologies, cat. no. 12500‐062)
  • Knockout Serum Replacement (Life Technologies, cat. no. 10828‐028)
  • Basic‐FGF (Peprotech, cat. no. 450‐33)
  • 5 mg/ml insulin (Sigma, cat. no. 19278)
  • 10 ng/ml human LIF (Millipore, cat. no. 1005)
  • Complete DMEM medium with 15% FBS (see recipe)
  • Phosphate‐buffered saline (PBS; prepare from PBS powder, Life Technologies, cat. no. 00‐3000)
  • 0.025% (w/v) trypsin (Life Technologies, cat. no. 27250‐018)
  • Stereomicroscope
  • Sterile blades
  • 6‐well plate
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Literature Cited

Literature Cited
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