Derivation of Multipotent Mesenchymal Stromal Cells from Ovine Bone Marrow

Daniel Vivas1, Marta Caminal2, Irene Oliver‐Vila2, Joaquim Vives3

1 Musculoskeletal Tissue Engineering Group, Vall d'Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona, Barcelona, 2 Servei de Teràpia Cellular, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Barcelona, 3 Departament de Medicina, Universitat Autònoma de Barcelona, Barcelona
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2B.9
DOI:  10.1002/cpsc.43
Online Posting Date:  February, 2018
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


In the field of orthopedics, translational research of novel therapeutic approaches involves the use of large animal models (such as sheep, goat, pig, dog, and horse) due to the similarities with humans in weight, size, joint structure, and bone/cartilage healing mechanisms. Particularly in the development of cell‐based therapies, the lack of manageable immunocompromised preclinical large animal models prevents the use of human cells, which makes it necessary to produce equivalent homologous cell types for the study of their pharmacodynamics, pharmacokinetics, and toxicology. The methods described herein allow for the isolation, expansion, manipulation, and characterization of fibroblastic‐like ovine bone marrow–derived multipotent mesenchymal stromal cells (BM‐MSC) that, similar to human BM‐MSC, adhere to standard plastic surfaces; express specific surface markers such as CD44, CD90, CD140a, CD105, and CD166; and display trilineage differentiation potential in vitro. Homogeneous cell cultures result from a 3‐week bioprocess yielding cell densities in the range of 2–4 × 104 MSC/cm2 at passage 2, which corresponds to ∼8 cumulative population doublings. Large quantities of BM‐MSC resulting from following this methodology can be readily used in proof of efficacy and safety studies in the preclinical development stage. © 2018 by John Wiley & Sons, Inc.

Keywords: bone marrow; cell culture; multipotent mesenchymal stromal cells; nonclinical development; sheep; preclinical animal model

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Ovine Bone Marrow Extraction and Subsequent Isolation of Mononucleated Cells
  • Basic Protocol 2: Isolation and Expansion of Ovine Mesenchymal Stromal Cells
  • Support Protocol 1: Ovine Serum Manufacturing
  • Support Protocol 2: Sterility Test
  • Basic Protocol 3: Characterization of Ovine Mesenchymal Stromal Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Ovine Bone Marrow Extraction and Subsequent Isolation of Mononucleated Cells

  • Sheep of interest
  • Anesthetic reagents for surgery (e.g., buprenorphine, midazolam, propofol, isoflurane)
  • Anticoagulant citrate dextrose solution, solution A (ACD‐A; e.g., Grifols, cat. no. 721781)
  • Phosphate‐buffered saline (PBS), sterile (e.g., Gibco, cat. no. 14190)
  • Histopaque, 1.077 g/ml (e.g., Sigma, cat. no. 10771 or equivalent)
  • Lysis solution, sterile (see recipe)
  • Complete culture medium (CCM; see recipe)
  • Surgery room and equipment including, at minimum:
  • Operating table
  • Anesthesia machine and cart
  • Electronic monitor
  • 11‐G trocar (e.g., Sterylab, cat. no. BEN 1110)
  • 15‐ml and 50‐ml conical tubes
  • Culture flasks, sterile
  • Centrifuge
  • Plastic Pasteur pipettes, sterile

Basic Protocol 2: Isolation and Expansion of Ovine Mesenchymal Stromal Cells

  • Isolated MNC sample (see protocol 1)
  • Complete culture medium (CCM; see recipe)
  • PBS (e.g., Gibco, cat. no. 14190)
  • 0.05% (w/v) trypsin (e.g., Gibco, cat. no. 25300‐054 or equivalent)
  • Hemocytometer or other method for counting cell number
  • Culture flask
  • 37°C, 95% humidity, 5% CO 2 incubator
  • Inverted microscope
  • 15‐ml and 50‐ml conical tubes
  • 1.5‐ml microcentrifuge tubes

Support Protocol 1: Ovine Serum Manufacturing

  • Sheep
  • 600‐ml sterile transfer bags (e.g., Terumo BCT, cat. no. BBT060CM) without anticoagulants
  • 20‐G needles (e.g., BD Biosciences, cat. no. 303007)
  • Tubing with Luer connectors (preferably Luer‐Lock ends)
  • Hair clippers
  • Scale, for weighing transfer bags
  • Centrifuge (e.g., Hermle Labortechnik Z513K)
  • Rectangular bucket rotor (e.g., Hermle Labortechnik, cat. no. 220.70 V06)
  • Class II biological safety cabinet
  • Sterile perforator
  • 50‐ml conical tubes
  • 56°C water bath
  • 150‐cm2 culture flasks
  • 50‐ml serological pipette

Support Protocol 2: Sterility Test

  • Sample to be tested (see protocol 1Basic Protocols 1 and protocol 22 or protocol 3)
  • Tryptose phosphate broth (e.g., Sigma, cat. no. T8159) or Dulbecco's Modified Eagle Medium (DMEM; e.g., Gibco, cat. no. 31885)
  • 25‐cm2 nonadherent culture flasks
  • CO 2 incubator
  • Inverted microscope
NOTE: Perform all steps in protocol 4 in a class II biological safety cabinet.

Basic Protocol 3: Characterization of Ovine Mesenchymal Stromal Cells

  • Cell suspension of known concentration
  • Complete culture medium (CCM; see recipe)
  • Differentiation media:
  • StemPro Osteogenesis Differentiation Kit (e.g., Gibco, cat. no. A1007201)
  • StemPro Adipogenesis Differentiation Kit (e.g., Gibco, cat. no. A1007001)
  • StemPro Chondrogenesis Differentiation Kit (e.g., Gibco, cat. no. A1007101)
  • PBS, sterile (e.g., Gibco, cat. no 14190)
  • 4% (w/v) formaldehyde in PBS
  • Staining reactive (see recipe)
  • Alizarin Red (AR) staining solution (e.g., Merck, cat. no. TMS‐008‐C)
  • Propylene glycol (e.g., Sigma, cat. no. 398039‐500ML)
  • Oil Red O staining solution (see recipe)
  • 1% (v/v) acetic acid in distilled water
  • 0.1% Safranin O (e.g., Sigma, cat. no. S8884‐25G) in distilled water
  • FACS flow solution (e.g., BD Biosciences, cat. no. 342003)
  • Antibodies for flow cytometry including:
  • FITC‐conjugated mouse anti‐human CD44 antibody (e.g., BD Biosciences, cat. no. 555478)
  • APC‐conjugated mouse anti‐human CD90 antibody (e.g., BD Biosciences, cat. no. 559869)
  • PE‐conjugated mouse anti‐human CD166 antibody (e.g., BD Biosciences, cat. no. 560903)
  • AF647‐conjugated mouse anti‐human CD140a antibody (e.g., BD Biosciences, cat. no. 562798)
  • PE‐conjugated mouse anti‐human CD105 antibody (e.g., Life Technologies, cat. no. MHCD10504)
  • Centrifuge
  • 24‐well plates
  • 37°C, 5% CO 2 humidified incubator
  • Flow cytometry tubes
  • Flow cytometer
PDF or HTML at Wiley Online Library



Literature Cited

  Bianco, P., Cao, X., Frenette, P. S., Mao, J. J., Robey, P. G., Simmons, P. J., & Wang, C.‐Y. (2013). The meaning, the sense and the significance: Translating the science of mesenchymal stem cells into medicine. Nature Medicine, 19, 35–42. doi: 10.1038/nm.3028.
  Caminal, M., Fonseca, C., Peris, D., Moll, X., Rabanal, R. M., Barrachina, J., … Vives, J. (2014). Use of a chronic model of articular cartilage and meniscal injury for the assessment of long‐term effects after autologous mesenchymal stromal cell treatment in sheep. New Biotechnology, 31, 492–498. doi: 10.1016/j.nbt.2014.07.004.
  Caminal, M., Moll, X., Codina, D., Rabanal, R. M., Morist, A., Barrachina, J., … Vives, J. (2014). Transitory improvement of articular cartilage characteristics after implantation of polylactide: polyglycolic acid (PLGA) scaffolds seeded with autologous mesenchymal stromal cells in a sheep model of critical‐sized chondral defect. Biotechnology Letters, 36, 2143–2153. doi: 10.1007/s10529‐014‐1585‐3.
  Caminal, M., Peris, D., Fonseca, C., Barrachina, J., Codina, D., Rabanal, R. M., … Vives, J. (2016). Cartilage resurfacing potential of PLGA scaffolds loaded with autologous cells from cartilage, fat, and bone marrow in an ovine model of osteochondral focal defect. Cytotechnology, 68, 907–919. doi: 10.1007/s10616‐015‐9842‐4.
  Caminal, M., Vélez, R., Rabanal, R. M., Vivas, D., Batlle‐Morera, L., Aguirre, M., … Vives, J. (2017). A reproducible method for the isolation and expansion of ovine mesenchymal stromal cells from bone marrow for use in regenerative medicine preclinical studies. Journal of Tissue Engineering and Regenerative Medicine, 11, 3408–3416. doi: 10.1002/term.2254.
  Chahla, J., Mannava, S., Cinque, M. E., Geeslin, A. G., Codina, D., & LaPrade, R. F. (2017). Bone marrow aspirate concentrate harvesting and processing technique. Arthroscopy Techniques, 6, 1–5. doi: 10.1016/j.eats.2016.10.024.
  Deans, R. J., & Moseley, A. B. (2000). Mesenchymal stem cells: Biology and potential clinical uses. Experimental Hematology, 28, 875–884. doi: 10.1016/S0301‐472X(00)00482‐3.
  Dominici, M., Le Blanc, K., Mueller, I., Slaper‐Cortenbach, I., Marini, F., Krause, D. S., … Horwitz, E. M. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8, 315–317. doi: 10.1080/14653240600855905.
  Hayflick, L. (1965). The limited in vitro lifetime of human diploid cell strains. Experimental Cell Research, 37, 614–636. doi: 10.1016/0014‐4827(65)90211‐9.
  Khan, M. R., Chandrashekran, A., Smith, R. K. W., & Dudhia, J. (2016). Immunophenotypic characterization of ovine mesenchymal stem cells. Cytometry Part A, 89, 443–450. doi: 10.1002/cyto.a.22849.
  Liu, T. M., Martina, M., Hutmacher, D. W., Hui, J. H. P., Lee, E. H., & Lim, B. (2006). Identification of common pathways mediating differentiation of bone marrow‐ and adipose tissue‐derived human mesenchymal stem cells into three mesenchymal lineages. Stem Cells, 25, 750–760. doi: 10.1634/stemcells.2006‐0394.
  O'Loughlin, P. F., Morr, S., Bogunovic, L., Kim, A. D., Park, B., & Lane, J. M. (2008). Selection and development of preclinical models in fracture‐healing research. The Journal of Bone and Joint Surgery, 90, 79–84. doi: 10.2106/JBJS.G.01585.
  Oliver‐Vila, I., Coca, M. I., Grau‐Vorster, M., Pujals‐Fonts, N., Caminal, M., Casamayor‐Genescà, A., … Vives, J. (2016). Evaluation of a cell‐banking strategy for the production of clinical grade mesenchymal stromal cells from Wharton's jelly. Cytotherapy, 18, 25–35. doi: 10.1016/j.jcyt.2015.10.001.
  Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J. D., … Marshak, D. R. (1999). Multilineage potential of adult human mesenchymal stem cells. Science, 284, 143–147. doi: 10.1126/science.284.5411.143.
  Prat, S., Gallardo‐Villares, S., Vives, M., Carreño, A., Caminal, M., Oliver‐Vila, I., … Vives, J. (2016). Clinical translation of a mesenchymal stromal cell‐based therapy developed in a large animal model and two case studies of the treatment of atrophic pseudoarthrosis. Journal of Tissue Engineering and Regenerative Medicine, [Epub ahead of print]. doi: 10.1002/term.2323.
  Reyes, B., Coca, M. I., Codinach, M., López‐Lucas, M. D., del Mazo‐Barbara, A., Caminal, M., … Vives, J. (2017). Assessment of biodistribution using mesenchymal stromal cells: Algorithm for study design and challenges in detection methodologies. Cytotherapy, 19, 1060–1069. doi: 10.1016/j.jcyt.2017.06.004.
  Sharpe, M., Leoni, G., Barry, J., Schutte, R., & Mount, N. (2016). Nonclinical studies for cell‐based medicines. In J. Vives & G. Carmona (Eds.), Guide to Cell Therapy GxP (pp. 49–106). Cambridge, MA: Academic Press.
  Ullah, I., Subbarao, R. B., & Rho, G. J. (2015). Human mesenchymal stem cells ‐ current trends and future prospective. Bioscience Reports, 35, 1–18. doi: 10.1042/BSR20150025.
  Vives, J., Oliver‐vila, I., & Pla, A. (2015). Quality compliance in the shift from cell transplantation to cell therapy in non‐pharma environments. Cytotherapy, 17, 1009–1014. doi: 10.1016/j.jcyt.2015.02.002.
Key References with Annotations
  Caminal et al. (2017). See above.
  This paper describes optimization of the protocol for the derivation of mesenchymal stem cells from ovine bone marrow.
PDF or HTML at Wiley Online Library