Preparation and Osteogenic Differentiation of Scaffold‐Free Mouse Adipose‐Derived Stromal Cell Microtissue Spheroids (ASC‐MT)

Ali Mirsaidi1, André N. Tiaden1, Peter J. Richards1

1 Bone and Stem Cell Research Group, CABMM, University of Zurich, Zurich, Switzerland
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2B.5
DOI:  10.1002/9780470151808.sc02b05s27
Online Posting Date:  November, 2013
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Abstract

In this unit, previously described methods are expanded upon, where procedures relating to the preparation, culturing, and osteogenic differentiation of scaffold‐free mouse adipose‐derived stromal cell microtissue spheroids (ASC‐MT) are outlined. Not only is a detailed methodology of how to engineer such spheroids are presented, but a full account of how to induce and analyze osteogenesis in these ASC‐MT constructs is given along with relevant figures to help better illustrate the methods described. Curr. Protoc. Stem Cell Biol. 27:2B.5.1‐2B.5.12. © 2013 by John Wiley & Sons, Inc.

Keywords: adipose‐derived stromal cells; osteogenesis; 3D‐culture

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: ASC Isolation and Expansion
  • Support Protocol 1: Induction of Osteogenic Differentiation in ASC 2‐D Cultures
  • Basic Protocol 2: Generation of Osteogenic ASC‐MTs
  • Basic Protocol 3: Harvesting and Processing of ASC‐MT for Histological Analysis of Osteogenic Differentiation
  • Support Protocol 2: Analysis of Osteogenesis in ASC‐MT Paraffin Wax Sections
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: ASC Isolation and Expansion

  Materials
  • Male mice (8‐ to 12‐weeks old)
  • 70% ethanol
  • Betadine (Mundipharma Medical Company)
  • Growth medium I (see recipe)
  • Dulbecco's phosphate‐buffered saline (D‐PBS, Life Technologies, cat. no. 14190‐094)
  • Digestion solution (see recipe)
  • 0.25% trypsin‐EDTA (Life Technologies, cat. no. 25200‐056)
  • Sterile surgical instruments (forceps, tweezers, clamps, micro‐scissors, scalpels)
  • 15‐ and 50‐ml tubes (Falcon)
  • Sterile petri dishes
  • Stereomicroscope
  • Shaker, 37°C
  • Centrifuge
  • 40‐µm cell strainer (BD Falcon, cat. no. 352340)
  • 25‐ and 75‐cm2 culture flasks (Nunclon, cat. nos. 734‐2064 and 734‐2066)
  • 37oC, 5% CO 2 humidified incubator
  • 5‐ and 10‐ml serological pipets, sterile

Support Protocol 1: Induction of Osteogenic Differentiation in ASC 2‐D Cultures

  Materials
  • ASCs (see protocol 1)
  • Growth medium II (see recipe)
  • Osteogenic induction medium (see recipe)
  • Dulbecco's phosphate‐buffered saline (D‐PBS, Life Technologies, cat. no. 14190‐169)
  • 10% (v/v) formaldehyde solution (Sigma, cat. no. F1635) in D‐PBS
  • Alizarin Red S working solution (see recipe)
  • 5‐ and 10‐ml serological pipets, sterile
  • 37°C, 5% CO 2 humidified incubator
  • Rocking platform
  • Light microscope

Basic Protocol 2: Generation of Osteogenic ASC‐MTs

  Materials
  • ASCs (see protocol 2)
  • Dulbecco's phosphate‐buffered saline (D‐PBS, Life Technologies, cat. no. 14190‐169)
  • 0.25% trypsin‐EDTA (Life Technologies, cat. no. 25200‐056)
  • Growth medium I or II (see recipe)
  • Osteogenic induction medium (see recipe)
  • Trypan blue solution (Sigma, cat. no. T8154)
  • Light microscope
  • 5‐ and 10‐ml serological pipets, sterile
  • 15‐ml tubes (Falcon)
  • Centrifuge
  • Hemacytometer
  • Terasaki plates (Nunc, cat. no. 734‐2079)
  • Reagent reservoir (Thermo Scientific, cat. no. 8093‐11)
  • Multichannel electronic pipettor (Thermo Scientific, cat. no. 2034)
  • 1250‐µl tips (Thermo Scientific, cat. no. 8041)
  • 37°C, 5% CO 2 humidified incubator

Basic Protocol 3: Harvesting and Processing of ASC‐MT for Histological Analysis of Osteogenic Differentiation

  Materials
  • Dulbecco's phosphate‐buffered saline (D‐PBS, Life Technologies, cat. no. 14190‐169)
  • Terasaki plates containing osteogenic ASC‐MTs (see protocol 3)
  • 10% (v/v) formaldehyde solution (Sigma, cat. no. F1635) in D‐PBS
  • Low‐melting‐point agarose (Sigma, cat. no. A9414)
  • Hematoxylin blue
  • Paraffin wax (Paraplast, Sigma, cat. no. P3558)
  • Sterile petri dishes (12‐cm diameter)
  • Sterile, non‐adhesive, stainless‐steel ASC‐MT harvesting device
  • Light microscope
  • Sterile 0.2‐ and 1.5‐ml microcentrifuge tubes (Eppendorf)
  • Tweezers
  • Microcentrifuge
  • Disposable needles
  • Tissue processing/embedding cassettes with lid (Sigma, cat. no. Z672122)
  • Tissue processor (Leica Histoprocessor, cat. no. ASP200S)
  • Paraffin embedding station (Leica, cat. no. EG1150H)
  • Embedding moulds (VWR Scientific)

Support Protocol 2: Analysis of Osteogenesis in ASC‐MT Paraffin Wax Sections

  Materials
  • Alizarin Red S working solution (see recipe)
  • DPX mountant (Sigma, cat. no. 44581)
  • Microtome (Leica, cat. no. RM2235)
  • Superfrost Plus slide (Thermo Scientific, cat. no. J1800AMNZ)
  • 37°C incubator
  • 60°C heating block
  • Light microscope
  • Additional reagents and equipment for hanging drop generation of ASC‐MTs (see protocol 3) and ASC‐MTs in paraffin wax (see protocol 4)
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Figures

Videos

Literature Cited

Literature Cited
  Bokel, C. and Brown, N.H. 2002. Integrins in development: Moving on, responding to, and sticking to the extracellular matrix. Dev. Cell 3:311‐321.
  Boudreau, N.J. 2003. Organized living: From cell surfaces to basement membranes. Sci STKE 2003:pe34.
  Dunlop, L.L. and Hall, B.K. 1995. Relationships between cellular condensation, preosteoblast formation and epithelial‐mesenchymal interactions in initiation of osteogenesis. Int. J. Dev. Biol. 39:357‐371.
  Friedrich, J., Seidel, C., Ebner, R., and Kunz‐Schughart, L.A. 2009. Spheroid‐based drug screen: Considerations and practical approach. Nat. Protoc. 4:309‐324.
  Goldmann, W.H. 2002. Mechanical aspects of cell shape regulation and signaling. Cell Biol. Int. 26:313‐317.
  Keller, G.M. 1995. In vitro differentiation of embryonic stem cells. Curr. Opin. Cell Biol. 7:862‐869.
  Kelm, J.M. and Fussenegger, M. 2010. Scaffold‐free cell delivery for use in regenerative medicine. Adv. Drug Deliv. Rev. 62:753‐764.
  Kelm, J.M., Timmins, N.E., Brown, C.J., Fussenegger, M., and Nielsen, L.K. 2003. Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol. Bioeng. 83:173‐180.
  Mirsaidi, A., Kleinhans, K.N., Rimann, M., Tiaden, A.N., Stauber, M., Rudolph, K.L., and Richards, P.J. 2012. Telomere length, telomerase activity and osteogenic differentiation are maintained in adipose‐derived stromal cells from senile osteoporotic SAMP6 mice. J. Tissue Eng. Regen. Med. 6:378‐390.
  Schenk, S. and Quaranta, V. 2003. Tales from the crypt[ic] sites of the extracellular matrix. Trends Cell Biol. 13:366‐375.
  Tapp, H., Hanley, E.N. Jr., Patt, J.C., and Gruber, H.E. 2009. Adipose‐derived stem cells: Characterization and current application in orthopaedic tissue repair. Exp. Biol. Med. 234:1‐9.
  Tarone, G., Hirsch, E., Brancaccio, M., De Acetis, M., Barberis, L., Balzac, F., Retta, S.F., Botta, C., Altruda, F., and Silengo, L. 2000. Integrin function and regulation in development. Int. J. Dev. Biol. 44:725‐731.
  Tiaden, A.N., Breiden, M., Mirsaidi, A., Weber, F.A., Bahrenberg, G., Glanz, S., Cinelli, P., Ehrmann, M., and Richards, P.J. 2012. Human serine protease HTRA1 positively regulates osteogenesis of human bone marrow‐derived mesenchymal stem cells and mineralization of differentiating bone‐forming cells through the modulation of extracellular matrix protein. Stem Cells 30:2271‐2282.
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