Reconstitution of a Bioengineered Salivary Gland Using a Three‐Dimensional Cell Manipulation Method

Miho Ogawa1, Takashi Tsuji1

1 RIKEN Center for Developmental Biology, Kobe, Hyogo
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 19.17
DOI:  10.1002/0471143030.cb1917s66
Online Posting Date:  March, 2015
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Abstract

One concept in regenerative therapy is the replacement of a lost or damaged organ with a regenerated, fully functional organ. Three‐dimensional cell manipulation techniques, designated “organ germ methods,” enable the normal development of a bioengineered organ germ in several types of ectodermal organs, such as teeth, hair follicles, and secretory glands. This method is useful for both organ regeneration technology and developmental biology, including cell kinetic analysis and the elucidation of gene regulation during organogenesis. Here, we describe a protocol for salivary gland reconstitution using the organ germ method to transplant a bioengineered salivary gland germ. © 2015 by John Wiley & Sons, Inc.

Keywords: organ germ method; bioengineered salivary gland; regeneration; organ culture; transplantation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Reconstruction of the Bioengineered Salivary Gland Germ
  • Basic Protocol 2: Transplantation of a Bioengineered Salivary Gland Germ
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Reconstruction of the Bioengineered Salivary Gland Germ

  Materials
  • Pregnant C57BL/6 mouse, E13 to E14
  • 70% ethanol
  • Dulbecco's phosphate‐buffered saline without Ca and Mg [D‐PBS(‐); Nacalai Tesque, cat. no. 14249‐95]
  • Culture medium, sterile (see recipe)
  • 50 U/ml Dispase (BD Bioscience, cat. no. 354235)
  • 70 U/μl deoxyribonuclease I (DNase) from bovine pancreas (Sigma, cat. no. D5025)
  • Reagent A (see recipe)
  • Reagent B (see recipe)
  • Reagent C (see recipe)
  • Silicon grease (Dow Corning Toray)
  • Collagen gel (see recipe)
  • Two straight scissors
  • Two curved forceps
  • Straight forceps
  • Dissecting microscope
  • 100‐mm and 35‐mm culture dish, sterile
  • 1‐ml syringes, sterile (Terumo, cat. no. SS‐01 T)
  • 25‐G needles (5/8; 0.50 × 16 mm), sterile (Terumo, cat. no.NN‐2516R)
  • 15‐ml centrifuge tubes, sterile (BD Bioscience, cat. no. 352196)
  • Centrifuge
  • Cotton swabs
  • 1.5‐ml microcentrifuge tubes, sterile (Eppendorf, cat. no. 0030 125.150)
  • Cell strainers (BD Bioscience, cat. no. 352235)
  • GELoader tips, 0.5 to 20 μl (Eppendorf, cat. no. 0030 001.222)
  • Polyglycolic acid (PGA) monofilament thread guide (9‐0 PGA absorbable suture; Gunze)
  • Cell culture insert/0.4‐μl pore size membrane (BD Biosciences, cat. no. 353090)
  • 6‐well plate
  • Additional reagents and equipment for euthanasia of mice (Donovan and Brown, )

Basic Protocol 2: Transplantation of a Bioengineered Salivary Gland Germ

  Materials
  • C57BL/6 mouse, female, 7 weeks of age
  • Dulbecco's phosphate‐buffered saline without Ca and Mg [D‐PBS(‐); Nacalai Tesque, cat. no. 14249‐95]
  • Animal clippers
  • Surgical tape
  • Two straight forceps
  • Straight scissors
  • Vannas scissors (Fine Science Tools Inc., cat. no. 15008‐08)
  • Surgical clips: the clips are handmade; referring to Figure 19.17.5B‐b; create clips with wire and a small rubber band to match the size of the mouse
  • Stereomicroscope
  • 8–0 nylon sutures
  • Additional reagents and equipment for anesthesia of rodents (Donovan and Brown, )
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Figures

Videos

Literature Cited

Literature Cited
  Avery, J.K. 2002. Oral Development and Histology. pp. 292‐330, Thieme Medical Publishers, New York.
  Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
  Hirayama, M., Ogawa, M., Oshima, M., Sekine, Y., Ishida, K., Yamashita, K., Ikeda, K., Shimmura, S., Kawakita, T., Tsubota, K., and Tsuji, T. 2013. Functional lacrimal gland regeneration by transplantation of a bioengineered organ germ. Nat. Commun. 4:2497.
  Ikeda, E., Morita, R., Nakao, K., Ishida, K., Nakamura, T., Takano‐Yamamoto, T., Ogawa, M., Mizuno, M., Kasugai, S., and Tsuji, T. 2009. Fully functional bioengineered tooth replacement as an organ replacement therapy. Proc. Natl. Acad. Sci. USA 106:13475‐13480.
  Jaskoll, T. and Melnick, M. 2004. Embryonic salivary gland branching morphogenesis. Madame Curie Bioscience Database 13‐14. Available at http://www.ncbi.nlm.nih.gov/books/NBK6103.
  Kagami, H., Wang, S., and Hai, B. 2008. Restoring the function of salivary glands. Oral Disease 14:15‐24.
  Knosp, M.W., Knox, M.S., and Hoffman, P.M., 2012. Salivary gland organogenesis. WIREs Dev. Biol. 1:69‐82.
  Nakao, K., Morita, R., Saji, Y., Ishida, K., Tomita, Y., Ogawa, M., Saitoh, M., Tomooka, Y., and Tsuji, T. 2007. The development of a bioengineered organ germ method. Nat. Methods 4:227‐230.
  Ogawa, M., Oshima, M., Imamura, A., Sekine, Y., Ishida, K., Yamashita, K., Nakajima, K., Hirayama, M., Tachikawa, T., and Tsuji, T., 2013. Functional salivary gland regeneration by transplantation of a bioengineered organ germ. Nat. Commun. 4:2498.
  Oshima, M., Mizuno, M., Imamura, A., Ogawa, M., Yasukawa, M., Yamazaki, H., Morita, R., Ikeda, E., Nakao, K., Takano‐Yamamoto, T., Kasugai, S., Saito, M., and Tsuji, T. 2011. Functional tooth regeneration using a bioengineered tooth unit as a mature organ replacement regenerative therapy. PLoS ONE 6:e21531.
  Patel, V.N., Rebustini, I.T., and Hoffman, M.P. 2006. Salivary gland branching morphogenesis. Differentiation 74:349‐364.
  Sakai, T., Larsen, M., and Yamada, M.K., 2003. Fibronectin requirement in branching morphogenesis. Nature 423:876‐881.
  Toyoshima, K., Asakawa, K., Ishibashi, N., Toki, H., Ogawa, M., Hasegawa, T., Irié, T., Tachikawa, T., Sato, A., Takeda, A., and Tsuji, T. 2012. Fully functional hair follicle regeneration through the rearrangement of stem cells and their niches. Nat. Commun. 3:784.
  Tucker, A.S., 2007. Salivary gland development. Semin. Cell. Dev. Biol. 18:237‐244.
  Wei, C., Larsen, M., Hoffman, P. M., and Yamada, M. K. 2007. Self‐Organization and branching morphogenesis of primary salivary epithelial cells. Tissue Engineering 13:721‐735.
Key References
  Nakao et al., 2007. See above.
  This is the first paper that describes a technique to reconstitute the organ germ induced by epithelial‐mesenchymal interactions.
  Ogawa et al., 2013. See above.
  This paper describes the protocol for transplanting the bioengineered salivary gland germ into an adult mouse and demonstrates that saliva is successfully secreted into the oral cavity. Please refer to this paper for the analysis method for the regenerated salivary glands.
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