Tracy C. Grikscheit, Erik R. Barthel, and Fr&eacute;d&eacute;ric G. Sala are supported by the California Institute for Regenerative Medicine (CIRM), grant numbers RN2-00946-1 (TCG) and TG2-01168 (ERB, FGS). Allison L. Speer is a Society of University Surgeons Ethicon scholar. Yashuhiro Torashima is funded by a Children's Hospital Los Angeles Saban Institute Research Career Development Fellowship.

1. Organoid Units Preparation
<ol>
 <li>Instruments appropriate for mouse dissection (scissors and forceps) should be sterilized by autoclave. </li>
 <li>Humanely euthanize the donor mouse according to local IACUC protocols. Ensure that the animal is dead before proceeding. </li>
 <li>Make a midline incision to gain access to the peritoneal cavity. Skin flaps can be reflected as needed to improve exposure.</li>
 <li>Eviscerate the small bowel and divide it just distal to the ligament of Treitz. Separate the smal...

<ol>
	<li>Grikscheit, T. C., Siddique, A., Ochoa, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+small+intestine+improves+recovery+after+massive+small+bowel+resection.">Tissue-engineered small intestine improves recovery after massive small bowel resection.</a> Ann. Surg. 240, 748-754 (2004).</li><li>Wales, P. W., Christison-Lagay, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+bowel+syndrome:+epidemiology+and+etiology.">Short bowel syndrome: epidemiology and etiology.</a> Sem. Ped. Surg. 19, 3-9 (2010).</li><li>Evans, G. S., Flint, N., Somers, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+a+method+for+the+preparation+of+rat+intestinal+epithelial+cell+primary+cultures.">The development of a method for the preparation of rat intestinal epithelial cell primary cultures.</a> J. Cell Sci. 101, 219-231 (1992).</li><li>Sala, F. G., Matthews, J. A., Speer, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicellular+approach+forms+a+significant+amount+of+tissue-engineered+small+intestine+in+the+mouse.">A multicellular approach forms a significant amount of tissue-engineered small intestine in the mouse.</a> Tiss. Eng. Part A. 17, 1841-1850 (2011).</li><li>Speer, A. L., Sala, F. G., Matthews, J. A., Grikscheit, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+tissue-engineered+stomach+demonstrates+epithelial+differentiation.">Murine tissue-engineered stomach demonstrates epithelial differentiation.</a> J. Surg. Res. 171, 6-14 (2011).</li><li>Haxhija, E. Q., Yang, H., Spencer, A. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+epithelial+cell+proliferation+is+dependent+on+the+site+of+massive+small+bowel+resection.">Intestinal epithelial cell proliferation is dependent on the site of massive small bowel resection.</a> Pediatr. Surg. Int. 23, 379-390 (2007).</li><li>Zhao, L., Cheng, Z., Dhall, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+corrective+pullthrough+surgery+in+a+mouse+model+of+Hirschsprung's+disease.">A novel corrective pullthrough surgery in a mouse model of Hirschsprung's disease.</a> J. Pediatr. Surg. 44, 759-766 (2009).</li><li>Petrosyan, M., Guner, Y. S., Williams, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+concepts+regarding+the+pathogenesis+of+necrotizing+enterocolitis.">Current concepts regarding the pathogenesis of necrotizing enterocolitis.</a> Ped. Surg. Int. 25, 309-318 (2009).</li><li>Shew, S. 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Hepatol. 7, 183-191 (2009).</li><li>Klempnauer, J., Grothues, F., Bektas, H., Pichlmayr, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+results+after+surgery+for+acute+mesenteric+ischemia.">Long-term results after surgery for acute mesenteric ischemia.</a> Surgery. , 121-239 (1997).</li><li>Fitzgibbons, S. C., Jones, B. A., Hull, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+biopsy-proven+parenteral+nutrition-associated+liver+fibrosis+and+biochemical+cholestasis+in+children+with+short+bowel+syndrome.">Relationship between biopsy-proven parenteral nutrition-associated liver fibrosis and biochemical cholestasis in children with short bowel syndrome.</a> J. Ped. Surg. 45, 95-99 (2010).</li><li>Spencer, A. 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No conflicts of interest declared.

We present a protocol for producing tissue-engineered intestine in the mouse using an organoid units-on-scaffold approach. The most critical steps are those of the organoid units preparation. Care must be taken to adequately clean and mechanically process the tissue, but equal care must be taken not to overdigest or overtriturate the organoid units after the digestion is performed (step 1.11). If this is done, the organoid units can be reduced to single cells, which can be lost in the supernatant of step 1.12, and are un...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Gibco</td>
 <td>114170-112</td>
 <td></td>
 </tr>
 <tr>
 <td>Antibiotic-Antimycotic 100X</td>
 <td>Invitrogen</td>
 <td>15240-062</td>
 <td></td>
 </tr>
 <tr>
 <td>Dispase</td>
 <td>Gibco</td>
 <td>17105-041</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase Type 1</td>
 <td>Worthington</td>
 <td>LS004194</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM High Glucose 1X</td>
 <td>Gibco</td>
 <td>11995-065</td>
 <td></td>
 </tr>
 <tr>
 <td>Heat inactivated FBS</td>
 <td>Invitrogen</td>
 <td>16140-071</td>
 <td></td>
 </tr>
 <tr>
 <td>Biofelt 100% PGA</td>
 <td>Concordia Medical</td>
 <td>FELT01-1005</td>
 <td>For polymer preparation as in Ref. 4 </td>
 </tr>
 <tr>
 <td>Poly-L-lactic acid</td>
 <td>Durect</td>
 <td>B6002-1</td>
 <td>For polymer preparation as in Ref. 4 </td>
 </tr>
 <tr>
 <td>Type I Collagen, rat tail</td>
 <td>Sigma-Aldrich</td>
 <td>C3867-1VL</td>
 <td>For polymer preparation as in Ref. 4 </td>
 </tr>
 <tr>
 <td>Ketoprofen 100 mg/ml</td>
 <td>Fort Dodge Animal Health</td>
 <td>71-KETOI-100-50</td>
 <td></td>
 </tr>
 <tr>
 <td>LabDiet 5001 rodent chow</td>
 <td>LabDiet</td>
 <td>5001</td>
 <td></td>
 </tr>
 <tr>
 <td>Septra 200 mg / 40 mg per 5 ml, USP</td>
 <td>Hi-Tech Pharmacal</td>
 <td>50383-824-16</td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane, USP</td>
 <td>Phoenix Pharmaceuticals</td>
 <td>57319-507-06</td>
 <td></td>
 </tr>
 </tbody>
</table>

tissue engineering of the intestine in a murine model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

Figure 1 shows an overall schema for the protocol documented here. The end result of this protocol is a globe or spherical structure of tissue-engineered murine intestine with a lumen, mucosa, submucosa, and surrounding muscularis. Figure 2A shows a typical globe in comparison to a starting polymer scaffold. Figure 2B displays the same construct sharply bivalved to reveal its lumen. Figure 3 demonstrates a hematoxylin/eosin-stained paraffin-mounted cross...

Watch this Scientific Journal Video about Tissue Engineering of the Intestine in a Murine Model at JoVE.com

Tissue Engineering of the Intestine in a Murine Model

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

tissue-engineering-of-the-intestine-in-a-murine-model

Research

JoVE Journal

Bioengineering

14.8K Görüntüleme.  Keck School of Medicine of the University of Southern California. Bu makalede ve beraberindeki video bir organoid üniteleri-on-iskele yaklaşımı kullanarak, fare doku mühendisliği bağırsak üretmek için bizim protokol sunuyoruz.