Name: Tissue Engineering of the Intestine in a Murine Model (Video) | JoVE Journal
Uploaded: 2012-12-01T14:00:00.000+00:00
Duration: 8 min 45 s
Description: Learn tissue engineering of the intestine using an organoid units-on-scaffold approach in mice. This protocol demonstrates isolation, digestion, and implantation techniques for regenerative medicine applications.

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5 minutes

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 small bowel from its mesentery using sharp and gentle blunt dissection. Identify the ileocecal junction and divide the small bowel 5 mm proximal to this. </li>
 <li>Using scissors, open the intestine lengthwise along the antimesenteric border in a Petri dish with 10 ml 4 &deg;C, sterile Hanks' buffered saline (HBSS, Invitrogen, Carlsbad CA) / 1X antibiotic-antifungal (Anti-Anti, Invitrogen) solution. Clear fecal matter from the opened intestine with gentle agitation then transfer opened intestine to a 15ml centrifuge tube with 10 ml of 4 &deg;C, sterile HBSS / 1X anti-anti.</li>
 <li>Wash the opened intestine three times in 10 ml of 4 &deg; C, sterile HBSS / 1X anti-anti in a test tube. Each wash can be performed with mild shaking of the 15 ml tube for 30 sec. After shaking, the intestinal tissue sinks to the bottom of the tube. Discard floating material, which is mesenchymal debris. Remove the washing solution carefully with a pipet.</li>
 <li>Mince the washed intestine in a Petri dish with 10 ml of 4 &deg;C, sterile HBSS / 1X anti-anti to less than 1 mm square pieces using a scissors. Gather the minced material up with an automatic pipet and place it into a test tube. </li>
 <li>Centrifuge the tube at 500 rpm for 8 min. Discard the supernatant, which contains fat and mesenchyme. </li>
 <li>Digest the minced, washed material with 10 ml of sterile HBSS / 1X anti-anti plus 0.125 mg/ml dispase (Invitrogen) and 800 units/ml collagenase type 1 (Worthington, Lakewood NJ). To prepare 40 ml of the digestion solution, weigh out 5 mg of dispase, 142 mg of collagenase, and add sterile HBSS up to a volume of 40 ml. Prepare this solution freshly each time organoid units are prepared, and keep at 4&deg; C until ready to use. Add the digestion solution directly to the pellet from step 1.8. </li>
 <li>Incubate the test tube containing the minced, washed material with the digestion solution at 37 &deg;C for 20 min.</li>
 <li>Retrieve the test tube and further disrupt the digested tissue by trituration with a 10 ml pipet. Repeat between 20 to 50 times until a uniform appearance is obtained. </li>
 <li>Centrifuge the test tube for 5 min at 800 rpm. Discard the supernatant, which contains single cells. </li>
 <li>Stop the digestion reaction with 10 ml of 4 &deg;C, sterile Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) plus 10% v/v heat-inactivated fetal bovine serum (HI-FBS, Invitrogen). Resuspend the pellet and shake the tube. </li>
 <li>Centrifuge the test tube for 5 min at 800 rpm. Remove the supernatant carefully with an automatic pipet until the last few drops. Use a disposable plastic pipet for the last few drops to avoid resuspending the pellet. </li>
</ol>
2. Loading of Polyglycolic Acid Scaffold
<ol>
 <li>Form 2-mm long, 5-mm outer diameter cylindrical scaffolds from nonwoven polyglycolic acid (2-mm sheet thickness, 60 mg cm-3 bulk density; porosity &gt; 95%, Concordia Fibers, Coventry RI) as described in Ref. 4. </li>
 <li>Trim the distal 2 mm of a disposable 1,000 microliter pipet tip with scissors prepared with 70% ethanol in distilled water. </li>
 <li>Place the scaffold into a 4-well culture plate. Load the organoid units onto the scaffold with the 1,000 microliter pipet, first into the lumen and then onto the outer surface. Use a forceps to ensure coating of the lumen. Do not disrupt or break open the cylindrical shape of the polymer. </li>
</ol>
3. Implantation into Host Mouse
<ol>
 <li>Use a syngeneic host mouse on the same background as the donor if available. Otherwise, employ an immunocompromised nonobese diabetic / severe combined immunodeficient or NOD / SCID animal (Jackson Laboratories, Sacramento CA).</li>
 <li>Induce general anesthesia with isoflurane. Shave, prep and drape the mouse's abdomen. </li>
 <li>Make a 5 mm midline incision to gain entrance to the peritoneal cavity. Identify and carefully eviscerate the greater omentum. Place the loaded polymer onto the omentum and wrap it with the tissue. Do not tear the omentum.</li>
 <li>Secure the polymer to the omentum with a 5-0 monocryl suture. Gently replace the omentum with the wrapped polymer into its anatomic position.</li>
 <li>Close the abdominal incision in layers using 4-0 vicryl sutures. Run the muscle closure and take care not to injure the abdominal viscera below the incision. Use interrupted sutures for the skin.</li>
 <li>Administer postoperative analgesia with 2 mg/kg ketoprofen (Ketofen, Fort Dodge Animal Health) in sterile water as a subcutaneous wheal adjacent to the incision. Animals should be evaluated daily and if the animal is demonstrating signs of pain or distress, an additional dose of ketoprofen may be administered on post operative day 2. By the third postoperative day, the animal should be fully recovered without evidence of pain or distress. If pain or distress continues on post operative day 3, this is considered abnormal and should be addressed in accordance with the IACUC and animal care facility protocols. </li>
 <li>Allow the mouse to recuperate and the tissue-engineered intestine to grow for four weeks. Give the animal ad libitum access to rodent chow (Lab Diet 5001, PMI Nutrition, St. Louis MO) and water with Septra 200 mg / 40 mg per 5 ml (Hi-Tech Pharmacal, Amityville NY) at 1:100 dilution. </li>
</ol>
4. Harvest
<ol>
 <li>Humanely euthanize the host animal four weeks after implantation.</li>
 <li>Reopen the original incision and reflect the skin cephalad to facilitate access to the peritoneal cavity.</li>
 <li>Open the muscle layer and identify the tissue-engineered construct as a globe of tissue.</li>
 <li>Take down adhesions to the construct from intraabdominal viscera using sharp dissection.</li>
 <li>Fix the construct in formalin for later paraffin mounting, or use the tissue fresh for biochemical assays such as real-time PCR or protein isolation. </li>
</ol>

<ol><li>Grikscheit, T. C., Siddique, A., Ochoa, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-engineered+small+intestine+improves+recovery+after+massive+small+bowel+resection%5BTitle%5D)+AND+%22Ann.+Surg%22%5BJournal%5D)">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/pubmed?term=((Short+bowel+syndrome%3A+epidemiology+and+etiology%5BTitle%5D)+AND+%22Sem.+Ped.+Surg%22%5BJournal%5D)">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/pubmed?term=((The+development+of+a+method+for+the+preparation+of+rat+intestinal+epithelial+cell+primary+cultures%5BTitle%5D)+AND+%22J.+Cell+Sci%22%5BJournal%5D)">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/pubmed?term=((A+multicellular+approach+forms+a+significant+amount+of+tissue-engineered+small+intestine+in+the+mouse%5BTitle%5D)+AND+%22Tiss.+Eng.+Part+A%22%5BJournal%5D)">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/pubmed?term=((Murine+tissue-engineered+stomach+demonstrates+epithelial+differentiation%5BTitle%5D)+AND+%22J.+Surg.+Res%22%5BJournal%5D)">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/pubmed?term=((Intestinal+epithelial+cell+proliferation+is+dependent+on+the+site+of+massive+small+bowel+resection%5BTitle%5D)+AND+%22Pediatr.+Surg.+Int%22%5BJournal%5D)">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/pubmed?term=((A+novel+corrective+pullthrough+surgery+in+a+mouse+model+of+Hirschsprung%27s+disease%5BTitle%5D)+AND+%22J.+Pediatr.+Surg%22%5BJournal%5D)">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/pubmed?term=((Current+concepts+regarding+the+pathogenesis+of+necrotizing+enterocolitis%5BTitle%5D)+AND+%22Ped.+Surg.+Int%22%5BJournal%5D)">Current concepts regarding the pathogenesis of necrotizing enterocolitis.</a> Ped. Surg. Int. 25, 309-318 (2009).</li>
<li>Shew, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surgical+concerns+in+malrotation+and+midgut+volvulus%5BTitle%5D)+AND+%22Ped.+Radiol%22%5BJournal%5D)">Surgical concerns in malrotation and midgut volvulus.</a> Ped. Radiol. 39, S167-S171 (2009).</li>
<li>Sampietro, G. M., Corsi, F., Maconi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prospective+study+of+long-term+results+and+prognostic+factors+after+conservative+surgery+for+small+bowel+Crohn%27s+disease%5BTitle%5D)+AND+%22Clin.+Gastroenterol.+Hepatol%22%5BJournal%5D)">Prospective study of long-term results and prognostic factors after conservative surgery for small bowel Crohn's disease.</a> Clin. Gastroenterol. 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/pubmed?term=((Long-term+results+after+surgery+for+acute+mesenteric+ischemia%5BTitle%5D)+AND+%22Surgery%22%5BJournal%5D)">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/pubmed?term=((Relationship+between+biopsy-proven+parenteral+nutrition-associated+liver+fibrosis+and+biochemical+cholestasis+in+children+with+short+bowel+syndrome%5BTitle%5D)+AND+%22J.+Ped.+Surg%22%5BJournal%5D)">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. U., Kovacevich, D., McKinney-Barnett, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pediatric+short+bowel+syndrome%3A+the+cost+of+comprehensive+care%5BTitle%5D)+AND+%22Am.+J.+Clin.+Nutr%22%5BJournal%5D)">Pediatric short bowel syndrome: the cost of comprehensive care.</a> Am. J. Clin. Nutr. 88, 1552-1559 (2008).</li>
<li>Kato, T., Tzakis, A. G., Selvaggi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intestinal+and+multivisceral+transplantation+in+children%5BTitle%5D)+AND+%22Ann.+Surg%22%5BJournal%5D)">Intestinal and multivisceral transplantation in children.</a> Ann. Surg. 243, 756-766 (2006).</li>
<li>Reyes, J., Bueno, J., Kocoshis, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+status+of+intestinal+transplantation+in+children%5BTitle%5D)+AND+%22J.+Ped.+Surg%22%5BJournal%5D)">Current status of intestinal transplantation in children.</a> J. Ped. Surg. 33, 243-254 (1998).</li>
</ol>

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><tbody><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-&lt;em&gt;L&lt;/em&gt;-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 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...

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 ...

This video will demonstrate our technique for generating tissue engineered small intestine in the mouse using an organoid units on scaffolded approach. In this experiment, animals were treated in accordance with institutional animal care and use committee and the Children's Hospital, Los Angeles Savon Research Institute Animal Care Facility guidelines. The first step is the procurement of whole mouse intestine.The humanely euthanized mouse and instruments are prepped with 70%ethanol in water, and a midline incision is made to gain entry to the peritoneal cavity. The small bowel is eviscerated and divided just distal to the ligament of trites. It is then sharply and bluntly dissected away from the mesentery and its vessels.Finally, the ileum has transected five millimeters Proximal to the ileocecal valve. The harvested small intestine is shown to scale. Here we proceed to open The intestine lengthwise along the anti mesenteric border in a hank's buffered saline with one X antibiotic antifungal solution.The intestine is then cleared a fecal Matter in the dish, followed by three repeat washings with HBSS and a test tube. The intestinal tissue sinks to the bottom of the tube and a floating material is Mesenchymal debris and can be discarded. The washing solution is removed carefully with a pipette.The intestine is then minced to less than one millimeter square Pieces using scissors. The minced material is gathered up with an automatic pipette. The minced wash material is now ready for digestion.The digestion solution contains DASE 0.125 milligrams per milliliter and collagenase 800 units per milliliter in HBSS with antibiotic antifungal. Note the brownish color of the digestion solution compared to the HBSS with antibiotic antifungal. It is freshly prepared and 10 milliliters are added to the mince material.This is incubated at 37 degrees Celsius for 20 minutes on a rotator. The tissue is further disrupted by iteration with a pipette as shown here. This is repeated from 20 to 50 times until a uniform appearance is obtained.The 10 milliliter pipette tip is optimally Sized. For this, the reaction is stopped With three milliliters of four degree DMEM with 10%fetal bovine serum. The material is centrifuged for 10 minutes.At 500 RPM, the supernatant is discarded and the pellet is resuspended in a wash of 10 milliliters of four degree DMEM with 10%fetal bovine serum. This is then centrifuge for five minutes at 800 RPM to recover all tissue except for single cells which remain in the S supernatant and are discarded. The pellet contains the organoid units, the lighter material at the top, indicated by the forceps and waist material.Below this layer, the DMEM with serum is removed with a pipette. Care is taken not to suspend the pellet in the pipette. The last few drops can be removed more safely with the disposable plastic pipette.Shown here is the polyglycolic acid or PGA polymer scaffold used in our experiments. The distal two millimeters of a disposable 1000 microliter pipette tip are trimmed with sterile scissors. We load the polymer with organoid units in four wall culture plates to minimize spread of the material away from the polymer.The organoid units are loaded onto the scaffold with the pipette first into the lumen, and then On the outer surface. A second forceps is used to ensure Adequate coating of the lumen care is taken not to disrupt the cylindrical shape of the polymer. The host mouse used in the case of a transgenic donor is an immunocompromised non-obese diabetic.Severe combined immunodeficient gamma chain deficient or nod skid gamma animal. Otherwise, syngeneic hosts can be implanted for implantation. After general anesthesia is induced, the abdomen is shaved, prepped and draped.A 0.5 centimeter Midline incision is made. The greater Omentum is identified and carefully eviscerated. The loaded polymer is wrapped in the omentum and secured with a five oh Monocryl suture.It is critically important that the omentum is not torn, or the cells may leak into the Peritoneum. The Loaded polymer Secured the omentum is replaced in the abdomen, which is closed in layers. A four oh Vicryl suture is used in an interrupted fashion to close.First the muscle layer and then the skin for Postoperative analgesia, two milligrams per kilogram. Ketoprofen in sterile water is injected as a subcutaneous wheel adjacent to the incision four weeks after implantation, the host animal is humanely euthanized and the original incision is reopened. The tissue engineered construct is identified as a globe and may be adherent to the stomach and intraabdominal viscera.The adhesions are sharply taken down and a steroid of tissue engineered Intestine is retrieved. Shown here is the harvested Steroid in direct comparison to the starting polymer. In this image, the lumen of the engineered intestine has been opened sharply to reveal its interior.The tissue can be fixed for histologic analysis or used freshly for various molecular biologic assays, as we have demonstrated in other publications. Shown here is a low power hematin and eosin stain micrograph of a typical successful tissue engineered small intestine construct. The left arrow indicates the lumen lined with intestinal mucosa, and the right arrow shows host pancreas.That has adhered to The construct.

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Learn tissue engineering of the intestine using an organoid units-on-scaffold approach in mice. This protocol demonstrates isolation, digestion, and implantation techniques for regenerative medicine applications.

Tissue Engineering of the Intestine in a Murine Model

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