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/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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+concerns+in+malrotation+and+midgut+volvulus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+study+of+long-term+results+and+prognostic+factors+after+conservative+surgery+for+small+bowel+Crohn's+disease.">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/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. U., Kovacevich, D., McKinney-Barnett, 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=Pediatric+short+bowel+syndrome:+the+cost+of+comprehensive+care.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+and+multivisceral+transplantation+in+children.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+intestinal+transplantation+in+children.">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 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

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JoVE Journal

Bioengineering

14.8K visualizações.  Keck School of Medicine of the University of Southern California. Este artigo eo vídeo que acompanha apresentar o nosso protocolo para gerar o tecido de intestino no mouse, utilizando uma abordagem de unidades-em-andaime organóide.

Vídeo: Tissue Engineering of the Intestine in a Murine Model

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.