We would like to thank Dr. George Commons and Dr. Dean Vistnes for their support and collaboration of our research. This work is supported by National Institute of Dental and Craniofacial Research grants 1 R21 DE019274-01, R01EB009689 and RC2 DE020771- 02, the Oak Foundation and Hagey Laboratory for Pediatric Regenerative Medicine to M.T.L. Dr. Hyun is supported by the Saint Joseph Mercy Hospital GME.

1. Cell Isolation &amp; Expansion
<ol>
 <li>All patient consent and experimental protocols were reviewed and approved by Stanford University Institutional Review Board (Protocol #2188 and #9999).</li>
 <li>Obtain human subcutaneous adipose tissue from elective lipoaspiration procedures under local/general anesthesia.</li>
 <li>There will be two layers in the lipoaspirate (Figure 1A). The supernatant contains the vast majority of the processed cellular material. The bottom layer is mostly injected saline. Adipose-derived stromal cells can be harvested from either layer, but the yield is much greater from the supernatant.</li>
 <li>To isolate the stromal vascular fraction (SVF), wash the lipoaspirate extensively with equal volumes of 1X phosphate-buffered saline (PBS) containing 2.5% betadine (x2), followed by equal volume of 1X PBS (x1) without betadine. Allow the wash to precipitate.</li>
 <li>Be careful to maintain sterile technique throughout the process as contamination with processed human tissue can happen.</li>
 <li>Aspirate the bottom layer and discard after each wash.</li>
 <li>Aliquot 15 ml of adipose tissue into 50 ml BD Falcon conical tubes.</li>
 <li>Digest the adipose tissue with an equal volume (15 ml) of filtered 0.075% Type II collagenase in Hank's Balanced Salt Solution (HBSS) at 37 &deg;C in a water bath for 60 min under constant agitation (approx. 180 shakes/min) - ventilate each tube every 15 min.</li>
 <li>Neutralize enzyme activity with 15 ml PBS containing 10% (Fetal Bovine Serum) FBS, and centrifuge at 1,000 rpm for 5 min at 4 &deg;C to obtain a high-density SVF pellet. The pellet will be a mix of adipose-derived stromal cells and erythrocytes.</li>
 <li>Discard the supernatant without disrupting the pellet.</li>
 <li>Resuspend the pellet in 10 ml of traditional growth media (Dulbecco's Modified Eagle Medium [DMEM]/10% FBS/1% penicillin-streptomycin solution).</li>
 <li>Filter suspension through a 100 &mu;m nylon cell strainer to remove cellular debris.</li>
 <li>Combine 3 tubes into one clean 50 ml conical. Centrifuge at 1,000 rpm for 5 min at 4 &deg;C.</li>
 <li>Discard the supernatant without disrupting the pellet.</li>
 <li>Resuspend the pellet in 5 ml of traditional growth media</li>
 <li>Combine two 50 ml conicals per one 10 cm plate or five 50 ml conicals for one 15 cm plate.</li>
 <li>Establish primary cultures overnight at 37 &deg;C/21% O2, 5% CO2 .</li>
 <li>Following overnight incubation, wash the plates extensively with PBS to remove residual non-adherent red blood cells. The resulting cell populations are adipose-derived stromal cells.</li>
 <li>Maintain the cells at sub-confluent levels to prevent spontaneous differentiation at 37 &deg;C/21% O2, 5% CO2 in growth media. The cells generally need to be split every three to four days in regular growth media when plated at 50% confluence.</li>
</ol>
2. Scaffold Preparation
<ol>
 <li>Scaffolds were made by Dr. Min Lee at University of California, Los Angeles - School of Dentistry.</li>
 <li>PLGA scaffolds were fabricated from 85/15 poly(lactic-co-glycolic acid) (inherent viscosity = 0.61 dl/g, Birmingham Polymers) by solvent casting and a particulate leaching process.</li>
 <li>PLGA/chloroform solutions were mixed with 200-300 &mu;m diameter sucrose to obtain 92% porosity (volume fraction), and compressed into thin sheets in a Teflon mold.</li>
 <li>After freeze-drying overnight, scaffolds were immersed in three changes of double-distilled (dd) H2O to dissolve the sucrose, and gently removed from the Teflon plate with a fine-tip spatula.</li>
 <li>After particulate leaching, all scaffolds were disinfected by immersion in 50%, 60% and 70% ethanol for 30 min each, followed by three rinses of ddH2O.</li>
 <li>All scaffolds were dried under a laminar flow hood.</li>
 <li>After scaffold fabrication, scaffolds were coated with apatite.
 <ol class="ualpha">
 <li>SBF (simulated body fluid) solution was prepared with ion concentrations that were 5 times that of human blood plasma. All solutions were sterile filtered through a 0.22 &mu;m PES membrane (Nalgene). Immediately before the coating process, dried PLGA scaffolds were subjected to glow discharge, argon-plasma etching (Harrick Scientific) to improve wetting and coating uniformity.</li>
 <li>Etched PLGA scaffolds were then incubated in SBF at 37 &deg;C inside a water-jacketed incubator for 12 hr, followed by Mg2+and HCO3--free SBF 2 for another 12&thinsp;hr at 37 &deg;C under gentle stirring.</li>
 <li>Coated PLGA scaffolds were rinsed gently with sterile ddH2O to wash away excess sodium chloride solution, dried in a laminar flow hood and disinfected with 70% ethanol.</li>
 <li>Integrity of the apatite coating was analyzed with a scanning electron microscope (SEM). Apatite-coated scaffolds were mounted on SEM stubs (Ted Pella) and coated with carbon to improve conductivity. The secondary electron mode was applied during SEM (FEI/Phillips XL-30) observation. Energy dispersion X-ray spectrum was obtained to confirm elemental composition of the apatite structures.</li>
 </ol>
 </li>
</ol>
3. Cell Seeding
<ol>
 <li>Upon reaching sub-confluence levels, wash the cells with PBS (x2) and trypsinize.</li>
 <li>Count the cells to quantify for seeding</li>
 <li>Seed onto pre-cut 4.0 mm diameter scaffolds with 1.5 x 105 cells.
 <ol class="ualpha">
 <li>Place individual scaffold into a well of a 96-well plate</li>
 <li>Re-suspend cells in regular growth media with a concentration of roughly 1.5 x 105 cells per 20 &mu;l of media</li>
 <li>Pipet 20 &mu;l directly onto the scaffold and place into cell culture incubator for 30 min.</li>
 <li>After 30 min, add 200 &mu;l of media and allow cells to incubate on the scaffold overnight prior to surgery</li>
 <li>It is not necessary to ensure adhesion of the cells onto the scaffold as seeding a sufficient number of cells will ensure that a majority will attach onto the scaffold. This has been validated in our lab in vivo with bioluminescence and histological analysis.</li>
 </ol>
 </li>
</ol>
4. Creation of Calvarial Defects and In Vivo Implantation
<ol>
 <li>Anesthetize adult (60 day-old) CD-1 nude mice with anesthetic cocktail. (ketamine/xylazine/acepromazine) at 50% concentration (1:1 dilution with 0.9% NaCl) or per recommended anesthetic regimen per institution protocols. These mice are athymic so the hASCs will not cause an immune reaction.
 <ol class="lalpha">
 <li>Dosage: Ketamine hydrochloride (80 mg/kg), Xylazine (2.5 mg/kg) Acepromazine (2.5 mg/kg)</li>
 <li>Duration of effect: approximately 30 min</li>
 </ol>
 </li>
 <li>Surgically drape the animal and sterilize the surgical site with betadine and alcohol (x3) and cover the mouse eyes with ointment prior to making a midline sagittal incision in the scalp of the animal to expose the right parietal bone. Remove the pericranium from the right parietal bone with blunt scraping (Figure 1B).</li>
 <li>Create a unilateral 4-mm full-thickness defect in the right non-suture associated parietal bone using a sterile diamond-coated trephine drill bit. Extreme caution must be taken not to disturb the underlying dura mater (Figure 1B) as the mouse calvarial thickness is &lt;.3 mm.</li>
 <li>Before implantation, rinse scaffolds with sterile PBS to prevent transfer of medium-derived growth factors.</li>
 <li>Place the scaffold into the defect.</li>
 <li>Finally, suture the skin closed and monitor the animal per established postoperative protocols.</li>
 <li>Use established analgesia as directed by your animal care protocols as needed post-operatively- e.g. buprenorphine 0.1 mg/kg.</li>
</ol>
5. Quantification of Osteoid Formation
<ol>
 <li>After MicroCT was performed on the mouse, the three-dimensional image was reconstructed using MicroView software as previously described1.</li>
 <li>Using Adobe Photoshop, the images were sized to a standard height.</li>
 <li>Using the Magic Wand feature, pixels were measured of the calvarial defect.</li>
 <li>Percentage healing of the defect was determined by subsequent CT scans measuring the calvarial defect area and determining pixel number and dividing it by the original defect's pixel number</li>
 <li>Consequently, if the MicroCT is unavailable, an alternative is using Adobe Photoshop and harvesting the calvaria at determined time points for histology.</li>
 <li>Using histological stains like Aniline Blue and Pentachrome that denote osteoid formation, multiple sections along the span of the defect should be stained</li>
 <li>Using Adobe Photoshop, crop out all the areas that are not in the calvarial defect</li>
 <li>Use the magic wand feature and determine pixel numbers of de novo bone formation in the area of the defect and compare to controls or other variables.</li>
</ol>
6. Representative Results
Adipose tissue has the potential to serve a vital role in the generation of progenitor cells for clinical application. Adipose tissue has a unique advantage in that there is a readily available supply that can be harvested in a relatively simple procedure that involves minimal morbidity and mortality. Once the tissue is harvested and collected, our protocol is outlined in Figure 2. Adipose-derived stromal cells are isolated through a series of washing steps, collagenase digestion, and centrifugation. Once the cells are plated in culture, they can be expanded, placed into various differentiation protocols in vitro, or placed directly in vivo.
The creation of the 4 mm critical-size calvarial defect provides a easily accessible and reproducible in vivo model to test the osteogenic differentiation ability of our adipose-derived stromal cells. Through the use of MicroCT, we are able to follow the formation of skeletal tissue in vivo and track the progress of our interventions (Figure 3). The critical-size calvarial defect will not heal within the lifetime of the animal and we see roughly 90% of the defect remain patent around 8 weeks. The scaffold itself (see discussion) has osteogenic inductive properties and has shown the ability to induce some bony regeneration. The typical results of scaffold placement without cells shows that around a one-third of the defect will have de novo bone formation at 8 weeks. With the augmentation of adipose-derived stromal cells, fully two-thirds or more of the defect will show osseous regeneration at around 8 weeks although there is variability between each animal and surgery. The formation of skeletal tissue can be quantified through histology and typical results show increased osteoid formation in samples with ASCs through Aniline Blue and Pentachrome staining (Figure 3C). In addition, we show that the implanted human cells contribute to the underlying de novo osseous formation through the use of GFP-labeled hASCs that show staining in vivo at 2 weeks near the area of de novo bone formation in the calvarial defect (Figure 3D). In addition, we use immunohistochemistry with human specific antibody to show human cell survival and contribution in the area of the defect (Figure 3E).
<img alt="Figure 1" src="/files/ftp_upload/4221/4221fig1.jpg" /> 
 Figure 1. A - The lipoaspirate has two layers. The top layer contains the adipocytes and the majority of the processes cellular material while the bottom layer contains the saline used during the lipoaspiration procedure. B - the creation of the calvarial defect through a midline incision over the pericranium to isolate the right parietal bone, subsequent creation of a 4 mm critical sized calvarial defect without disruption of the underlying dura mater.
<img alt="Figure 2" src="/files/ftp_upload/4221/4221fig2.jpg" /> 
 Figure 2. Overview of the harvesting and application of lipoaspirate from the isolation of adipose-derived stromal cells to their expansion, differentiation and use in vitro and in vivo.
<img alt="Figure 3" src="/files/ftp_upload/4221/4221fig3.jpg" /> 
 Figure 3. A -MicroCT showing in vivo healing of the critical size calvarial defect with the application of ASCs through a hydroxyapatitie scaffold (Bottom Row). Controls include no scaffold and defect (Top row) and defect with placement of the scaffold without cells (Middle Row) B - Quantification of osseous healing from the MicroCT showing significantly increased healing in the ASC group. C - Histology showing increased osteoid formation of the ASC group (bottom row) through Aniline Blue and Pentachrome staining. For Aniline Blue, the osteoid stains dark blue and for Pentachrome, the osteoid stains yellow. D - hASCS labeled with GFP were seeded onto a scaffold and placed into a calvarial defect and sacrificed at 2 weeks. Staining was done with a GFP labeled antibody to show the human cells contributing to regeneration in the area of the defect. E - Human nuclear antigen immunohistochemistry showing prevalence of human cells in the area of the defect at 2 weeks. <a href="https://www.jove.com/files/ftp_upload/4221/4221fig3large.jpg" target="_blank"> Click here to view larger figure</a>.

<ol>
	<li>Levi, B., James, A. W., Nelson, 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=Human+adipose-derived+stromal+cells+heal+critical+size+mouse+calvarial+defect.">Human adipose-derived stromal cells heal critical size mouse calvarial defect.</a> PLoS One. 5, (2010).</li><li>Zuk, P. A., Zhu, M., Ashjia, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adipose+tissue+is+a+source+of+multipotent+stem+cells.">Human adipose tissue is a source of multipotent stem cells.</a> Mol. Biol. Cell. 13, 4279-4295 (2002).</li><li>Keefe, M. S., Keefe, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+the+effectiveness+of+different+techniques+for+intraoperative+antibiotics+into+alloplastic+implants+for+use+in+facial+reconstruction.">An evaluation of the effectiveness of different techniques for intraoperative antibiotics into alloplastic implants for use in facial reconstruction.</a> Arch Facial Plastic Surg. 11, 246-251 (2009).</li><li>Mitchell, J. B., McIntosh, K., Zvonic, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunophenotype+of+human+adipose-derived+cells:+Temporal+changes+in+stromal-associated+and+stem+cell-associated+markers.">Immunophenotype of human adipose-derived cells: Temporal changes in stromal-associated and stem cell-associated markers.</a> Stem Cells. 24, 376-385 (2006).</li><li>Dominici, M., Blanc, K. L. e., Mueller, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+criteria+for+defining+multipotent+mesenchymal+stroma+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stroma cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8, 315-317 (2006).</li><li>Cowan, C. M., Shi, Y. Y., Aalami, O. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+adult+stromal+cells+heal+critical-size+calvarial+defects.">Adipose-derived adult stromal cells heal critical-size calvarial defects.</a> Nat Biotechnol. 22, 560-567 (2004).</li><li>Levi, B., Nelson, E. R., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dura+mater+stimulates+human+adipose-derived+stromal+cells+to+undergo+bone+formation+in+mouse+calvarial+defects.">Dura mater stimulates human adipose-derived stromal cells to undergo bone formation in mouse calvarial defects.</a> Stem Cells. 29, 1241-1255 (2011).</li><li>Phipps, M. C., Clem, W. C., Catledge, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+responses+to+bone-mimetic+electrospun+matrices+composed+of+polycaprolactone,+collagen+I+and+nanoparticulate+hydroxyapatite.">Mesenchymal stem cells responses to bone-mimetic electrospun matrices composed of polycaprolactone, collagen I and nanoparticulate hydroxyapatite.</a> PLoS One. 6, (2011).</li><li>Yuan, H., Zang, Z., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoinduction+by+calcium+phosphate+biomaterials.">Osteoinduction by calcium phosphate biomaterials.</a> J. Mater. Sci. Mater. Med. 9, 723-726 (1998).</li><li>Wei, G., Jun, Q., Giannobile, W. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enchancement+of+osteogenesis+by+nano-fibrous+scaffolds+incorporating+rhBMP-7+nanospheres.">The enchancement of osteogenesis by nano-fibrous scaffolds incorporating rhBMP-7 nanospheres.</a> Biomaterials. 28, 2087-2096 (2007).</li><li>Li, C., Verpari, C., Jin, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+silk-BMP-2+scaffolds+for+bone+tissue+engineering.">Electrospun silk-BMP-2 scaffolds for bone tissue engineering.</a> Biomaterials. 27, 3115-3124 (2006).</li><li>Zhang, Y., Fan, W., Nothdurft, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+evaluation+of+adenovirus+combined+silk+fibroin+scaffolds+for+bone+morphogenetic+protein-7+gene+delivery.">In vitro and in vivo evaluation of adenovirus combined silk fibroin scaffolds for bone morphogenetic protein-7 gene delivery.</a> Tissue Eng Part C Methods. 17, 789-797 (2011).</li><li>Levi, B., Hyun, J. S., Nelson, 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=Non-integrating+knockdown+and+customized+scaffold+design+enhances+human-adipose-derived+stem+cells+in+skeletal+repair.">Non-integrating knockdown and customized scaffold design enhances human-adipose-derived stem cells in skeletal repair.</a> Stem Cells. 29, 21028-21029 (2011).</li></ol>

No conflicts of interest declared.

Since the isolation of adipose-derived stromal cells2 from lipoaspirate, these cells have been differentiated into a wide variety of cellular lineages. Adipose tissue is from mesodermal origins and therefore, multipotent adipose-derived stromal cells will likely be most effective with application towards a mesodermal lineage. The ability to generate skeletal tissue is especially critical given the shortage of donor sites for an autograft and the inherent limitations of synthetic material including infection, r...

<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></td>
 <td></td>
 <td></td>
 <td>Lipoaspirate Harvest</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Gibco</td>
 <td>10010-023</td>
 <td></td>
 </tr>
 <tr>
 <td>Hank's Balanced Salt Solution</td>
 <td>Cellgro</td>
 <td>21-023-CV</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase</td>
 <td>Sigma</td>
 <td>C6885-500MG</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Strainer 100 &mu;m </td>
 <td>BD Falcon</td>
 <td>352360</td>
 <td></td>
 </tr>
 <tr>
 <td>Steri-top 500 ml .22 &mu;m filter </td>
 <td>Millipore</td>
 <td>SCGPT05RE</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Calvarial Defect</td>
 </tr>
 <tr>
 <td>Z500 Brushless MicromotorsUM50C </td>
 <td>NSK </td>
 <td>NSKZ500</td>
 <td></td>
 </tr>
 <tr>
 <td>Circular Knife 4.0 mm </td>
 <td>Xemax Surgical </td>
 <td>CK40</td>
 <td></td>
 </tr>
 </tbody>
</table>

repair of a critical-sized calvarial defect model using adipose-derived stromal cells harvested from lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

Watch this Scientific Journal Video about Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate at JoVE.com

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

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15.8K Views.  Stanford University. This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

Video: Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens4. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice 5-7, after application of bioactive molecules 8,9, after altering physiological parameters of healing 10, after modifying the amount or time of stabilization 11, after distraction osteogenesis 12, after creation of a non-union 13, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.

This article describes a method for stabilizing long bone fractures that is based on the application of modified Ilizarov external fixators 1-3. After application of the fixators and creation of the bone injury, healing can be assessed, distraction osteogenesis can be performed, or non-union or critical sized defect can be created and used to study therapeutic interventions.

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem cells can be obtained from different tissues and several methods for isolation have been described. The presented method for the isolation of adipose-derived regenerative cells (ADRCs) can be used within many therapeutic areas because the method is a general procedure and, therefore, not limited to erectile dysfunction (ED) therapy. ED is a common and serious side effect to radical prostatectomy (RP) since ED often is not well treated with conventional therapy. Using ADRC&#8217;s as treatment for ED has attracted great interest due to the initial positive results after a single injection of cells into the corpora cavernosum. The method used for the isolation of ADRC&#8217;s is a simple, automated process, that is reproducible and ensures a uniform product. Furthermore, the sterility of the isolated product is ensured because the entire process takes place in a closed system. It is important to minimize the risk of contamination and infection since the stem cells are used for injection in humans. The whole procedure can be done within 2.5-3.5 hours and does not require a classified laboratory which eliminates the need for shipping tissue to an off-site. However, the procedure has some limitations since the minimum amount of drained lipoaspirate for the isolation device to function is 100 g.

Precise disclosure of methods and protocols is crucial for large scale uptake of stem cell therapies. Here, we present a protocol to isolate adipose-derived regenerative cells, used for a single intracavernous injection as treatment of erectile dysfunction (ED) following radical prostatectomy (RP).

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...

Mechanical Stromal Vascular Fraction in Fibrin Hydrogel for Regenerative Medicine

The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the past, the extraction of these cells from adipose tissue required a multistep enzyme-based process, resulting in a heterogenous cell mix consisting of ACSs and other cells, which are jointly termed the stromal vascular fraction (SVF). More recently introduced mechanical SVF (mSVF) isolation protocols are less time-consuming and bypass regulatory concerns. We recently proposed a protocol that generates mSVF rich in stromal cells based on a combination of emulsification and centrifugation. One current issue in mSVF application for wound therapy application is the lack of a scaffold providing protection from mechanical manipulation and desiccation. Fibrin hydrogels have been shown to be a useful adjunct in cell transfer for wound healing purposes in the past. In the work herein, we delineate the preparation steps of an mSVF-fibrin hydrogel construct as a novel approach for translational research and clinical application.

Author Spotlight: Exploring the Potential of Fat-Derived Stromal Vascular Fraction for Wound Healing

Mechanically isolated stromal vascular fraction (SVF) in combination with a fibrin hydrogel offers an easy and efficient carrier for viable adipose-derived stromal cells for various indications, including tissue engineering and or wound healing purposes. Here, we present the preparation of a mechanical SVF (mSVF)-fibrin hydrogel construct for translational research and clinical application.

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the ...