This study was supported and funded by the Avenir Foundation (Fondation de l'Avenir, 255 rue de Vaugirard, 75719 Paris cedex 15, Paris, France). This study would never have been conducted without the precious help of the veterinary team of the Laboratory of Biosurgical Research from the Alain Carpentier Foundation.

All animals were treated according to the Animal Research Ethics Committee (guidelines of the French Ministry of Agriculture). The protocol received the approval of the local ethics committee authorized for animal experimentations at the Paris Descartes University (registered number MESR 2035.02; Faculty of Medicine Paris Descartes, Paris, France).
1. ADSC Culture and Labeling
<ol>
	<li>Obtain confirmed ADSCs from a private institution. Culture allogenic ADSCs at 37 &#176;C and 5% CO2 with alpha minimum essential medium including 10% fetal veal serum and 1% antibiotics (penicillin and streptomycin).</li>
	<li>Obtain around 24 x 106 ADSCs per animal using standard cell expansion procedures (37 &#176;C and 5% CO2). Harvest the ADSCs using a standard trypsin solution (10 mL for a T150 dish). Perform cell counting with a hemocytometer.</li>
	<li>Perform cell labeling the day before transplantation using the PKH67 staining procedure described below. 
	NOTE: The goal of the staining procedure is to allow cell tracking after transplantation.
	<ol>
		<li>In order to label 2 x 106 ADSCs, prepare Solution A (2 x 106 cells with 100 &#181;L of Diluent C) and Solution B (4 &#181;L of PKH67 with 1 mL of Diluent C).</li>
		<li>Incubate Solutions A and B for 2 min. Then, add 100 &#181;L of fetal veal serum for 1 min.</li>
		<li>Rinse the solution with 300 &#181;L of citrated RPMI (298.5 &#181;L of RPMI and 1.5 &#181;L of citrate) and centrifuge at 1,500 x g for 5 min. Repeat this operation 2 times. Maintain the labeled cells and protect them from light until sheet construction.</li>
	</ol>
	</li>
</ol>
2. Double ADSC-sheet Construct
<ol>
	<li>The day before transplantation, prepare a 12-well temperature-responsive cell culture dish with 4 mL per well of the cell culture medium, described above, before PKH-labeling. Seed the dish with 1.5 x 106 PKH-labeled cells per well and incubate for 12 h (37 &#176;C and 5% CO2).</li>
	<li>On the day of transplantation, detach the confluent cell sheet by incubating the dish at room temperature for 30 min.</li>
	<li>Gently aspirate one ADSC sheet with a pipette and layer it on a hydrophobic paper (1.5-cm diameter). Use this paper as a support membrane.</li>
	<li>Grasp another ADSC sheet and gently deposit it on top of the other one to obtain a double-layer construct.</li>
	<li>Repeat these procedures to obtain as many ADSC-sheet constructs as needed (4 per animal).</li>
</ol>
3. Esophageal Endoscopic Submucosal Dissection (ESD)
<ol>
	<li>On the day of transplantation, pre-medicate the animals with 10 mg/kg of intramuscular ketamine and induce them with 8 mg/kg of intravenous propofol. Then, perform endotracheal intubation and maintain the anesthesia with isoflurane 2.5% inhalation.</li>
	<li>Once the animal is under general anesthesia, perform ESD with a gastroscope, a videoscope, and an electrosurgery unit.
	<ol>
		<li>Use an endoscopic knife and soft coagulation to obtain hemi-circumferential dorsal marks ranging from 40 cm to 45 cm from the dental arch.</li>
		<li>Inject a glycerol solution containing indigo carmine dye into the submucosal layer for the separation of the mucosal layer from the muscular layer.</li>
		<li>Use an endoscopic knife with endocut I mode to obtain circumferential incisions.</li>
		<li>Use an endoscopic knife with forced coagulation mode to perform the submucosal dissection, moving from the proximal incision to the distal incision.</li>
	</ol>
	</li>
	<li>At the end of the ESD procedure, observe the hemi-circumferential, 5 cm-long esophageal scar exposing the muscular layer.</li>
</ol>
4. Endoscopic Transplantation
<ol>
	<li>Immediately after the ESD, endoscopically transplant animals with 4 double ADSC-sheet constructs.
	<ol>
		<li>Grasp an ADSC-sheet construct with endoscopic forceps and protect it during transportation to the wound site by using a large, transparent endoscopic cap.</li>
		<li>Apply the entire surface of the ADSC-sheet construct to the ulcer bed.</li>
		<li>Gently apply the ADSC-sheet construct for 10 min to obtain a complete and stable engraftment. For application, use endoscopic forceps or an endoscopic cap and apply the whole surface of the ADSC construct onto the esophageal submucosal layer.</li>
	</ol>
	</li>
	<li>Repeat this procedure four times for each animal in order to obtain four transplanted and adherent double cell-sheet constructs. 
	NOTE: The goal is to cover approximatively half of the wounded area.</li>
</ol>
5. Postoperative Evaluation and Follow-up
<ol>
	<li>For each animal, ensure post-ESD analgesia with the intramuscular injection of 0.2 mg/kg of morphine three times a day for the first day and with a 28-day anti-acid treatment of esomeprazole (40 mg/day). Perform antibiotic prophylaxis with amoxicillin (1 g/day) for 7 days. Authorize liquids on day 1 and solids on the following day.</li>
	<li>Follow up with animals for 28 days. Obtain daily clinical evaluations using Mellow Pinkas dysphagia scores16 and weight variation measurements.</li>
	<li>Multimodal stricture evaluation: under general anesthesia, on scheduled days 3, 14, and 28, perform a multimodal evaluation of stricture occurrence.
	<ol>
		<li>Perform an endoscopic evaluation using scar description, stricture measurement, and paper support membrane detection. Measure the stricture using the gastroscope to cross through the stricture (the diameter of the gastroscope is 8 mm). Observe the scar, paying attention to the inflammatory aspect (mucosal erythema, mucosal edema, and mucosal spontaneous bleeding) and the absence or presence of the paper support membrane within the scar.</li>
		<li>Apply pCLE green probe through the gastroscope operator canal directly into the scar bed. Search for a spontaneous and organized green signal compatible with PKH67-labeled ADSC-sheet construct engraftment.</li>
		<li>Perform 2 orthogonal incidences of baryte esophagography with a radiological hoop (front and left-sided incidences). Keep the tightest incidence for stricture evaluation (degree of stricture (%) = [1-(length of the short axis under stenosis/length of the normal axis under stenosis) x 100)])17,18.</li>
	</ol>
	</li>
	<li>Animal sacrifice.
	<ol>
		<li>On day 28, at the end of the follow-up period, perform the animal sacrifice. While the animal is still under general anesthesia, inject 100 mg/kg of intravenous phenobarbital. Ensure animal death using clinical examination for the absence of a heartbeat and of breathing movement.</li>
		<li>After a sternotomy and organ exposure, remove the esophagus from each sacrificed animal (with 10% buffered formalin fixation, paraffin embedding, and 4 &#181;m-thick sections). Stain the slides with Hematoxylin and Saffron15. Digitalize the slides for computerized analysis and compare the animal groups15.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

In this pig model, the ADSC-sheet transplantation was technically successful, and the in vivo pCLE evaluation allowed for cell engraftment monitoring. Clinical, endoscopical, radiological, and histological evaluations demonstrated the effectiveness of the endoscopic ADSC sheet at esophageal stricture prevention after extended ESD.
The endoscopic transplantation of an ADSC glycerol solution containing an indigo carmine dye sheet is an innovative approach in regenerative medicine. Ohki et al. first described the endoscopic transplantation procedure. In their studies, cell-sheet constructs were composed of oral mucosal epithelial cell sheets layered on a thin sheet of polyvinylidene difluoride, used as a support membrane10,11,19. The support membrane was grasped with endoscopic forceps and carefully placed on the ulcer bed. Cell-sheet adherence was obtained after 10 min of gentle application. This procedure has not yet been reproduced. In this study, double ADSC-sheet constructs were transplanted for multiple reasons. The choice of ADSC was based on several studies that demonstrated their anti-inflammatory abilities, especially in the skin-healing process. ADSCs are known to modulate keratinocyte and fibroblast interactions7. The mechanism of fibrosis development is complex and only partially understood. The paracrine effect seems to play a major role in ADSC action, including anti-inflammatory factor production, such as TGF&#946; and pro-angiogenic factors20.
Endoscopic ADSC-sheet transplantation has several limitations. First, cell sheets were applied for 10 min to the esophageal scar to obtain a stable adhesion. This part of the procedure was challenging, technically difficult, and had to be performed by an experienced endoscopist. Nevertheless, all animals were successfully transplanted, confirming the feasibility of the endoscopic procedure. Recently, the use of a 3D-printed device to improve the success of cell-sheet transplantation was reported21. Second, this technique is associated with an unknown survival rate. An esophageal scar is an aggressive environment for cells. A temperature-responsive dish allows for the production of the cell-sheet construct through the extracellular matrix and for the formation of cell connections. Thus, to improve the survival rate and the ADSC interactions, a double ADSC-sheet construct was chosen22,23. This study was not designed to assess either the cell survival rate or the dose effect of transplantation on esophageal stricture prevention, which are both important questions. Third, as in Ohki et al., the use of a support membrane was necessary to carry the ADSC-sheet construct. After transplantation, the paper support membrane could not be removed without construct destruction. This intra-esophageal foreign material was responsible for the increased inflammation. An optimized technique would allow for the removal of the paper support membrane.
Transplantation monitoring with pCLE was also challenging. This new endoscopic imaging technique allows in vivo histological analysis, including visualization of the glandular pattern, micro-vascularization, and cellular alteration. pCLE was evaluated in many digestive diseases, such as inflammatory bowel disease, Barrett&#39;s esophagus, and mucinous pancreas cysts24,25. The pCLE-compatible PKH67 fluorophore was used for cell labeling. A fluorescent signal compatible with the presence of the ADSC sheet was observed in 4 animals on day 3. This positive result reflected the success of the ADSC-sheet transplantation. However, because PKH67 has a decreased signal over time and after cell division, pCLE evaluation was possible only a few days after transplantation26. Ohki et al. and Kanai et al. monitored cell sheet transplantation through immunostaining, and they confirmed the presence of cells until day 8 after transplantation. Mature cells were used in this study, which have the advantage of specific immunostaining.
Esophageal stricture is one of the most serious adverse events following ESD, thereby limiting technique development. Optimal techniques to prevent stricture formation are still lacking. In clinical practice, patients with extended esophageal ESD are prescribed different preventive treatments, such as corticosteroids, endoscopic balloon dilatations, or esophageal stenting27. Local application of biomaterial has been conducted, with varying results28,29. The aim of regenerative medicine is to reconstruct a tissue or organ using various approaches, such as tissue replacement or local immunomodulation. For tissue replacement, the use of biological scaffolds with or without epithelial cells was evaluated in the literature30,31. A precocious and increased re-epithelialization was observed in the esophageal scar. For local immunomodulation, little data are available in the literature about ADSC injection or amniotic membrane application32,33. A delayed stricture formation and a decreased stricture rate were observed.
In this pig model, the endoscopic cell-sheet transplantation procedure was successfully performed. Transplanted ADSCs organized in double cell sheets demonstrated their ability to prevent esophageal stricture formation after extended ESD. Mechanisms involved in the ADSC healing process are poorly understood and must be investigated further in future studies. Thus, in view of these promising results, a clinical trial should be conducted to assess the effectiveness of the ADSC-sheet construct after extended ESD in patients.

The management of superficial esophageal tumors has changed with the development of new endoscopic techniques. Nowadays, endoscopic resection is the first-line treatment. Indeed, it is associated with lower morbidity and mortality rates than a surgery with equal oncological results1,2,3. Endoscopic mucosal resection (EMR) and endoscopic submucosal resection (ESD) are the most widely-used techniques. In the case of an extended superficial tumor, ESD is preferred. Compared to EMR, ESD allows en bloc resectioning, regardless of lesion size and shape4,5,6. The main delayed complication of ESD is esophageal stricture formation, which generally occurs between one and two weeks after resection. Recent published studies have shown that stricture formation is correlated to the size of the resection. The Japanese Endoscopic Society recommends avoiding ESD sizes larger than &#190; of the esophageal circumference because they are associated with stricture development in more than 90% of cases and are responsible for severe feeding troubles and major deterioration in quality of life.
The prevention of esophageal stricture remains challenging. Mechanisms involved in stricture formation are only partially known. Stricture formations seems to result from the association of two different mechanisms: (1) pro-inflammatory cellular recruitment and (2) excessive fibrosis development7. Several preventive treatments have been proposed. However, results were unsatisfactory, with little benefit and severe side effects8,9. Recently, a Japanese team, Ohki et al., proposed to transplant a single-layer cell sheet of autologous oral mucosal cells into the esophageal scar. Transplantation was performed immediately after ESD10,11. They demonstrated the effectiveness of this innovative approach, first in a canine model and then in patients.
Adipose tissue-derived stromal cells (ADSCs) are promising in regenerative medicine. Their application in several fields has shown interesting results, especially in the wound-healing process. ADSC therapy offers several advantages, because the cells are easily isolated and are associated with anti-inflammatory properties, local immunomodulating effects, neovascularization induction, and differentiation abilities into mesenchymal and non-mesenchymal lineages12,13,14.
In a previous study, our team demonstrated the effectiveness of double ADSC-sheet endoscopic transplantation for esophageal stricture prevention after extended ESD in a swine model15. In this article, reports of ADSC-sheet construction and endoscopical transplantation technique are presented.

<table><tbody><tr><td>Transparent endoscopic cap Q180 compatible</td><td>Olympus Optical Co</td><td></td><td></td></tr><tr><td>GIF-Q180 gastroscope&amp;nbsp;</td><td>Olympus Optical Co</td><td></td><td></td></tr><tr><td>Videoscope System Exera II&amp;nbsp;</td><td>Olympus Optical Co</td><td></td><td></td></tr><tr><td>Injection needle 18 G</td><td>Olympus Optical Co</td><td></td><td></td></tr><tr><td>Electrosurgery unit ERBE ICC 350&amp;nbsp;</td><td>ERBE Technology</td><td></td><td></td></tr><tr><td>Indigo carmin 1%</td><td>Life</td><td></td><td></td></tr><tr><td>Endoscopic hybrid knife</td><td>Life</td><td></td><td></td></tr><tr><td>Minisonde Z pCLE green probe</td><td>Mauna Kea Technology</td><td></td><td>You must learn how to use the probe. The manipulation could be difficult.</td></tr><tr><td>Fetal bovine serum</td><td>Sigma Aldrich</td><td>12105C</td><td></td></tr><tr><td>Trypsin</td><td>Sigma Aldrich</td><td>T146</td><td></td></tr><tr><td>Alpha minimum essential medium</td><td>Thermo Fisher</td><td>22561-021</td><td></td></tr><tr><td>Phosphate-Buffered Salines</td><td>Thermo Fisher</td><td>10010-023</td><td></td></tr><tr><td>PKH67 dye kit</td><td>Sigma Aldrich</td><td>Mini67-KT</td><td></td></tr><tr><td>12-well temperature responsive cell culture dish</td><td>Upcell Thermo Scientific</td><td>174900</td><td>Feel the weel with 4 mL standard medium culture 30 min before seeding cells</td></tr><tr><td>Esomeprazole 40 mg&amp;nbsp;</td><td>Biogaran</td><td></td><td></td></tr><tr><td>Moprhine sulfate 50 mg/mL</td><td>Lavoisier</td><td></td><td></td></tr><tr><td>Amoxicilline 1 g</td><td>Biogaran</td><td></td><td></td></tr><tr><td>Ketamine 250 mg/5 mL</td><td>Panpharma</td><td></td><td></td></tr><tr><td>Propofol 10 mg/mL</td><td>Fresenius</td><td></td><td></td></tr><tr><td>Hydrophobic paper</td><td>Carrefour</td><td></td><td></td></tr></tbody></table>

adsc-sheet transplantation to prevent stricture after extended esophageal endoscopic submucosal dissection

In past years, the cell-sheet construct has spurred wide interest in regenerative medicine, especially for reconstructive surgery procedures. The development of diversified technologies combining adipose tissue-derived stromal cells (ADSCs) with various biomaterials has led to the construction of numerous types of tissue-engineered substitutes, such as bone, cartilage, and adipose tissues from rodent, porcine, or human ADSCs. Extended esophageal endoscopic submucosal dissection (ESD) is responsible for esophageal stricture formation. Stricture prevention remains challenging, with no efficient treatments available. Previous studies reported the effectiveness of mucosal cell-sheet transplantation in a canine model and in humans. ADSCs are attributed anti-inflammatory properties, local immune modulating effects, neovascularization induction, and differentiation abilities into mesenchymal and non-mesenchymal lineages. This original study describes the endoscopic transplantation of an ADSC tissue-engineered construct to prevent esophageal stricture in a swine model. The ADSC construct was composed of two allogenic ADSC sheets layered upon each other on a paper support membrane. The ADSCs were labeled with the PKH67 fluorophore to allow probe-based confocal laser endomicroscopy (pCLE) monitoring. On the day of transplantation, a 5-cm and hemi-circumferential ESD known to induce esophageal stricture was performed. Animals were immediately endoscopically transplanted with 4 ADSC constructs. The complete adhesion of the ADSC constructs was obtained after 10 min of gentle application. Animals were sacrificed on day 28. All animals were successfully transplanted. Transplantation was confirmed on day 3 with a positive pCLE evaluation. Compared to transplanted animals, control animals developed severe strictures, with major fibrotic tissue development, more frequent alimentary trouble, and reduced weight gain. In our model, the transplantation of allogenic ADSCs, organized in double cell sheets, after extended ESD was successful and strongly associated with a lower esophageal stricture rate.

The culture of ADSCs and the procedure to obtain the ADSC sheet is shown in Figure 1. Figure 2 shows the construction of the graft, composed of two ADSC sheets layered upon each other on their paper support membrane. ADSCs were previously labeled with the PKH67 fluorophore to allow in vivo graft monitoring with pCLE. Figure 3 shows the different steps of extended esophageal endoscopic submucosal dissection, resulting in a 5-cm and hemi-circumferential esophageal scar. Figure 4 shows the endoscopic transplantation procedure. The transplantation was successful for all animals after 10 min of gentle application. Figure 5 shows the positive pCLE evaluation on day 3, confirming the success of the transplantation procedure.
The follow-up period allowed for multimodal stricture analysis. The different assessments are showed in the following figures and table. Compared to control animals, the transplanted animal group showed less frequent alimentary trouble and greater weight gain on day 28. One animal from the transplanted group developed a severe esophageal stricture compared to all animals in the control group. Compared to transplanted animals, control animals developed significant fibrotic tissue. Table 1 shows clinical and endoscopical findings during the follow-up period. Figure 6 shows the endoscopic and radiological findings at the end of the follow-up period in transplanted and non-transplanted animals. Figure 7 shows the different histological findings between transplanted and control animals. These data confirm the effectiveness of ADSC-sheet transplantation for esophageal stricture prevention.
<img alt="Figure 1" src="/files/ftp_upload/55018/55018fig1.jpg" /> 
Figure 1. ADSC-sheet Construct. A and B. Early-passage ADSCs in standard culture dishes with different magnifications. ADSC sheets were obtained by culturing PKH-labeled ADSCs on commercial temperature-responsive culture dishes. C. When the incubation temperature was reduced to 20 &#176;C (room temperature), all cells spontaneously detached from the dish surfaces as intact ADSC sheets, without the need for enzymatic treatment. D. An ADSC sheet harvested before graft construction using a transfer membrane. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55018/55018fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55018/55018fig2.jpg" /> 
Figure 2. Double ADSC-sheet Construct. A, B, and C. The day before transplantation, PKH-labeled ADSCs were harvested at early passage number (P4 - P5), seeded on a 12-well temperature-responsive cell culture, and cultured at 37 &#176;C and 5% CO2 with alpha minimum essential medium including 10% fetal veal serum and 1% antibiotics (penicillin and streptomycin). Each well was coated with a poly-N-isopropylacrylamide membrane (at the bottom of each well) and seeded with 1.5 x 106 cells. When the incubation temperature was reduced to 20 &#176;C (room temperature), all cells spontaneously detached as intact ADSC sheets without the need for enzymatic treatment. D. Theoretical schema of the double cell-sheet construct composed of two ADSC sheets layered upon each other and applied to a paper support membrane. E. Macroscopic view of a completed double cell-sheet construct. Modified from Reference 15. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55018/55018fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55018/55018fig3.jpg" /> 
Figure 3. Endoscopic Submucosal Dissection Procedure. A. Endoscopic marks were created to delineate the resection area, ranging from 40 to 45 cm from the dental arch and involving half of the circumference. B. The submucosal injection of a glycerol solution containing indigo carmine dye. The goal of this injection was to separate the mucosal layer from the muscular layer in order to expose the submucosal layer. C. Peripheral incisions were performed externally to the endoscopic marks, revealing the injected submucosal layer. D. The endoscopic view at the end of the submucosal dissection showing a 5 cm-long and hemi-circumferential esophageal scar. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55018/55018fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55018/55018fig4.jpg" /> 
Figure 4. Double ADSC-sheet Endoscopic Transplantation Procedure. A. Four ADSC constructs ready to be transplanted. B. Protection of the ADSC construct under a large endoscopic cap before transplantation. C. Endoscopic view of the crossing of the animal pharynx, showing the ADSC construct protected under the endoscopic cap. D. 10 min of application was sufficient to obtain sheet adherence. Application was performed using the endoscopic cap or endoscopic forceps. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55018/55018fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55018/55018fig5.jpg" /> 
Figure 5. pCLE Findings. pCLE on day 3 shows spontaneous and intense signals similar to those obtained in vitro, compatible with a successful cell-sheet transplantation. No signal was found on days 14 and 28. A. The in vitro pCLE signal of ADSCs in suspension. B. The in vitro pCLE signal of ADSCs organized in a cell sheet. C. The in vivo pCLE probe application to the esophageal scar. D. The in vivo pCLE signal of ADSCs visualized on day 3 after transplantation. Modified from Reference 15. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55018/55018fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/55018/55018fig6.jpg" /> 
Figure 6. Results of the Morphological Analysis on Day 28. Compared to the control group, the endoscopic and radiological findings on day 28 showed a short and light stricture without upstream dilatation and a completely re-epithelialized mucosa. Modified from Reference 15. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55018/55018fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/55018/55018fig7.jpg" /> 
Figure 7. Histological Evaluation of the Stricture Areas. The histological analysis was performed after HES-labeling and slide digitalization. In transplanted animals (A), the healing process was improved, with increased re-epithelialization and decreased fibrosis development, compared to non-transplanted animals (B). Intense fibrotic tissue development was observed due to muscularis mucosae destruction, fibrotic inner muscularis layer invasion, and large mucosal defects. Modified from Reference 15. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55018/55018fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/55018/55018table1.jpg" /> 
Table 1. Clinical Assessments during the Follow-up Period. Clinical assessments were performed using a daily clinical evaluation according to the Mellow Pinkas score and with weight variation. (*) Mellow Pinkas score16. Modified from Reference 15.

Watch this Scientific Journal Video about ADSC-sheet Transplantation to Prevent Stricture after Extended Esophageal Endoscopic Submucosal Dissection at JoVE.com

ADSC-sheet Transplantation to Prevent Stricture after Extended Esophageal Endoscopic Submucosal Dissection

This study reports a successful method of endoscopical adipose tissue-derived stromal cell (ADSC)-sheet transplantation for esophageal stricture prevention after an extended endoscopic submucosal dissection (ESD) in a swine model.

In past years, the cell-sheet construct has spurred wide interest in regenerative medicine, especially for reconstructive surgery procedures. The ...

adsc-sheet-transplantation-to-prevent-stricture-after-extended

Nanyang Technological University

Research

JoVE Journal

Medicine

8.4K Views.  Université Paris Descartes Sorbonne Paris Cité.  Deze studie meldt een succesvolle methode van endoscopische vetweefsel afgeleide stromale cellen (ADSC)-plaat transplantatie voor oesofageale vernauwing preventie na een verlengde endoscopische submucosale dissectie (ESD) in een varkens model. 

Video: ADSC-sheet Transplantation to Prevent Stricture after Extended Esophageal Endoscopic Submucosal Dissection

An Ivor Lewis Esophagectomy Designed to Minimize Anastomotic Complications and Optimize Conduit Function

We describe a novel esophagogastric anastomotic technique (&ldquo;side-to-side: staple line-on-staple line&rdquo;, STS) for intrathoracic anastomoses designed to create a large diameter anastomosis while simultaneously maintaining conduit blood supply. This technique aims to minimize the incidence of anastomotic leaks and strictures, which is a frequent source of morbidity and occasional mortality after esophagectomy. We analyze the results of this STS technique on 368 patients and compared outcomes to 112 patients who underwent esophagogastric anastomoses using an end-to-end stapler (EEA) over an 8-year time interval at our institution.
The STS technique involves aligning the remaining intrathoracic esophagus over the tip of the lesser curve staple line of a stomach tube, created as a replacement conduit for the esophagus. A linear stapling device cuts through and restaples the conduit staple line to the lateral wall of the esophagus in a side-to-side fashion. The open common lumen is then closed in two layers of sutures.
There was a total of 12 (3.8%) anastomotic leaks in patients who underwent STS esophagogastric anastomosis. Two of eight patients (25%) had anastomotic leaks after esophagectomy for end-stage achalasia as compared to a 2.8% leak rate (10/336) after esophagectomy for other conditions. Eighteen (5.2%) patients required a median of 2 dilatations for anastomotic stricture after STS anastomosis. Supplemental jejunostomy feedings were required in only 11.1% of patients undergoing STS anastomoses following hospital discharge. In contrast, patients undergoing EEA anastomoses demonstrated anastomotic leak and stricture rates of 16.1% and 14.3% respectively (p&lt;0.01). Time analysis of postoperative contrast studies following the STS technique typically demonstrated a straight/uniform diameter conduit with essentially complete contrast emptying into the small bowel within 3 minutes in 88.4% of patients.
The incidence of esophagogastric anastomotic leaks and strictures were extremely low using this novel anastomotic technique. Additionally we believe that based on time and qualitative analyses of postoperative contrast studies, this technique appears to optimize postoperative upper gastrointestinal tract function; however, further comparative studies are needed.

We describe a novel intrathoracic esophagogastric anastomotic technique designed to create a large diameter anastomosis while simultaneously maintaining conduit blood supply to minimize the incidence of anastomotic leaks and strictures. Construction and orientation of the stomach conduit designed to optimize upper gastrointestinal tract function is also described.

We describe a novel esophagogastric anastomotic technique (&ldquo;side-to-side: staple line-on-staple line&rdquo;, STS) for intrathoracic anastomoses ...

Cancer Research

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

Expansion of human pediatric esophageal epithelial cells utilizing conditional reprogramming provides investigators with a patient-specific population of cells that can be utilized for engineering esophageal constructs for autologous implantation to treat defects or injury and serve as a reservoir for therapeutic screening assays.

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

Developmental Biology

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 ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Bioengineering

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

Vessel-sparing Excision and Primary Anastomosis

Urethroplasty is considered to be the standard treatment for urethral strictures since it provides excellent long-term success rates. For isolated short bulbar or posterior urethral strictures, urethroplasty by excision and primary anastomosis (EPA) is recommended. As EPA only requires the excision of the narrowed segment and the surrounding spongiofibrosis, a full-thickness transection of the corpus spongiosum, as performed in the traditional transecting EPA (tEPA), is usually unnecessary. Jordan et al. introduced the idea of a vessel-sparing approach in 2007, aiming to reduce surgical trauma, especially to the dual arterial blood supply of the urethra, and, thus, potentially reducing the risk of postoperative erectile dysfunction or glans ischemia. This approach could also be beneficial for subsequent urethral interventions such as redo urethroplasty using a free graft, in which a well-vascularized graft bed is imperative. Nevertheless, these potential benefits are only assumptions as prospective studies comparing the functional outcome of both techniques with validated questionnaires are currently lacking. Moreover, vessel-sparing EPA (vsEPA) should at least be able to provide similar surgical outcomes as tEPA. The aim of this paper is to give an elaborate, step-by-step overview of how to manage patients with isolated short bulbar or posterior urethral strictures with vsEPA. The main objective of this manuscript is to outline the surgical technique and to report the representative surgical outcome. A total of 117 patients were managed according to the described protocol. The analysis was performed on the entire patient cohort and on the bulbar (n = 91) and posterior (n = 26) vsEPA group separately. Success rates were 93.4% and 88.5% for the bulbar and posterior vsEPA, respectively. To conclude, vsEPA, as outlined in the protocol, provides excellent success rates with low complication rates for isolated short bulbar and posterior urethral strictures.

Here, we present an elaborate and efficient protocol to treat isolated short bulbar or posterior urethral strictures with vessel-sparing excision and primary anastomosis.

Urethroplasty is considered to be the standard treatment for urethral strictures since it provides excellent long-term success rates. For isolated short ...

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into &#39;adult&#39; rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. ...

Neuroscience

Use of the Scissor-Type Knife During the Peroral Endoscopy Myotomy Procedure for the Treatment of Achalasia

Peroral endoscopic myotomy (POEM) is one of the first-line treatment modalities along with pneumatic dilation and Heller myotomy for patients with achalasia. Endoscopists, especially trainees during the learning phase, commonly face difficulty in tissue plane dissection and selective myotomy while working near the esophagogastric junction, with increased risks of inadvertent injury, unexpected bleeding, and inadequate myotomy. To minimize the technical difficulty and improve the safety of POEM, we describe a protocol for using a scissor-type knife for the main steps of POEM, including mucosal incision, submucosal tunneling, myotomy, and hemostasis. The standard techniques used with the scissor-type knife involve grasping the target tissue, and then dissection or coagulation. The confirmation of the cutting line after grasping improves the accuracy and reliability of dissection, which is particularly useful for the selective myotomy of the internal circular muscle. Meanwhile, the scissor-type knife provides enhanced hemostatic capability and enables hemostasis and pre-coagulation without the device exchange for hemostatic forceps. Evaluation of the clinical outcomes in three patients who successfully received POEM using the scissor-type knife revealed no perioperative adverse events. At the 3-month follow-up, all patients achieved clinical success with postoperative Eckardt scores ranging from 0 to 1. In conclusion, the use of a scissor-type knife could minimize the technical difficulty and improve the safety of the POEM procedures, which may be suitable for trainees during the learning phase.

To minimize the technical difficulty and improve the safety of peroral endoscopic myotomy (POEM), we describe a protocol for using a scissor-type knife for the main steps of POEM, including mucosal incision, submucosal tunneling, myotomy, and hemostasis.

Peroral endoscopic myotomy (POEM) is one of the first-line treatment modalities along with pneumatic dilation and Heller myotomy for patients with ...

Robotic Myotomy and Partial Fundoplication for Achalasia

Laparoscopic Heller myotomy is currently considered the standard definitive treatment of achalasia. With the advancements in technology, robotic Heller myotomy has emerged as an alternative approach to traditional laparoscopy due to three-dimensional (3D) visualization, fine motor control, and improved ergonomics provided by the robot. 
Although there is a lack of randomized controlled trials, robotic-assisted Heller myotomy seems to be associated with lower rates of intraoperative perforations compared to the laparoscopic approach. A robotic approach may also improve surgical outcomes by providing a more complete myotomy. 
Here, we describe the detailed steps of robotic myotomy and partial fundoplication for achalasia.

Surgical myotomy with a partial fundoplication may be used in selected patients as a definitive treatment for achalasia. This article provides a step-by-step description of a robotic myotomy and partial fundoplication in a 32-year-old patient with megaesophagus.

Laparoscopic Heller myotomy is currently considered the standard definitive treatment of achalasia. With the advancements in technology, robotic Heller ...

Robotic D3 Partial Duodenal Resection with Primary Side-to-Side Anastomosis

Duodenal stenosis is a condition that can be related to several diseases, being either intrinsic, such as neoplasm and inflammatory stenosis, or extrinsic, such as pancreatic pseudocyst, superior mesenteric artery syndrome, and foreign bodies. Current treatments range from endoscopic approaches, such as endoscopic resection and stent placement, to surgical approaches, including duodenal resection, pancreaticoduodenectomy, and gastrointestinal bypass. Minimally invasive robot-assisted surgery is gaining importance due to its potential to decrease surgical stress, intraoperative blood loss, and postoperative pain, while its instruments and 3D-vision facilitate fine dissection and intra-abdominal suturing, all leading to a reduced time to functional recovery and shorter hospital stay. We present a case of a 75-year-old female who underwent robotic D3 partial duodenal resection with primary side-to-side duodeno-jejunal anastomosis for a 5 cm adenoma with focal high-grade dysplasia.

This protocol presents a case of a robotic partial duodenal resection with primary side-to-side duodeno-jejunal reconstruction in a patient with a 5 cm duodenal stenosis. This is done at the third duodenal segment (D3) after an endoscopic mucosal resection (EMR) for a duodenal polyp.

ADSC-sheet Transplantation to Prevent Stricture after Extended Esophageal Endoscopic Submucosal Dissection

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