Transplantation af fedtvæv-afledte stamceller ark til at reducere udsivningen efter delvis kolektomi i en rotte Model
Summary
Forberedelse og transplantation af fedtvæv afledte stamceller (ASC) plader på utilstrækkeligt syet kolorektal anastomoser i en rotte model præsenteres. Dette nye program viser, at ASC ark kan reducere kolorektal anastomose lækage.
Abstract
Anastomotiske lækage er en katastrofal komplikation efter kolorektal kirurgi. Selv om aktuelle metoder til forebyggelse af lækage har forskellige niveauer af klinisk effekt, er de indtil nu ufuldkommen løsninger. Stamcelleterapi bruger ASC plader kunne være en løsning på dette problem. ASCs betragtes som lovende kandidater til fremme af væv, healing på grund af deres trofiske og immunmodulerende egenskaber. Her, leverer vi metoder til at producere high-density ASC ark, der kan transplanteres til en kolorektal anastomose i en rotte model til at reducere udsivningen. ASCs dannet celle ark i thermo-responderende kultur retter, der nemt kunne afmonteres. På dagen for transplantation, blev en delvis kolektomi med en 5-sutur kolorektal anastomose udført. Dyrene blev straks transplanteret med 1 ASC ark pr. rotte. ASC ark overholdt spontant anastomose uden nogen lim, sutur eller enhver biomateriale. Dyregrupper blev ofret 3-7 dage efter operationen. I forhold til transplanterede dyr, var forekomsten af Anastomotiske bylder og lækage højere i kontroldyr. I vores model, transplantation af ASC plader efter kolorektal anastomose var vellykket og forbundet med en lavere lækage.
Introduction
Delvis kolektomi med en primær anastomose er en almindeligvis udføres kirurgi, der kan gøres for mange sygdomme, der påvirker tyktarmen som kolorektal cancer, Crohns sygdom og diverticulitis1,2. Mest frygtede komplikation efter kolorektal anastomose er Anastomotiske lækage3. Selv om flere risikofaktorer forbundet med Anastomotiske lækager er blevet identificeret, forblive løsninger for at forhindre lækage ukendt4,5.
Fedtvæv-afledte stromale celler (ASCs) er forbundet med anti-inflammatoriske og trofiske egenskaber6,7, hvilket gør disse celler lovende kandidater til regenerativ terapier8. Effektiviteten af ASCs til fremme af væv healing blev vist i forskellige væv såsom hjertemuskulaturen, hud og spiserøret9,10,11,12,13. Men der er få rapporter om anvendelsen af ASCs til fremme af intestinal healing. Lokale transplantation af ASCs til eksperimentelle kolorektal anastomoser via ASC-belagt biosutures eller serosal injektioner af ASCs viste enten ingen forbedring i healing14 eller forhindrede ikke Anastomotiske lækage på trods af et gunstigere Anastomotiske Healing15.
Lokale transplantation af ASCs i suspension eller kombineret med biomaterialer kan være forbundet med utilstrækkelig celle fastholdelse eller en mangelfuld fordeling af transplanterede celler11. Celle ark teknologi16,17 tilbyder en innovativ metode til ASC levering18,19. Derfor, i en tidligere undersøgelse, en ny tilgang blev foreslået i hvilke ASCs organiseret i en celle ark kan anvendes på eksperimentelle kolorektal anastomose20. Denne undersøgelse viste, at ASC ark transplantation har succes med at reducere kolorektal anastomose lækage efter delvis kolektomi i en rotte model. Denne artikel rapporter ASC ark forberedelse og kirurgisk transplantation teknik.
Protocol
Representative Results
Discussion
Anastomotiske lækage er den mest alvorlige hændelse efter kolon resektion med en primær anastomose. Optimal teknikker til at forhindre Anastomotiske forstyrrelser og lækage stadig mangler. Lokal applikation af en matrix med biomaterialer er gennemført med varierende resultater25,26,27. Celle terapier sigter mod at lette væv reparation af væv udskiftning eller stimulation af lokale helbredelse gennem paracrine sekretion.</…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Forfatterne er taknemmelig for, at Prof. Dr. ser Hovius, Dr. M.A.M. Mureau og alle kirurger plastikkirurgi departement for indsamling af subkutane fedtvæv. P. Sukho er støttet af en bevilling fra The Holland Fellowship program (NFP-12/435) under forsøgets gennemførelse. Y.M. Bastiaansen-Jenniskens understøttes af tilskud fra hollandske Arthritis Foundation (LP11).
Materials
| LG DMEM | Gibco, Life technologies | 22320022 | ASC isolation and culture |
| Collagenase type I | Gibco, Life technologies | 17100-01 | ASC Isolation |
| Bovine Serum Albumin | Sigma-Aldrich | A9418 | ASC Isolation |
| Fetal bovine serum | Gibco, Life technologies | 10270-106 | FBS, ASC isolation and culture |
| 3% acetic acid with methylene blue | Stemcell Technologies | 7060 | ASC Isolation |
| Gentamicin | Gibco, Life technologies | 15750-037 | ASC isolation and culture |
| Ampothericin B | Gibco, Life technologies | 15290-018 | ASC isolation and culture |
| Shaker (Gallenkamp Environmental Shaker Model 10X 400) | Akribis Scientific | F240 | ASC Isolation |
| Centrifuge | Hettichlab | Rotina380 | ASC isolation and culture |
| Phosphate buffer saline | Gibco, Life technologies | 14190-094 | PBS, ASC isolation and culture |
| Ascorbic acid-2-phosphate | Sigma-Aldrich | A8960 | ASC isolation and culture |
| Human recombinant fibroblast growth factor 2 | AbD Serotec | AF-100-15 | FGF2, ASC isolation and culture |
| Trypsin EDTA | Gibco, Life technologies | 25200-056 | ASC culture |
| Dimethyl sulfoxide | Sigma-Aldrich | D2650-5x | DMSO, ASC freezing |
| Thermo-responsive culture dishes | Nunc Thermo scientific | 174904 | ASC sheet preperation |
| Non-absorbable braided silk 4/0 | B Braun | 21151042 | surgery |
| Non-absorbable monofilament polyamide 8/0 | B Braun | G1118170 | surgery |
| Absorbable braided polyglycolic acid 5/0 | B Braun | C1048207 | surgery |
References
- Kawahara, H., et al. Single-incision laparoscopic right colectomy for recurrent Crohn’s disease. Hepatogastroenterology. 57 (102-103), 1170-1172 (2010).
- Saia, M., Buja, A., Mantoan, D., Agresta, F., Baldo, V. Colon Cancer Surgery: A Retrospective Study Based on a Large Administrative Database. Surg Laparo Endo Per. 26 (6), e126-e131 (2016).
- Snijders, H. S., et al. Meta-analysis of the risk for anastomotic leakage, the postoperative mortality caused by leakage in relation to the overall postoperative mortality. Eur J Surg Oncol. 38 (11), 1013-1019 (2012).
- Daams, F., Luyer, M., Lange, J. F. Colorectal anastomotic leakage: aspects of prevention, detection and treatment. World J Gastroentero. 19 (15), 2293-2297 (2013).
- Testini, M., et al. Bovine pericardium patch wrapping intestinal anastomosis improves healing process and prevents leakage in a pig model. PLoS One. 9 (1), e86627 (2014).
- Kilroy, G. E., et al. Cytokine profile of human adipose-derived stem cells: expression of angiogenic, hematopoietic, and pro-inflammatory factors. J Cell Physiol. 212 (3), 702-709 (2007).
- Mussano, F., et al. Growth Factor Profile Characterization of Undifferentiated and Osteoinduced Human Adipose-Derived Stem Cells. Stem Cells Int. , 6202783 (2017).
- Hassan, W. U., Greiser, U., Wang, W. Role of adipose-derived stem cells in wound healing. Wound Repair Regen. 22 (3), 313-325 (2014).
- Shudo, Y., et al. Addition of mesenchymal stem cells enhances the therapeutic effects of skeletal myoblast cell-sheet transplantation in a rat ischemic cardiomyopathy model. Tissue Eng Pt A. 20 (3-4), 728-739 (2014).
- Otsuki, Y., et al. Adipose stem cell sheets improved cardiac function in the rat myocardial infarction, but did not alter cardiac contractile responses to beta-adrenergic stimulation. Biomed Res. 36 (1), 11-19 (2015).
- Hamdi, H., et al. Epicardial adipose stem cell sheets results in greater post-infarction survival than intramyocardial injections. Cardiovasc Res. 91 (3), 483-491 (2011).
- Cerqueira, M. T., et al. Human adipose stem cells cell sheet constructs impact epidermal morphogenesis in full-thickness excisional wounds. Biomacromolecules. 14 (11), 3997-4008 (2013).
- Perrod, G., et al. Cell Sheet Transplantation for Esophageal Stricture Prevention after Endoscopic Submucosal Dissection in a Porcine Model. PLoS One. 11 (3), e0148249 (2016).
- Pascual, I., et al. Adipose-derived mesenchymal stem cells in biosutures do not improve healing of experimental colonic anastomoses. Brit J Surg. 95 (9), 1180-1184 (2008).
- Yoo, J. H., et al. Adipose-tissue-derived Stem Cells Enhance the Healing of Ischemic Colonic Anastomoses: An Experimental Study in Rats. J Korean Soc Coloproctol. 28 (3), 132-139 (2012).
- Okano, T. Cell sheet engineering. Rinsho Shinkeigaku. 46 (11), 795-798 (2006).
- Chen, G., et al. Application of the cell sheet technique in tissue engineering. Biomed Rep. 3 (6), 749-757 (2015).
- Labbe, B., Marceau-Fortier, G., Fradette, J. Cell sheet technology for tissue engineering: the self-assembly approach using adipose-derived stromal cells. Methods Mol Biol. 702, 429-441 (2011).
- Sukho, P., et al. Adipose Tissue-Derived Stem Cell Sheet Application for Tissue Healing In Vivo: A Systematic Review. Tissue Eng Part B-Re. , (2017).
- Sukho, P., et al. Effects of adipose stem cell sheets on colon anastomotic leakage in an experimental model: Proof of principle. Biomaterials. 140, 69-78 (2017).
- Verseijden, F., et al. Angiogenic capacity of human adipose-derived stromal cells during adipogenic differentiation: an in vitro study. Tissue Eng Pt A. 15 (2), 445-452 (2009).
- Sukho, P., et al. Effect of Cell Seeding Density and Inflammatory Cytokines on Adipose Tissue-Derived Stem Cells: an in Vitro Study. Stem Cell Rev. 13 (2), 267-277 (2017).
- . Cell Harvesting by Temperature Reduction Available from: https://tools.thermofisher.com/content/sfs/brochures/AppNote-Nunc-UpCell-N20230.pdf (2008)
- Wu, Z., et al. Colorectal anastomotic leakage caused by insufficient suturing after partial colectomy: a new experimental model. Surg Infect (Larchmt). 15 (6), 733-738 (2014).
- Wu, Z., et al. Reducing colorectal anastomotic leakage with tissue adhesive in experimental inflammatory bowel disease. Inflamm Bowel Dis. 21 (5), 1038-1046 (2015).
- Wu, Z., et al. Reducing anastomotic leakage by reinforcement of colorectal anastomosis with cyanoacrylate glue. Eur Surg Res. 50 (3-4), 255-261 (2013).
- Aysan, E., Dincel, O., Bektas, H., Alkan, M. Polypropylene mesh covered colonic anastomosis. Results of a new anastomosis technique. Int J Surg. 6 (3), 224-229 (2008).
- Suarez-Grau, J. M., et al. Fibrinogen-thrombin collagen patch reinforcement of high-risk colonic anastomoses in rats. World J Gastrointest Surg. 8 (9), 627-633 (2016).
- Kobayashi, J., Okano, T. Fabrication of a thermoresponsive cell culture dish: a key technology for cell sheet tissue engineering. Sci Technol Adv Mat. 11 (1), 014111 (2010).
- Elloumi-Hannachi, I., Yamato, M., Okano, T. Cell sheet engineering: a unique nanotechnology for scaffold-free tissue reconstruction with clinical applications in regenerative medicine. J Intern Med. 267 (1), 54-70 (2010).
- Miyahara, Y., et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med. 12 (4), 459-465 (2006).
- Makarevich, P. I., et al. Enhanced angiogenesis in ischemic skeletal muscle after transplantation of cell sheets from baculovirus-transduced adipose-derived stromal cells expressing VEGF165. Stem Cell Res Ther. 6, 204 (2015).
