Name: co-transplantation of human ovarian tissue with engineered endothelial cells: a cell-based strategy combining accelerated perfusion with direct paracrine delivery
Uploaded: 2018-05-16T14:00:00
Description: Watch this Scientific Journal Video about Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Deli

Omar Alexander Man för illustrationerna. L.M. stöddes av en Pilot Award från Cornell kliniska och Translational Science Center och en ASRM forskning bidrag. Författarna vill tacka James lab medlemmar för kritisk läsning av manuskriptet.

Alla förfaranden som berör animaliska ämnen har godkänts av den institutionella djur vård och användning kommittén (IACUC) vid Weill Cornell Medical College. Alla xenotransplantation experiment med hjälp av äggstocksvävnad utfördes i enlighet med tillämpliga riktlinjer och förordningar. Mänskliga äggstocksvävnad samlades in från patienter som planeras för kemoterapi eller strålbehandling för cancerbehandling eller tidigare benmärgstransplantation. Den institutionella Granskningsnämnden (IRB) kommitt...

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+ischemic+tissue+damage+in+ovarian+cortical+tissue+with+or+without+antioxidant+(ascorbic+acid)+treatment.">Quantitative assessment of ischemic tissue damage in ovarian cortical tissue with or without antioxidant (ascorbic acid) treatment.</a> Fertil Steril. 82 (3), 679-685 (2004).</li><li>Abir, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+posttransplantation+survival+of+human+ovarian+tissue+by+treating+the+host+and+graft.">Improving posttransplantation survival of human ovarian tissue by treating the host and graft.</a> Fertil Steril. 95 (4), 1205-1210 (2011).</li><li>Friedman, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+improvements+in+human+ovarian+grafting+by+various+host+and+graft+treatments.">Possible improvements in human ovarian grafting by various host and graft treatments.</a> Hum Reprod. 27 (2), 474-482 (2012).</li><li>Shikanov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+encapsulation+and+vascular+endothelial+growth+factor+delivery+promotes+ovarian+graft+survival+in+mice.">Fibrin encapsulation and vascular endothelial growth factor delivery promotes ovarian graft survival in mice.</a> Tissue Eng Part A. 17 (23-24), 3095-3104 (2011).</li><li>Soleimani, R., Heytens, E., Oktay, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+neoangiogenesis+and+follicle+survival+by+sphingosine-1-phosphate+in+human+ovarian+tissue+xenotransplants.">Enhancement of neoangiogenesis and follicle survival by sphingosine-1-phosphate in human ovarian tissue xenotransplants.</a> PLoS One. 6 (4), e19475 (2011).</li><li>Israely, T., Dafni, H., Nevo, N., Tsafriri, A., Neeman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+ectopic+ovarian+xenotransplantation:+multiparameter+characterization+of+the+neovasculature+by+dynamic+contrast-enhanced+MRI.">Angiogenesis in ectopic ovarian xenotransplantation: multiparameter characterization of the neovasculature by dynamic contrast-enhanced MRI.</a> Magn Reson Med. 52 (4), 741-750 (2004).</li><li>Buratini, J., Price, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicular+somatic+cell+factors+and+follicle+development.">Follicular somatic cell factors and follicle development.</a> Reprod Fertil Dev. 23 (1), 32-39 (2011).</li><li>Dunlop, C. E., Anderson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+and+assessment+of+follicular+growth.">The regulation and assessment of follicular growth.</a> Scand J Clin Lab Invest Suppl. 244, 13-17 (2014).</li><li>Durlinger, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+primordial+follicle+recruitment+by+anti-M&#252;llerian+hormone+in+the+mouse+ovary.">Control of primordial follicle recruitment by anti-M&#252;llerian hormone in the mouse ovary.</a> Endocrinology. 140 (12), 5789-5796 (1999).</li><li>Schmidt, K. L., Ernst, E., Byskov, A. G., Nyboe Andersen, A., Yding Andersen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+primordial+follicles+following+prolonged+transportation+of+ovarian+tissue+prior+to+cryopreservation.">Survival of primordial follicles following prolonged transportation of ovarian tissue prior to cryopreservation.</a> Hum Reprod. 18 (12), 2654-2659 (2003).</li><li>Jensen, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outcomes+of+transplantations+of+cryopreserved+ovarian+tissue+to+41+women+in+Denmark.">Outcomes of transplantations of cryopreserved ovarian tissue to 41 women in Denmark.</a> Hum Reprod. 30 (12), 2838-2845 (2015).</li><li>Oktay, K., Newton, H., Aubard, Y., Salha, O., Gosden, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+immature+human+oocytes+and+ovarian+tissue:+an+emerging+technology?.">Cryopreservation of immature human oocytes and ovarian tissue: an emerging technology?.</a> Fertil Steril. 69 (1), 1-7 (1998).</li><li>Shultz, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+lymphoid+and+myeloid+cell+development+in+NOD/LtSz-scid+IL2R+gamma+null+mice+engrafted+with+mobilized+human+hemopoietic+stem+cells.">Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.</a> J Immunol. 174 (10), 6477-6489 (2005).</li><li>Ramalingam, R., Rafii, S., Worgall, S., Brough, D. E., Crystal, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E1(-)E4(+)+adenoviral+gene+transfer+vectors+function+as+a+&amp;#34;pro-life&amp;#34;+signal+to+promote+survival+of+primary+human+endothelial+cells.">E1(-)E4(+) adenoviral gene transfer vectors function as a &amp;#34;pro-life&amp;#34; signal to promote survival of primary human endothelial cells.</a> Blood. 93 (9), 2936-2944 (1999).</li><li>Seandel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+functional+and+durable+vascular+niche+by+the+adenoviral+E4ORF1+gene.">Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene.</a> Proc Natl Acad Sci U S A. 105 (49), 19288-19293 (2008).</li><li>Meirow, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+fibrosis+and+blood-vessels+damage+in+human+ovaries+exposed+to+chemotherapy.+Potential+mechanisms+of+ovarian+injury.">Cortical fibrosis and blood-vessels damage in human ovaries exposed to chemotherapy. Potential mechanisms of ovarian injury.</a> Hum Reprod. 22 (6), 1626-1633 (2007).</li><li>Assidi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+potential+markers+of+oocyte+competence+expressed+in+bovine+cumulus+cells+matured+with+follicle-stimulating+hormone+and/or+phorbol+myristate+acetate+in+vitro.">Identification of potential markers of oocyte competence expressed in bovine cumulus cells matured with follicle-stimulating hormone and/or phorbol myristate acetate in vitro.</a> Biol Reprod. 79 (2), 209-222 (2008).</li><li>Thakur, S. C., Datta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Higher+expression+of+hyaluronan+binding+protein+1+(HABP1/p32/gC1qR/SF2)+during+follicular+development+and+cumulus+oocyte+complex+maturation+in+rat.">Higher expression of hyaluronan binding protein 1 (HABP1/p32/gC1qR/SF2) during follicular development and cumulus oocyte complex maturation in rat.</a> Mol Reprod Dev. 75 (3), 429-438 (2008).</li><li>Dolmans, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+transplantation+of+isolated+human+ovarian+follicles+and+cortical+tissue+into+nude+mice.">Short-term transplantation of isolated human ovarian follicles and cortical tissue into nude mice.</a> Reproduction. 134 (2), 253-262 (2007).</li><li>Amorim, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+freezing+and+thawing+of+human+ovarian+tissue+on+follicular+growth+after+long-term+xenotransplantation.">Impact of freezing and thawing of human ovarian tissue on follicular growth after long-term xenotransplantation.</a> J Assist Reprod Genet. 28 (12), 1157-1165 (2011).</li><li>Kawamura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippo+signaling+disruption+and+Akt+stimulation+of+ovarian+follicles+for+infertility+treatment.">Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.</a> Proc Natl Acad Sci U S A. 110 (43), 17474-17479 (2013).</li><li>Suzuki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+fertility+preservation+following+ovarian+tissue+vitrification+in+patients+with+primary+ovarian+insufficiency.">Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency.</a> Hum Reprod. 30 (3), 608-615 (2015).</li><li>Campbell, B. K., Clinton, M., Webb, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+anti-M&#252;llerian+hormone+(AMH)+during+follicle+development+in+a+monovulatory+species+(sheep).">The role of anti-M&#252;llerian hormone (AMH) during follicle development in a monovulatory species (sheep).</a> Endocrinology. 153 (9), 4533-4543 (2012).</li><li>Donnez, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+ovarian+activity+and+pregnancy+after+transplantation+of+cryopreserved+ovarian+tissue:+a+review+of+60+cases+of+reimplantation.">Restoration of ovarian activity and pregnancy after transplantation of cryopreserved ovarian tissue: a review of 60 cases of reimplantation.</a> Fertil Steril. 99 (6), 1503-1513 (2013).</li><li>Ferreira, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+sample+size+on+the+outcome+of+ovarian+tissue+cryopreservation.">The effects of sample size on the outcome of ovarian tissue cryopreservation.</a> Reprod Domest Anim. 45 (1), 99-102 (2010).</li><li>Gavish, Z., Peer, G., Roness, H., Cohen, Y., Meirow, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicle+activation+and+'burn-out'+contribute+to+post-transplantation+follicle+loss+in+ovarian+tissue+grafts:+the+effect+of+graft+thickness.">Follicle activation and 'burn-out' contribute to post-transplantation follicle loss in ovarian tissue grafts: the effect of graft thickness.</a> Hum Reprod. 30 (4), 1003 (2015).</li><li>Donnez, J., Dolmans, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertility+Preservation+in+Women.">Fertility Preservation in Women.</a> N Engl J Med. 377 (17), 1657-1665 (2017).</li><li>Salama, M., Woodruff, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+advances+in+ovarian+autotransplantation+to+restore+fertility+in+cancer+patients.">New advances in ovarian autotransplantation to restore fertility in cancer patients.</a> Cancer Metastasis Rev. 34 (4), 807-822 (2015).</li><li>Donnez, J., Dolmans, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cortex+transplantation:+60+reported+live+births+brings+the+success+and+worldwide+expansion+of+the+technique+towards+routine+clinical+practice.">Ovarian cortex transplantation: 60 reported live births brings the success and worldwide expansion of the technique towards routine clinical practice.</a> J Assist Reprod Genet. 32 (8), 1167-1170 (2015).</li><li>Meirow, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantations+of+frozen-thawed+ovarian+tissue+demonstrate+high+reproductive+performance+and+the+need+to+revise+restrictive+criteria.">Transplantations of frozen-thawed ovarian tissue demonstrate high reproductive performance and the need to revise restrictive criteria.</a> Fertil Steril. 106 (2), 467-474 (2016).</li><li>Kalich-Philosoph, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclophosphamide+triggers+follicle+activation+and+"burnout";+AS101+prevents+follicle+loss+and+preserves+fertility.">Cyclophosphamide triggers follicle activation and "burnout"; AS101 prevents follicle loss and preserves fertility.</a> Sci Transl Med. 5 (185), 185ra162 (2013).</li><li>Kano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMH/MIS+as+a+contraceptive+that+protects+the+ovarian+reserve+during+chemotherapy.">AMH/MIS as a contraceptive that protects the ovarian reserve during chemotherapy.</a> Proc Natl Acad Sci U S A. 114 (9), E1688-E1697 (2017).</li></ol>

Här visar vi att samtidig transplantation av chefer ger en betydande fördel för äggstocksvävnad livskraft och funktion efter xenografts i möss. Standarder för klinisk tillämpning för äggstocksvävnad auto-transplantation för fertilitet bevarande har inte set och de optimala parametrarna (storlek, transplantation webbplats, varaktigheten av transplantat, etc.) 32 , 33 , 34 för förbättrad återvinning av fol...

Cancer är fortfarande bland de ledande dödsorsakerna i industriländerna, men decennier av forskning har gett betydande framsteg för de flesta typer av cancer, och i vissa fall nästan fördubblad överlevnad priser1. Kemoterapeutika är tyvärr ofta gonadotoxic, ozonnedbrytande reserven av primordial folliklar i äggstockarna och minska fertilitet2. Denna växande befolkning kan dra nytta av olika metoder för fertilitet bevarande inklusive äggcell eller embryo frysförvaring, men patienter som kräver snabbt insättande av cancerbehandling och prepubertala patienter är berättigade till dessa alternativ. Alternativt kan har vissa patienter valt att frysa äggstocksvävnad innan deras terapeutiska regimen, och på återkrav och eftergift, auto-transplantation vävnad att återställa fertilitet3. Ännu, hittills har överlevnaden för njurtransplantaten och follikulär utdata efter auto-transplantation förblir relativt låg4, främst på grund av vävnad ischemi och hypoxi5,6,7. Trots många insatser för att förbättra lönsamheten för äggstockscancer kortikala grafter med anti-oxidanter8,9, proangiogena cytokiner10,11,12,1 3eller mekaniska manipulationer14, transplantat ischemi i en 5-7 dag fönster efter transplantation undergräver lönsamhet och överlevnad av transplantat7. För att lösa detta, utvecklat vi en cell-baserad strategi för att underlätta anastomos värd och transplantat fartyg och därmed påskynda reperfusion för äggstocksvävnad.
Förutom den ischemisk förolämpningen mot ympade äggstocksvävnad i fönstret efter transplantation, kan störningar av Inter follikulära signalering bidra till utarmning av pool15,16. Eftersom exogena endotelceller (chefer) bidrar till stabil och fungerande fartyg i peripheryen av graften, presenterar de en unik möjlighet att förmedla en definierad molekylär ingång transplanterad vävnad. Som ett bevis på principen, var chefer konstruerad för att snabb Super fysiologiska nivåer av Anti-Mullerian hormon (AMH), medlem av omvandla tillväxtfaktor beta (TGFβ) superfamiljen som har visat att begränsa follikeltillväxten17. Jämförelse av follikulära distribution i ympkvistar Co transplanteras med kontroll och AMH-uttryckande celler kontrollerar biologisk aktivitet och styrkan av bakåtkompilerade chefer.
Sammanfattningsvis, kan genom att förbättra transplantat livskraft och undertrycka tidig mobilisering av follikulära poolen, detta tillvägagångssätt öka produktiviteten i auto-transplanterade äggstocksvävnad hos patienter som genomgår fertilitet bevarande. Dessutom kan den ExEC-baserad plattformen experimentella förhör av molekylär tillsynsmyndigheter som har varit inblandade i follikulär utveckling.

<table>
	<tbody>
		<tr>
			<td>Leibovitz&#8217;s L-15 medium</td>
			<td>Gibco</td>
			<td>11415064</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td>Anti-Anti X100</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S 1888</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Sigma</td>
			<td>F 8630</td>
			<td>from bovine plasma</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T 1063</td>
			<td>from human plasma</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D 2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>12491015</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzyme Cell Detachment Medium</td>
			<td>Invitrogen</td>
			<td>00-4555-56</td>
			<td>Accutase</td>
		</tr>
		<tr>
			<td>Plastic paraffin film</td>
			<td>Bemis NA</td>
			<td></td>
			<td>Parafilm M</td>
		</tr>
		<tr>
			<td>Surgical paper tape 2.5 cm</td>
			<td>3M</td>
			<td>1530-1</td>
			<td>Micropore</td>
		</tr>
		<tr>
			<td>Surgical Paper tape 1.25 cm</td>
			<td>3M</td>
			<td>1530-0</td>
			<td>Micropore</td>
		</tr>
		<tr>
			<td>Perforated plastic Surgical tape 1.25 cm</td>
			<td>3M</td>
			<td>1527-0</td>
			<td>Transpore</td>
		</tr>
		<tr>
			<td>Monofilament Absorbable Suture</td>
			<td>Covidien</td>
			<td>UM-203</td>
			<td>Biosyn</td>
		</tr>
		<tr>
			<td>Braided Absorbable Suture</td>
			<td>Covidien</td>
			<td>GL-889</td>
			<td>Polysorb</td>
		</tr>
		<tr>
			<td>Povidone-iodine Solution USP 10%</td>
			<td>Purdue Products</td>
			<td>67618-153-01</td>
			<td>Betadine Solution Swab Stick</td>
		</tr>
		<tr>
			<td>Cryoviales</td>
			<td>Nunc</td>
			<td>377267</td>
			<td>CryoTube</td>
		</tr>
		<tr>
			<td>sterile ocular lubricant</td>
			<td>Dechra</td>
			<td>17033-211-38</td>
			<td>Puralube</td>
		</tr>
		<tr>
			<td>1.7 ml micro-centrifuge tube</td>
			<td>Denville</td>
			<td>C-2172</td>
			<td>Eppendorf</td>
		</tr>
		<tr>
			<td>Anasthesia system</td>
			<td>VetEquip</td>
			<td>V-1 table top system with scavenging</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial cells</td>
			<td>Angiocrine Biosciences, Inc., San Diego, CA, USA</td>
			<td></td>
			<td>Isolated, transfected with E4-ORF- 1 and labeled endothelial cells</td>
		</tr>
		<tr>
			<td>Trichrome stain</td>
			<td>Sigma</td>
			<td>HT15-1kt</td>
			<td>Trichrome Stain (Masson) Kit</td>
		</tr>
		<tr>
			<td>Isolectin</td>
			<td>Invitrogen</td>
			<td>I32450</td>
			<td>isolectin GS-IB4 From Griffonia simplicifolia, Alexa Fluor&#8482; 647 Conjugate</td>
		</tr>
	</tbody>
</table>

co-transplantation of human ovarian tissue with engineered endothelial cells: a cell-based strategy combining accelerated perfusion with direct paracrine delivery

Infertilitet är en vanlig biverkning av kemoterapi eller strålbehandling och för vissa patienter, Frysförvaring av oocyter eller embryon är inte ett alternativ. Som ett alternativ, ett ökande antal dessa patienter väljer att frysa äggstocksvävnad för autograft efter återkrav och eftergift. Trots förbättringar i resultat bland patienter som genomgår auto-transplantation av frysförvarade äggstocksvävnad, förblir effektiv revaskularisering ympade vävnad ett stort hinder. För att mildra ischemi och därmed förbättra utfall hos patienter som genomgår auto-transplantation, utvecklade vi en vaskulära cell-baserad strategi för att påskynda perfusion för äggstocksvävnad. Vi beskriver en metod för samtidig transplantation av exogena endotelceller (chefer) med frysförvarade äggstocksvävnad i en musmodell xenograft. Vi förlänga denna strategi att anställa chefer som har konstruerats för att konstitutivt express Anti-Mullerian hormon (AMH), vilket möjliggör ihållande parakrin signalering indata till äggstockscancer ympkvistar. Samtidig transplantation med chefer ökat follikulära volym och förbättrad antral follikeln utveckling och AMH-uttryckande chefer främjas lagring av quiescent primordial folliklar. Denna kombinerade strategi kan vara ett användbart verktyg för förmildrande ischemi och modulerande follikulära aktivering inom ramen av fertilitet bevarande och/eller infertilitet i stort.

För att fastställa om samtidig transplantation av chefer ger en förmån till patienternas vävnad, var tinade cystor kortikala band uppdelad i lika stora bitar och rekonstituerades bilateralt i immunförsvar, nicka scid gamma (NSG), möss. Med ena sidan inbäddade i en fibrin propp ensam (ingen ECs) och de andra innehåller chefer (figur 1a), tjänade varje mus som sin egen kontroll. Chefer erhölls via isolering av primära endotel från mänskliga navels...

Watch this Scientific Journal Video about Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Deli

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

För vissa patienter är det enda alternativet för fertilitet bevarande frysförvaring för äggstocksvävnad. Tyvärr undergräver försenad revaskularisering follikulära livskraft. Här presenterar vi ett protokoll för att co transplantera mänskliga äggstocksvävnad med endotelceller för utnyttjande som cell-baserad strategi kombinerar accelererade perfusion med en direkt parakrin leverans av bioaktiva molekyler.

Samtidig transplantation av mänskliga äggstocksvävnad med bearbetade Endothelial celler: en Cell-baserad strategi kombinera accelererade Perfusion med direkta parakrin leverans

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. ...

The overall goal of this protocol is to xenograft cryopreserved human ovarian tissue in combination with engineered endothelial cells that accelerate re-perfusion and provide paracrine stimulus that modulates follicular activation within the graft. This method provides a cell-based means of improving post-transplant viability of cryopreserved ovarian tissue and more broadly, establishes a robust model system for interrogating molecular regulation of human folliculogenesis. The main advantage of this technique is that it uses a single vehicle that of endothelial cells to both mitigate ischemia and provide a sustained source of secreted factors to engraft the tissue.So this method provide global benefit for tissue viability. It can also provide insight into molecular regulators that have been implicated in follicular development. To make a fibrin clot, work quickly and add 360 microliters of HEPES buffer to 200 microliters of the fibrinogen stock solution.Gently pipette to mix and keep the tube on ice. In the second microfuge tube, prepare thrombin working solution by adding 148.7 microliters of DMEM to 50 microliters of thrombin stock solution. Gently pipette to mix.Then add 1.3 microliters of one mole per liter of calcium chloride. Gently pipette to mix and keep the sample on ice. Using the enzyme cell detachment medium Accutase, detach previously prepared engineered endothelial cells from the plate and transfer them to a third microfuge tube.Then, spin down the cells. Next, using a hemocytometer and a covered glass, count the cells. To calculate the number of cells needed for the engraftment, convert a 20, 000 times area of ovarian tissue into millimeter squared by using a ruler to measure the dimensions.The multiply the area by 20, 000 to determine the number of cells needed for the engraftment. Spin down the required cells. And use DMEM to re-suspend the pellet for a total volume of 16.8 microliters.Keep the cell suspension on ice. Next, place a piece of ovarian tissue on a sterile gauze sponge for a few seconds to dry it. Then transfer the tissue onto a previously prepared plastic paraffin film in a 50 millimeter petri dish.Add 39.2 microliters of the fibrinogen solution into the prepared cell suspension. Mix by gentle pipetting, and keep the suspension on ice. Then working quickly, add 14 microliters of the thrombin solution to the tube and gently pipette once to mix.Pipette the mixture on top of the ovarian tissue in the shape of an elongated droplet. Incubate the petri dish with the clot at 37 degrees Celsius. Work quickly once you have added the thrombin solution to the fibrinogen cell suspension.Synchronizing the future preparation in surgery is a challenge and it may be prudent to perform a practice run prior to expending precious human tissue. After anesthetizing the 10 to 14 week old female NSG mouse, and preparing it for surgery according to the text protocol, heat the animal and monitor the body temperature throughout the procedure. With a povidone iodine swab stick, clean the trimmed area and then use sterile alcohol to wipe it off.Next, place a drop of sterile ocular lubricant vet ointment over each of the mouse's eyes to protect them from dehydration and damage to the cornea. After performing a bilateral oophorectomy according to the text protocol, inject buprenorphine subcutaneous. Then make a horizontal incision in the fascia above the gluteus maximus at a length that fits the clot dimensions.Create a pocket within this virtual space by opening the scissors underneath the fascia. Pick up one piece of previously prepared encapsulated cortical tissue and place it in the pocket. Then use six O braided absorbable suture to suture the fascia before repeating the co-transplant on the contra-lateral side.Close the dorsal wall with four O monofilament absorbable suture in simple interrupted stitches. Inject lidocaine subcutaneous at the incision site. Then use a sterile alcohol prep to clean the skin and carry out post-operative care according to the text protocol.As seen here, two weeks after bilateral engraftment of E4-ORF1 treated human umbilical vein endothelial cells into NSG female mice, functionally perfused vessels formed from GFP labeled exogenous ECs or ExECs at the interface of host tissue and the graft. ExECs formed functional vessels when co-transplanted with ovarian tissue in cortical pieces co-transplanted with the mouse or human ExECs, increase the number of surviving follicles relative to control grafts. This increase in follicle development was associated with a decreased fibrotic area in ExEC assisted grafts.Final fibrotic area values were calculated for each graft as the average of three sections analyzed. Importantly, grafts that were co-transplanted with human foreskin fibroblasts showed poor tissue viability and relatively few follicles. 14 weeks after engraftment, mice were stimulated with menotropins.And the progression of follicle growth was monitored via MRI. Developing follicles were noted in both control and ExEC assisted grafts. However, more and larger sized follicles were noticed in the ExEC assisted grafts.Compared to antral follicles derived from the same patient's tissue, the granulosa cell layer in the more advanced follicle displayed increased expression of the ovulatory markers CD44 and HABP1, as well as increased cell death and reduced proliferation. After watching this video, you will understand how to perform co-transplantation of cryopreserved ovarian tissue with endothelial cells in a fibrin clot. And once mastered, you should be able to complete this protocol in less than one hour.Building on this approach, endothelial cells can be engineered to express any combination of secreted factors, thereby enabling interrogation of signaling pathways that may be relevant to fertility preservation, folliculogenesis, and/or tissue transplantation.

Research

JoVE Journal

Bioengineering

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

Avkänning av Barrier Tissue Störningar med en organisk Elektro Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

Tissue Engineering av tumör Stromal mikromiljö med Ansökan till Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Generation och Nagelvävnadstekniska Fartyg i en musmodell

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

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

Produktion, karakterisering och potentiella användningsområdena för en 3D vävnadstekniska Human Esophageal Mucosal Modell

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

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Leverans av proteiner, peptider eller cellimpermeabel Små molekyler i levande celler genom inkubation med Endosomolytic Reagens dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Automatiserad Contraction analys av Human Engineered hjärtvävnad för Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Lokalt av primära nedsatt vävnad segment och inkorporering med romanen byggnadsställning-fri konstruktion teknik

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

En ny tenorrhaphy sutur teknik med vävnadskonstruerad kollagen graft för att reparera stora sena defekter

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Three-dimensional Patterning of Engineered Biofilms with a Do-it-yourself Bioprinter

Tredimensionell Patterning av Engineered bio filmer med en gör-det-själv Bioprinter

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Skapa encellig Co-kulturer på ett deterministiskt sätt med ett Microfluidic chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...