Omar Alexander Man for the illustrations. 
L.M. was supported by a Pilot Award from the Cornell Clinical and Translational Science Center and an ASRM research grant. 
The authors would like to thank James lab members for critical reading of the manuscript.

All procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at Weill Cornell Medical College. All xenotransplantation experiments using ovarian tissue were performed in accordance with relevant guidelines and regulations. Human ovarian tissue was collected from patients scheduled for chemotherapy or radiotherapy for cancer treatment or prior bone marrow transplantation. The institutional review board (IRB) Committee of Weill Cornell Medical College approved the collection of tissue for potential autologous use, and upon the patient&#39;s informed consent a donation of up to 10% of their ovarian tissue for research use was performed.
1. Collection of Human Ovarian Tissue
NOTE: When an ovarian tissue is transported from a remote facility transit, time should not exceed 5 h18,19.
<ol>
	<li>Collect the ovarian tissue and rinse with a sterile saline solution.</li>
	<li>Place the ovary in a sterile container.</li>
	<li>Pour Leibovitz&#39;s L-15 medium until the ovary is completely immersed in the medium.</li>
	<li>Close and seal the container.</li>
	<li>Transport the container on ice.</li>
</ol>
2. Processing the Procured Ovarian Tissue, Adapted from Schmidt et al.18
<ol>
	<li>Preparations for ovarian processing and freezing
	<ol>
		<li>Prepare 100 mL of medium for processing: Leibovitz&#39;s L-15 medium 99 mL + 1 mL antibiotic-antimycotic solution. Filter media using 0.2 &#181;m filter. Keep the medium refrigerated.</li>
		<li>Prepare 100 mL of freezing solution using DMSO as a cryo-protectant. Add 69.64 mL Leibovitz&#39;s L-15 medium, 17.66 mL fetal bovine serum (FBS), 3.42 g of sucrose (to create a final concentration of 0.1 mol/L), 10.65 mL DMSO (to create a final concentration of 1.5 mol/L), and 1 mL antibiotic-antimycotic solution. Filter the medium using a 150 mL filter unit, 0.2 &#181;m. Store the medium at 4 &#176;C.</li>
		<li>Sterilize the surgical tools using an autoclave programmed for a wrapped solids sterilization cycle.</li>
	</ol>
	</li>
	<li>Upon arrival of the tissue
	<ol>
		<li>Set the sterile surgical tools in the biosafety cabinet: scalpel with blade number 21, sharp fine curved scissors and forceps at varied sizes: long (about 150 mm length) and 2 medium-sized (about 110 mm length).</li>
		<li>Wear sterile gloves, open the container within the biosafety cabinet. Take the ovary and place it in a 150 mm Petri dish and pour cold medium prepared in step 2.1.1 on top of the ovary to prevent dehydration of the ovarian tissue.</li>
		<li>Isolate the ovary from any residual tissue and rinse it with cold medium until it&#39;s devoid of tissue and blood.</li>
		<li>Place the ovary in a clean 150 mm Petri dish, add cold medium, prepared in step 2.1.1 until the ovary is half submerged in it.</li>
	</ol>
	</li>
	<li>Dissection of the ovarian tissue
	<ol>
		<li>Bisect the ovary and remove the medulla first by sharp dissection, using curved fine scissors. Then scrape the medulla away, using a sterile scalpel with a blade number 21. Precede until the cortical tissue is 1-1.5 mm in thickness.</li>
		<li>Cut the cortical tissue into slivers of 2-3 mm in width, the length of the strip will be the entire length of the ovarian piece that was processed.</li>
	</ol>
	</li>
</ol>
3. Ovarian Tissue Slow Freezing, Adapted from Newton et al. and Oktay et al.6,20
<ol>
	<li>Preparation for freezing
	<ol>
		<li>Label cryovials (1.8 mL).</li>
		<li>Add 1.5 mL of the freezing solution prepared in step 2.1.2 to each vial.</li>
		<li>Transfer one cortical strip per cryogenic vial containing the freezing solution.</li>
		<li>Equilibrate the cortical strip for 20 min on a rotating plate, apply gentle agitation at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Slow freezing of ovarian tissue
	<ol>
		<li>Load the cryovials into a programmable planer freezer starting at 0 &#176;C.</li>
		<li>Cool at 2 &#176;C/min to -7 &#176;C.</li>
		<li>Keep the tissues at this constant temperature for 10 min.</li>
		<li>Perform manual seeding for ice crystal nucleation induction, by touching each cryovial with a cotton tip immersed in liquid nitrogen (LN2).</li>
		<li>Continue to cool at 0.3 &#176;C/min until sample temperature reaches -40 &#176;C.</li>
		<li>Cool at a faster rate of 10 &#176;C/min to -140 &#176;C.</li>
		<li>Transfer cryovials to the dewar for storage in LN2 (-196 &#176;C).</li>
	</ol>
	</li>
</ol>
4. Preparations for the Surgeries (Bilateral Oophorectomy and Co-transplantation)
<ol>
	<li>Preparation of plates
	<ol>
		<li>A day prior to thawing the ovarian tissue for transplantation
		<ol>
			<li>Place a piece of a plastic paraffin film at the bottom of a 50 mm Petri dish, spray it with 70% Ethanol until it will be completely covered.</li>
			<li>Leave the Petri dishes overnight in the biosafety cabinet. Prepare 2 Petri dishes per mouse.</li>
		</ol>
		</li>
		<li>At the day of surgeries
		<ol>
			<li>Aspirate the ethanol from the Petri dish containing the plastic paraffin film within the biosafety cabinet.</li>
			<li>Leave the Petri dish lead half open until ethanol evaporates completely.</li>
			<li>Close the lid of the Petri dish when the plastic paraffin film is completely dry. Keep it within the biosafety cabinet.</li>
			<li>Label wells of a 6 well plate accordingly, 0.1 mol/L Sucrose+ 1 mol/L DMSO, 0.1 mol/L Sucrose+ 0.5 mol/L DMSO, 0.1 mol/L Sucrose, Basic Thawing Solution (BTS), Medium.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of the solutions
	<ol>
		<li>Prepare 100 mL of BTS: Leibovitz&#39;s L-15 medium 79 mL + FBS 20 mL + Antibiotic-Antimycotic solution 1 mL. Filter with a 0.22 &#181;m filter system.</li>
		<li>Prepare 10 mL of BTS with 0.1 mol/L Sucrose. Scale 0.342 g of Sucrose and add 10 mL of BTS prepared in step 4.2.1, Agitate gently until the Sucrose is completely dissolved. Filter the solution using a syringe filter 0.22 &#181;m.</li>
		<li>Prepare the solutions according to Table 1. Cover tubes containing DMSO with aluminum foil. Keep refrigerated until use.</li>
	</ol>
	</li>
</ol>
5. Ovarian Tissue Rapid Thawing
<ol>
	<li>Take out of the LN2 dewar a vial containing frozen ovarian tissue and keep it at room temperature for 30 s.</li>
	<li>Wipe the vial clean using a tissue paper.</li>
	<li>Immerse the vial in a 30 &#176;C water bath for 1-2 min until its&#39; content is thawed.</li>
	<li>Open the vial and place the cortical strip in the first well that contains 3ml of the first solution (0.1 mol/L Sucrose+ 1 mol/L DMSO, prepared in step 4.2.3). 
	NOTE: All steps involving opening the lid of the plate will be done under laminar flow.</li>
	<li>Incubate the 6 well plate on a rotating plate at 4 &#176;C for 5 min. Keep the plate covered with aluminum foil for this step.</li>
	<li>Transfer the cortical strip into the second well containing 3 mL of the second solution (0.1 mol/L Sucrose+ 0.5 mol/L DMSO, prepared in step 4.2.3).</li>
	<li>Incubate the 6 well plate on a rotating plate for gentle agitation at 4 &#176;C for 5 min. Keep the plate covered with aluminum foil for this step.</li>
	<li>Transfer the cortical strip into the third well, containing 4 mL of solution (BTS with 0.1 mol/L Sucrose, prepared in step 4.2.2).</li>
	<li>Incubate on a rotating plate for gentle agitation at 4 &#176;C for 5 min.</li>
	<li>Transfer the cortical strip into the fourth well containing 4 mL of solution (BTS, prepared in step 4.2.1).</li>
	<li>Incubate on a rotating plate for gentle agitation at 4 &#176;C for 5 min.</li>
	<li>Transfer the cortical strip into the last well containing 4 mL of cold medium (prepared in step 2.1.1). Keep thawed ovarian tissue on ice until performing the transplantation.</li>
</ol>
6. Encapsulation of the Ovarian Tissue
<ol>
	<li>Prepare the following solutions
	<ol>
		<li>Prepare 25 mL of 20 mmol/L HEPES buffer in 0.9% saline. Filter and store at 4 &#176;C.</li>
		<li>Prepare 1 mL of CaCl2 at the concentration of 1 mol/L. Filter and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare a Fibrinogen 50 mg/mL stock solution
	<ol>
		<li>Add 1 g of Fibrinogen into 20 mL of 20 mmol/L HEPES buffer in 0.9% saline by slowly mixing fibrinogen into the HEPES buffered saline over several hours at 37 &#176;C.</li>
		<li>Label 1.7 mL micro-centrifuge tubes.</li>
		<li>Filter the solution through a 0.45 &#181;m syringe filter and then through a 0.2 &#181;m syringe filter.</li>
		<li>Aliquot the solution into 200 &#181;L into micro-centrifuge tube and store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare a Thrombin 100 U/mL stock solution 
	NOTE: Thrombin solutions adsorb to glass, aliquot the solution in plastic tubes/vials.
	<ol>
		<li>Add 2.5 mL of 0.9% sterile saline to 250 U of Thrombin.</li>
		<li>Gentle shake until the solution to completely dissolve.</li>
		<li>Label 1.7 mL micro-centrifuge tubes.</li>
		<li>Filter through a 0.2 &#181;m syringe filter, aliquot 50 &#181;L into micro-centrifuge tubes and store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Make a Fibrin clot of 70 &#181;L, obtaining a final concentration of 10 mg/mL fibrinogen and 5 U/mL Thrombin 
	NOTE: Make sure to work fast, the solution clots in a few seconds. Also, avoid the addition of bubbles to the mixture.
	<ol>
		<li>Prepare a Fibrinogen solution in a 1.7 mL micro-centrifuge tube.
		<ol>
			<li>Add 360 &#181;L of 20 mmol/L HEPES Buffer in 0.9% saline solution to the aliquoted 200 &#181;L of the Fibrinogen stock, prepared in step 6.2.4.</li>
			<li>Mix by gentle pipetting.</li>
			<li>Keep it on ice.</li>
		</ol>
		</li>
		<li>Prepare a Thrombin solution in a second micro-centrifuge tube.
		<ol>
			<li>Add 148.7 &#181;L of DMEM to the aliquoted 50 &#181;L of the Thrombin stock, prepared in step 6.3.4.</li>
			<li>Mix by gentle pipetting.</li>
			<li>Add 1.3 &#181;L of 1 mol/L CaCl2.</li>
			<li>Mix by gentle pipetting.</li>
			<li>Keep it on ice.</li>
		</ol>
		</li>
		<li>Prepare a single cell suspension, In a third micro-centrifuge tube.
		<ol>
			<li>Detach the engineered endothelial cells from the plate using an enzyme cell detachment medium.</li>
			<li>Spin down the and count the cells using a hemocytometer with a cover glass.</li>
			<li>Use 20,000 cells per every 1 mm2 of ovarian tissue. Calculate 20,000x area of ovarian tissue in mm2.</li>
			<li>Spin down the engineered endothelial cells and re-suspend it in basic medium (DMEM) to reach a total volume of 16.8 &#181;L.</li>
			<li>Keep it on ice.</li>
		</ol>
		</li>
		<li>Place a piece of ovarian tissue on a sterile gauze sponge to dry it for a few seconds.</li>
		<li>Place the piece of ovarian tissue on top of the plastic paraffin film within the 50 mm Petri dish.</li>
		<li>Add 39.2 &#181;L of the Fibrinogen solution, prepared in step 6.4.1.2, into the cell suspension prepared in step 6.4.3.4, mix by gentle pipetting. Keep it on ice.</li>
		<li>Add 14 &#181;L of the Thrombin solution prepared in step 6.4.2.4. Mix gently by pipetting once. Work fast.</li>
		<li>Place the mixture on top of the ovarian tissue, pipet it in the form of an elongated droplet.</li>
		<li>Place the Petri dish with the clot in the incubator at 37 &#176;C.</li>
	</ol>
	</li>
</ol>
7. Bilateral Oophorectomy and Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells to NSG Mice
NOTE: Ten to fourteen-week-old female NSG mice21 were used (Jackson Labs).
<ol>
	<li>Prepare all surgical tools, as elaborated in step 2.1.3, make sure you have all sutures, pads, and surgical tapes handy.</li>
	<li>Anesthetize the mouse using an anesthesia system with Isoflurane.
	<ol>
		<li>Place the mouse in the induction chamber, close lid and open the appropriate stopcock.</li>
		<li>Open flowmeter to 1,000 mL/min. Turn on the vaporizer and set it to deliver 2-3.5%.</li>
		<li>Observe effects of anesthesia; loss of consciousness, slow and regular breaths.</li>
		<li>Transfer to the nose cone to maintain anesthesia. Turn the vaporizer to deliver 1-3%, depending on vital signs.</li>
		<li>Verify absence of pedal or tail reflex. If the reflex is present, increase isoflurane until it&#39;s absent.</li>
	</ol>
	</li>
	<li>Trim the mouse&#39;s back hair, from the tail up to the clavicles line, using an electric hair trimmer.</li>
	<li>Place the mouse in a prone position on the surgical platform.</li>
	<li>Fix the nose cone to the surgical platform using a surgical tape.</li>
	<li>Tape the mouse&#39;s limbs gently to the surgical platform using a 1.25 cm-wide surgical paper tape.</li>
	<li>Use a sterile lubricant jelly and insert a thermometer PR. Fix the thermometer by taping it to the surgical platform using a 1.25 cm wide perforated plastic surgical tape.</li>
	<li>Tape the tail to the surgical platform by using a 2.5 cm wide surgical paper tape.</li>
	<li>Heat, using an infrared or a hot water heating pad, and monitor the mouse&#39;s body temperature throughout the procedure. Keep body temperature within the range of 35-37 &#176;C.</li>
	<li>Clean the trimmed area with a sterile Povidone-Iodine Solution swab stick and wipe using sterile alcohol prep.</li>
	<li>Repeat step 6.9 twice more.</li>
	<li>Place a drop of sterile ocular lubricant vet ointment over each of the mouse&#39;s eyes to protect from dehydration and damage to the cornea.</li>
	<li>Cover the mouse using a sterile surgical drape.</li>
	<li>Inject Buprenorphine (1 mg/Kg) sub-cutaneous (S/C).</li>
	<li>Perform a longitudinal medial dorsal incision using a scalpel (blade #21).</li>
	<li>Perform a bilateral oophorectomy, use a dorsal approach.
	<ol>
		<li>Free the subcutaneous connective tissue by blunt dissection using the scissors.</li>
		<li>Pinch the fascia laterally to the midline make an incision and reach the peritoneal cavity using the sharp scissors.</li>
		<li>Grab the ovarian fat pad and the ovary gently with tweezers and pull it out of the abdominal cavity.</li>
		<li>Grasp the ovary and the fat pad with a vascular clamp.</li>
		<li>Ligate the ovary and the fat pad using a 4/0 monofilament absorbable suture at the base of the ovary, distal to the fallopian tube.</li>
		<li>Clip the ovary distal to the tie. Make sure there is no bleeding from the base of the stump.</li>
		<li>Place the tissue back into the abdominal cavity and suture the fascia using a 6/0 braided absorbable suture.</li>
		<li>Repeat stages 7.15.1-7.15.7 at the contralateral side.</li>
	</ol>
	</li>
	<li>Co-transplant the ovarian tissue.
	<ol>
		<li>Perform a horizontal incision in the fascia above the Gluteus Maximus, at the length that fits the clots dimensions. Create a pocket within this virtual space by opening the scissors underneath the fascia.</li>
		<li>Pick up one piece of encapsulated cortical tissue, as prepared in step 6.4.8 and place it in this pocket.</li>
		<li>Suture the fascia using a 6/0 braided absorbable suture.</li>
	</ol>
	</li>
	<li>Repeat stages 7.16.1-7.16.3 at the contralateral side as well.</li>
	<li>Close the dorsal wall using simple interrupted stitches with a 4/0 monofilament absorbable suture.</li>
	<li>Inject Lidocaine 0.5% (1.2 mL/Kg) S/C at the incision site.</li>
	<li>Use a sterile alcohol prep to clean the skin.</li>
	<li>Turn off the isoflurane while monitoring the mouse&#39;s temperature.</li>
	<li>Provide 1-2 min of O2, without Isoflurane. Keep on warming the mouse until it starts to move and return to consciousness.</li>
	<li>Place the mouse in a clean recovery cage and later house the mouse in a separate cage until the complete healing of the surgical wound.</li>
	<li>Inject Buprenorphine (1 mg/Kg) S/C as needed every 8-12 h, for 24 h postoperatively.</li>
	<li>Keep the mice in separate cages when all food, water, bedding, and cages are autoclaved, until the end point of the experiment. Keep the cages in a pressurized ventilated room. Use personal protective equipment whenever handling the mice.</li>
</ol>

<ol>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Statistics,+2017.">Cancer Statistics, 2017.</a> CA Cancer J Clin. 67 (1), 7-30 (2017).</li><li>Magelssen, H., Melve, K. K., Skjaerven, R., Fossa, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parenthood+probability+and+pregnancy+outcome+in+patients+with+a+cancer+diagnosis+during+adolescence+and+young+adulthood.">Parenthood probability and pregnancy outcome in patients with a cancer diagnosis during adolescence and young adulthood.</a> Hum Reprod. 23 (1), 178-186 (2008).</li><li>Donnez, J., Dolmans, M. M., Diaz, C., Pellicer, A. <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:+time+to+move+on+from+experimental+studies+to+open+clinical+application.">Ovarian cortex transplantation: time to move on from experimental studies to open clinical application.</a> Fertil Steril. 104 (5), 1097-1098 (2015).</li><li>Stoop, D., Cobo, A., Silber, S. <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+for+age-related+fertility+decline.">Fertility preservation for age-related fertility decline.</a> Lancet. 384 (9950), 1311-1319 (2014).</li><li>Aubard, Y., 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=Orthotopic+and+heterotopic+autografts+of+frozen-thawed+ovarian+cortex+in+sheep.">Orthotopic and heterotopic autografts of frozen-thawed ovarian cortex in sheep.</a> Hum Reprod. 14 (8), 2149-2154 (1999).</li><li>Newton, H., Aubard, Y., Rutherford, A., Sharma, V., Gosden, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+temperature+storage+and+grafting+of+human+ovarian+tissue.">Low temperature storage and grafting of human ovarian tissue.</a> Hum Reprod. 11 (7), 1487-1491 (1996).</li><li>Van Eyck, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+paramagnetic+resonance+as+a+tool+to+evaluate+human+ovarian+tissue+reoxygenation+after+xenografting.">Electron paramagnetic resonance as a tool to evaluate human ovarian tissue reoxygenation after xenografting.</a> Fertil Steril. 92 (1), 374-381 (2009).</li><li>Nugent, D., Newton, H., Gallivan, L., 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=Protective+effect+of+vitamin+E+on+ischaemia-reperfusion+injury+in+ovarian+grafts.">Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts.</a> J Reprod Fertil. 114 (2), 341-346 (1998).</li><li>Kim, S. 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>

Michael Ginsberg is an employee of Angiocrine Biosciences, Inc., San Diego, CA, 92130, United States, that isolated, transfected with E4-ORF- 1 and labeled the endothelial cells we used.

Here we demonstrate that co-transplantation of exECs provides a significant benefit to ovarian tissue viability and function following xenograft in mice. Standards for clinical application of ovarian tissue auto-transplantation for fertility preservation have not been set and the optimal parameters (size, transplantation site, duration of graft, etc.)32,33,34 for enhanced recovery of the follicular pool remain undefined...

Cancer remains among the leading causes of death in the developed world, yet decades of research have yielded significant progress for most types of cancer, and in some cases nearly doubled survival rates1. Unfortunately, chemotherapeutic agents are often gonadotoxic, depleting the reserve of primordial follicles in ovaries and reducing fertility2. This growing population can benefit from various methods of fertility preservation including oocyte and/or embryo cryopreservation, however, patients requiring prompt initiation of cancer therapy and pre-pubertal patients are ineligible for these options. As an alternative, some patients have chosen to cryopreserve ovarian tissue before undertaking their therapeutic regimen, and upon recovery and remission, auto-transplanting tissue to restore fertility3. Yet, to date, graft survival and follicular output following auto-transplantation remain relatively low4, mainly due to tissue ischemia and hypoxia5,6,7. Despite numerous efforts to improve the viability of ovarian cortical grafts using anti-oxidants8,9, pro-angiogenic cytokines10,11,12,13, or mechanical manipulations14, graft ischemia in a 5 to 7 day window post-transplant undermines the viability and survival of the graft7. To address this, we developed a cell-based strategy to facilitate anastomosis of host and graft vessels and thus hasten reperfusion of ovarian tissue.
In addition to the ischemic insult to grafted ovarian tissue in the post-transplant window, the disruption of inter-follicular signaling may contribute to depletion of the pool15,16. Because exogenous endothelial cells (ExECs) contribute to stable and functioning vessels in the periphery of the graft, they present a unique opportunity to convey a defined molecular input to transplanted tissue. As a proof of principle, ExECs were engineered to express super-physiological levels of Anti-Mullerian hormone (AMH), a member of the transforming growth factor beta (TGF&#946;) superfamily that has been shown to restrict follicular growth17. Comparison of follicular distribution in grafts co-transplanted with control and AMH-expressing cells verifies the biological activity and potency of engineered exECs.
In summary, by improving graft viability and suppressing premature mobilization of the follicular pool, this approach can increase the productivity of auto-transplanted ovarian tissue in patients undergoing fertility preservation. Moreover, the ExEC-based platform enables experimental interrogation of molecular regulators that have been implicated in follicular development.

<table><tbody><tr><td>Leibovitz&amp;rsquo;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&amp;trade; 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

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. As an alternative, an increasing number of these patients are choosing to cryopreserve ovarian tissue for autograft following recovery and remission. Despite improvements in outcomes among patients undergoing auto-transplantation of cryopreserved ovarian tissue, efficient revascularization of grafted tissue remains a major obstacle. To mitigate ischemia and thus improve outcomes in patients undergoing auto-transplantation, we developed a vascular cell-based strategy for accelerating perfusion of ovarian tissue. We describe a method for co-transplantation of exogenous endothelial cells (ExECs) with cryopreserved ovarian tissue in a mouse xenograft model. We extend this approach to employ ExECs that have been engineered to constitutively express Anti-Mullerian hormone (AMH), thus enabling sustained paracrine signaling input to ovarian grafts. Co-transplantation with ExECs increased follicular volume and improved antral follicle development, and AMH-expressing ExECs promoted retention of quiescent primordial follicles. This combined strategy may be a useful tool for mitigating ischemia and modulating follicular activation in the context of fertility preservation and/or infertility at large.

To determine whether co-transplantation of ExECs provides a benefit to patients&#39; tissue, thawed ovarian cortical strips were divided into equal sized pieces and engrafted bilaterally into immuno-compromised, NOD scid gamma (NSG), mice. With one side embedded in a fibrin clot alone (no ECs) and the other containing ExECs (Figure 1a), each mouse served as its own control. ExECs were obtained via isolation of primary endothelium from human umbilical cords an...

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

For some patients, the only option for fertility preservation is cryopreservation of ovarian tissue. Unfortunately, delayed revascularization undermines follicular viability. Here, we present a protocol to co-transplant human ovarian tissue with endothelial cells for utilization as a cell-based strategy combining accelerated perfusion with a direct paracrine delivery of bioactive molecules.

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

co-transplantation-human-ovarian-tissue-with-engineered-endothelial

Research

JoVE Journal

Bioengineering

7.4K Views.  Weill Cornell Medical College. Voor sommige patiënten is de enige optie voor behoud van de vruchtbaarheid cryopreservatie van eierstokweefsel. Helaas ondermijnt vertraagde revascularisatie folliculaire levensvatbaarheid. Hier presenteren we een protocol om mede de transplantatie van menselijke eierstokweefsel met endotheliale cellen voor gebruik als een cel-gebaseerde strategie combineren versnelde perfusie met een directe paracrine levering van biologische actieve moleculen.

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

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

Cancer Research

Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is being used to investigate the health-protective effects of young ovaries in young females. The current procedures employ an orthotopic transplantation method, where allogenic ovaries are transplanted back to their original position in the ovarian bursa. This is in contrast to the more commonly used heterotopic transplantation of ovaries/ovarian tissue subcutaneously or under the kidney capsule. All three locations provide efficient revascularization of the transplanted tissues. However, orthotopic transplantation provides the ovary with the most natural signaling environment and is the only procedure that provides the opportunity for the animal to reproduce naturally post-operatively. One must take care to remove all endogenous ovarian tissue during the ovariectomy procedure. If any endogenous tissue remains or if only one ovary is removed, the transplanted tissue will remain dormant until the existing tissue becomes senescent. While revascularization of the transplanted ovaries occurs very quickly, the transplant recipient can take a considerable amount of time to adapt to a new hypothalamic/pituitary/gonadal/adrenal (HPG/A) axis signaling regime associated with the transplanted tissue. This normally takes about 100 days in the mouse. Therefore, transplantation experiments should be designed to accommodate this adaptation period. Typical results with ovarian transplantation will include changes in the health of the recipient that reflect the age of the transplanted ovary, rather than the chronological age of the recipient.

Described here are orthotopic ovarian transplantation protocols for modifying ovarian influence on the physiology of the recipient mouse. This surgical procedure is used to expose female mice to various types of ovarian influence and may include heterochronic transplantations and transplantation of ovaries that have undergone various ex vivo treatments.

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is ...

Developmental Biology

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

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

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

Human Ovarian Epithelium Organoids: A Model for Tissue Repair and Drug Testing

Human Ovarian Surface Epithelium Organoids as a Platform to Study Tissue Regeneration

The ovarian surface epithelium (OSE), the outermost layer of the ovary, undergoes rupture during each ovulation and plays a crucial role in ovarian wound healing while restoring ovarian integrity. Additionally, the OSE may serve as the source of epithelial ovarian cancers. Although the OSE regenerative properties have been well studied in mice, understanding the precise mechanism of tissue repair in the human ovary remains hampered by limited access to human ovaries and suitable in vitro culture protocols. Tissue-specific organoids, miniaturized in vitro models replicating both structural and functional aspects of the original organ, offer new opportunities for studying organ physiology, disease modeling, and drug testing. 
Here, we describe a method to isolate primary human OSE (hOSE) from whole ovaries and establish hOSE organoids. We include a morphological and cellular characterization showing heterogeneity between donors. Additionally, we demonstrate the capacity of this culture method to evaluate hormonal effects on OSE-organoid growth over a 2-week period. This method may enable the discovery of factors contributing to OSE regeneration and facilitate patient-specific drug screenings for malignant OSE.

Author Spotlight: Advancing Human Ovarian Repair and Cancer Studies with Surface Epithelium Organoids

This protocol describes establishing three-dimensional (3D) tissue organoids from primary human ovarian surface epithelium (hOSE) cells. The protocol includes isolation of hOSE from freshly collected ovaries, cellular expansion of the hOSE, cryopreservation-thawing procedures, and organoid derivation. Immunofluorescence, quantitative analysis, and showcasing utility as a screening platform are included.

The ovarian surface epithelium (OSE), the outermost layer of the ovary, undergoes rupture during each ovulation and plays a crucial role in ovarian wound ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Medicine

Characterization of Blood Outgrowth Endothelial Cells (BOEC) from Porcine Peripheral Blood

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, inflammation, and homeostasis. The endothelium also plays an important role in pathophysiologies such as atherosclerosis, hypertension, and diabetes. Endothelial cells form the inner lining of blood and lymphatic vessels and display heterogeneity in structure and function. Various groups have evaluated the functionality of endothelial cells derived from human peripheral blood with a focus on endothelial progenitor cells derived from hematopoietic stem cells or mature blood outgrowth endothelial cells (or endothelial colony-forming cells). These cells provide an autologous resource for therapeutics and disease modeling. Xenogeneic cells may provide an alternative source of therapeutics due to their availability and homogeneity achieved by using genetically similar animals raised in similar conditions. Hence, a robust protocol for the isolation and expansion of highly proliferative blood outgrowth endothelial cells from porcine peripheral blood has been presented. These cells can be used for numerous applications such as cardiovascular tissue engineering, cell therapy, disease modeling, drug screening, studying endothelial cell biology, and in vitro co-cultures to investigate inflammatory and coagulation responses in xenotransplantation.

Characterization of Blood Outgrowth Endothelial Cells BOEC from Porcine Peripheral Blood

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, ...

Transplantation of Bioengineered Lung Using Decellularized Mouse Lungs and Primary Human Endothelial Cells

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor shortages and posttransplant complications persist. Bioengineered lungs, integrating patient-specific cells into decellularized animal scaffolds, present a promising alternative. Despite progress in using bioengineered lungs in animal models, functionality and structure remain immature. This protocol addresses a critical barrier in organ bioengineering: the need for a cost-effective experimental platform. By using mouse models instead of larger animals like rats or swine, researchers can significantly reduce the resources required for each experiment, accelerating research progress. 
The protocol outlines a detailed procedure for lung bioengineering using mouse heart-lung blocks and human primary cells, focusing on isolation strategy for the mouse heart-lung block, decellularization, bioreactor setup, perfusion-based organ culture, and orthotopic transplantation of bioengineered lungs. This mouse-scale platform not only reduces experimental costs but also provides a viable framework for optimizing cell types and numbers for recellularization, testing different cell types using histological and molecular methods, and ensuring blood flow post-transplantation. The method holds potential for broad applications, including studying cell interactions in three-dimensional culture conditions, cell-matrix interactions, and ex vivo cancer modeling, thereby advancing the field of organ bioengineering.

This paper describes how to create bioengineered mouse lungs using decellularization and recellularization methods. It also details subsequent orthotopic lung transplantation.

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Biology

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts ...

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

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