<meta charset="utf8"></head><body>הערכת כדאיות זקיקים ברקמות קליפת המוח השחלתית האנושית המבחנה

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.8 mL vials</td>
			<td>VWR International GmbH</td>
			<td>479-6837</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipette</td>
			<td>Sarstedt</td>
			<td>86.1254.001</td>
			<td></td>
		</tr>
		<tr>
			<td>4 well plate</td>
			<td>Gynemed</td>
			<td>GYOOPW-FW04</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Tube</td>
			<td>Sarstedt</td>
			<td>62.559.001</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Sarstedt</td>
			<td>83.3920</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacillol AF</td>
			<td>Hartmann</td>
			<td>973385</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein AM</td>
			<td>Merck</td>
			<td>17783</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type 1A</td>
			<td>Merck</td>
			<td>C2674</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryosure DMSO</td>
			<td>WAK Chemie</td>
			<td>WAK-DMSO-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Custodiol</td>
			<td>Dr. Franz K&#246;hler Chemie</td>
			<td>00867288</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS CTS</td>
			<td>Gibco Life technologies</td>
			<td>A12856-01</td>
			<td></td>
		</tr>
		<tr>
			<td>ErgoOne pipette aid</td>
			<td>Starlab</td>
			<td>S7166-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene glycol</td>
			<td>Sigma Aldrich</td>
			<td>102466</td>
			<td></td>
		</tr>
		<tr>
			<td>Euronda sterilization container</td>
			<td>euronda</td>
			<td>282021</td>
			<td></td>
		</tr>
		<tr>
			<td>G-MOPS+</td>
			<td>Vitrolife</td>
			<td>10130</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal meshes</td>
			<td>Sigma Aldrich</td>
			<td>S0770</td>
			<td></td>
		</tr>
		<tr>
			<td>Metzenbaum scissors</td>
			<td>world precision instruments</td>
			<td>501262102</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Bath System</td>
			<td>Nterilizer</td>
			<td>N-Bath 3.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone (PVP)</td>
			<td>SAGE</td>
			<td>ART-4005</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum substitute supplement (SSS)</td>
			<td>Fujifilm Irvine scientific</td>
			<td>99193</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cup</td>
			<td>Sarstedt</td>
			<td>75.562.105</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile forceps</td>
			<td>Carl Roth</td>
			<td>KL05.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Merck</td>
			<td>S0389</td>
			<td></td>
		</tr>
	</tbody>
</table>

vitrification of ovarian cortex tissue to achieve a glassy state of aggregation

Cryopreservation of ovarian tissue is an important option for fertility preservation. Explanted tissue containing ovarian follicles, in which oocytes are embedded, is cryopreserved. After storage, the ovarian tissue can be thawed, warmed, and reimplanted in the patient. For viable cells or tissue, two cryopreservation methods are available: slow freezing and vitrification1.
Vitrification is used to preserve biological materials, such as embryos and oocytes, with superior survival rates compared to the slow freezing protocol1,2,3,4. Slow freezing has limitations, such as ice crystal formation, which can potentially damage cell and tissue structures. However, slow freezing is an important cryopreservation approach that facilitates the long-term storage of biological samples, and the functionality of this method has been widely proven5. Vitrification induces a glassy state of aggregation, preventing ice crystal formation6,7. On a technical level, vitrification significantly simplifies the cryopreservation procedure by reducing equipment maintenance, decreasing the likelihood of technical errors, and shortening the duration of the cryopreservation process8,9. In female fertility preservation, ovarian tissue cryopreservation is a decisive approach prior to cancer treatment10. Different groups have successfully demonstrated the concept of cryopreservation, thawing, and transplantation of tissue based on the slow freezing protocol11,12,13,14, which is currently regarded as the standard approach15.
Vitrification of ovarian tissue is regarded as a promising alternative method16,17,18,19,20,21, in terms of resource-saving22, follicular survival rates, DNA fragmentation levels, and balanced angiogenic potential23,24,25,26,27. This is substantiated by successful deliveries in Japan28, the USA29, and Germany30.
Comparing the two options for ovarian tissue cryopreservation (OTC)-vitrification versus the standard procedure of slow freezing-results are partially conflicting in current meta-analyses16. Several factors may have contributed to this, as current vitrification protocols vary greatly. These differences include the choice of cryoprotectant or combination of protectants, their concentration, the composition of the OTC media, the size of tissue fragments, and the device used as a tissue carrier. Accordingly, there is no standardized warming protocol.
As the authors found a method that yields convincing results in terms of handling, viability, apoptosis onset, release of angiogenic factors, and even a report of a birth after reimplantation9,27, a very detailed description of the protocol is provided. The described method offers a valid and effective protocol that may contribute to the standardization of vitrification of ovarian tissue.

<meta charset="utf8"></head><body>מחבר זרקור: קידום שימור פוריות אצל חולות סרטן צעירות באמצעות ויטריפיקציה של רקמת השחלות וצמיחה זקיקית חוץ גופית

ויטריפיקציה של רקמת קליפת המוח השחלתית להשגת מצב זגוגי של צבירה

The scope of our research group is fertility protection in young female cancer patient. It is important to improve existing protocols to increase the chances of a later pregnancy. That includes the vitrification of ovarian tissue and also the in vitro growth of follicles With the in vitro growth of ovarian follicular.The crucial point is the multi-step tissue culture approach prior to the follicular isolation procedure as ovarian cortex tissue is very dense and limits follicular growth. Tissue vitrification is more complex than the slow freezing procedure, but it simplifies the whole process and has other advantages as well. We have shown that vitrification is a significant alternative to the common slow freezing protocol, which is supported by the previously reported five successful deliveries.The stress of freezing and thawing as well as the undersupply after reimplantation, together with the reperfusion damage impacts follicular survival and leads to a significant decrease in the number of the follicles. Limiting the success of the therapy option, vitrification is thought to preserve the follicles with a similar or even greater success compared to slow freezing approaches. We aim, therefore, to bring vitrification to a pre-clinical setting, that will provide all cryobank the possibility for tissue cryo-preservation, despite any personal and financial limitations.

Ovarian tissue cryopreservation (OTC) is an important option for fertility preservation. For patients whose gonadotoxic treatments cannot be postponed or ...

vitrification-ovarian-cortex-tissue-to-achieve-glassy-state

Research

JoVE Journal

Medicine

Vitrification of Ovarian Cortex Tissue to Achieve a Glassy State of Aggregation

826 Views.  University Hospital of Bonn. היקף קבוצת המחקר שלנו הוא הגנה על פוריות אצל נשים צעירות חולות סרטן. חשוב לשפר את הפרוטוקולים הקיימים כדי להגדיל את הסיכויים להריון מאוחר יותר. זה כולל את הויטריפיקציה של רקמת השחלה וגם את הצמיחה במבחנה של זקיקים עם הצמיחה במבחנה של זקיקי השחלות.הנקוד...

Video: מחבר זרקור: קידום שימור פוריות אצל חולות סרטן צעירות באמצעות ויטריפיקציה של רקמת השחלות וצמיחה זקיקית חוץ גופית

Ovarian tissue cryopreservation (OTC) is an important option for fertility preservation. For patients whose gonadotoxic treatments cannot be postponed or for pre-pubertal girls, it is often the only option for fertility protection. Cryopreservation can be performed either by vitrification or by slow freezing. Slow freezing is currently the standard approach. An increasing number of studies indicate that vitrification can replace slow freezing in the state-of-the-art in vitro fertilization (IVF) laboratories, significantly improving thawing survival rates and simplifying the technical aspects of cryopreservation. A metal grid-based, high-throughput protocol for rapid vitrification of ovarian cortex tissue, suitable for clinical routine, is described. The sterilization of metal grids and liquid nitrogen ensures high quality, meeting good manufacturing practice (GMP) standards. Vitrification was conducted to ensure ultra-rapid cooling rates. Instead of slowly thawing, samples were rapidly warmed. To assess follicular viability, calcein staining was performed both prior to cryopreservation and after rapid warming. The successful application of vitrification and rapid warming using metal grids is reported. No significant differences in follicular viability were observed prior to vitrification and after rapid warming. These results substantiate the high capacity of tissue vitrification for clinical routine applications as a potential substitute for the widely used slow-freezing method.

A protocol for the vitrification of ovarian tissue, as an alternative cryopreservation method to the widely used slow freezing protocol, is presented.

Vitrification and Rapid Warming of Human Ovarian Cortex Tissue for Follicular Viability Assessment

To begin, cut the metal meshes into strips measuring 25 by 8 millimeters. Place the customized meshes in a sterilization container and autoclave for two hours. After autoclaving, place the sterilization container and 1.8 milliliter vials under the laminar flow bench.Open the sterilization container and remove the metal grid. Fit the grid into the cap of the 1.8 milliliter vial and close the vial. After collecting human ovarian cortex tissue, remove the medulla and cut the tissue into desired shapes.Add five milliliters of vitrification solution 1 into the first well of the six-well plate. Using the cell strainer, equilibrate the ovarian cortex tissue for five minutes in well 1 of the plate. Move the cell strainer with the cortex tissue to well 2 containing five milliliters of vitrification solution 2 and equilibrate for five minutes.Then, place the strainer containing cortex tissue into well 3 of a six-well plate, containing five milliliters of vitrification solution 3 for six minutes. Fill the prepared cryo vial with sterilized liquid nitrogen and place it into the cryo dewar vessel filled with liquid nitrogen. Load the tissue samples onto the metal grid of the vitrification device within one minute.Then insert the metal grid into the liquid nitrogen in the grid-based cryo vial. Add six milliliters of equilibration solution to well 1 of a sterile six-well plate, then transfer six milliliters of RS, each to wells 2 and 3. Incubate for one hour at room temperature.Warm the rapid warming solution in a sterile sample beaker for one hour on a heating plate set at 37 degrees Celsius. Under the laminar flow bench, open the cryo-preserved vials containing vitrified ovarian cortical tissue. Rapidly transfer the mesh into the rapid warming solution.Using sterile forceps transfer the tissue to the equilibration solution and incubate for three minutes on a rocking shaker. Then, rinse the tissue at room temperature for 10 minutes each with RS1 and RS2 on a rocking shaker. Dissolve calcein in 100 microliters of DMSO.Transfer three microliters of calcein into the bottom of a 1.5 milliliter tube and store at minus 20 degrees Celsius. Add 0.007 grams of collagenase in a 1.5 milliliter tube, and store the tube at minus 20 degrees Celsius. Add 997 microliters of DPBS to the frozen three microliters of calcein and re-suspend to dissolve the calcein.Then add cold collagenase powder to obtain 1000 microliters of working solution. Then add 500 microliters of working calcein solution and transfer two pieces of two millimeters of ovarian cortex fragments into the wells of a four well dish. Then incubate for 90 minutes at 37 degrees Celsius, protecting from light.Finally, under fluorescence microscopy, determine the follicular viability.