Name: Video: Ovarian Fat Pad Transplantation Assay: An In Vivo Technique to Introduce Cells into Ovarian Fat Pad in Mouse Models - Concept
Uploaded: 2023-04-30T13:27:18.000+00:00
Duration: 1 min 29 s
Description: 1.0K Views. Source: Flesken-Nikitin, A. et al. Transplantation Into the Mouse Ovarian Fat Pad. J. Vis. Exp. (2016) In this video, we demonstrate an assay for transplantation of experimental cells and tissue fragments into the mouse ovarian fat pad. This method can be used to monitor cell regeneration and tumor transformation via non-invasive techniques.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Ovarian Fat Pad Transplantation
NOTE: Disinfect instruments between each animal surgery. Prepare and arrange all equipment in advance. Dorsal transplantations to the right ovarian fat pad are preferential as this avoids the spleen. However, recipients may have transplants in both ovarian fat pads.

<ol>
	<li>Use syngeneic mice or severe combined immunodeficient (SCID/NCr) mice as recipients of primary cells. For human cells, such as HIO118 cells, use Nod/SCID/gamma (NSG) mice or SCID mice.</li>
	<li>Anesthetize the recipient mouse with a single intraperitoneal injection of 2,2,2-Tribromoethanol at a dosage of 0.15 mL/10 g body weight. Place the animal on a heating pad in the animal hood and confirm proper anesthetization by loss of pedal reflex (toe pinch). Apply vet ointment on the eyes to prevent dryness while the animal is under anesthesia. Inject the animal subcutaneously with the analgesic Ketoprofen at a 4 mg/ g body weight dosage to treat post-surgical pain. 
	NOTE: As an alternative, use isoflurane for anesthesia. Place the animal in the induction chamber and adjust the oxygen flowmeter to 0.8-1.5 L/min and the isoflurane vaporizer to 2-3%. Remove the anesthetized animal from the chamber, let it breathe isoflurane from a mask and set the oxygen flowmeter to 0.4-0.8 L/min and the isoflurane vaporizer to 1.5%, then start the surgery.</li>
	<li>Shave the surgical area with #40 clippers; remove hair from a site two times the surgical area, prepare the shaved skin with three antiseptic scrubs of povidone-iodine, followed by 70% ethanol, and cover with a sterile drape.</li>
	<li>Move the animal under a dissection microscope located in a Biosafety cabinet. Expose the reproductive tract by an incision in the dorsomedial position directly above the ovarian fat pad. 
	CRITICAL STEP: Precise skin incision facilitates wound healing; the use of a scalpel is recommended in step 1.4.</li>
	<li>Using fine blunt forceps, pull the ovarian fat pad carefully through the incision towards the midline, minimizing damage to the nerves and major blood vessels. 
	CRITICAL STEP: If bleeding occurs, stop the surgery and consider transplanting to a different host animal.</li>
	<li>Under the control of the dissection microscope with the help of a 28 gauge beveled needle, make a 2-4 mm deep incision into the ovarian fat pad, 3-4 mm above the ovary. 
	CRITICAL STEP: Ensure that the incision only goes through half of the fat pad; puncturing through to the bottom side causes leakage. Be swift and proceed without delay after this step.</li>
	<li>For cell transplantations, fill a syringe (30-gauge needle) with 10-20 &#181;L of the cell-basement membrane matrix mixture and inject it into the fat pad incision. For uterine tube transplantation, pick up the uterine tube with fine forceps and place it in a 10-20 &#181;L basement membrane matrix kept on ice. Using a 0.1-10/20 &#181;L XL graduated filter tip (shortened by 3 mm), pick up the tissue and basement membrane matrix suspension and release it into the fat pad incision.</li>
	<li>Wait 5 minutes for the basement membrane matrix to solidify and place the reproductive tract back into the peritoneum. Close the muscles with two stitches of surgical suture and the skin with two small wound clips or surgical sutures.</li>
	<li>Repeat steps 1.4-1.8 to transplant a PBS-basement membrane matrix mixture into the animal&#39;s contra-lateral fat pad, which will serve as a control. 
	CRITICAL STEP: After the surgery, place the animal on a heating pad because heat loss is rapid in anesthetized mice.</li>
	<li>Let the animal recover on a heating pad in the native cage and observe the animal until fully awake from the anesthesia. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until fully recovered.</li>
	<li>Place a handful of pre-wet food pellets inside the cage in addition to dry food on the cage after surgery. Apply post-surgical analgesics if needed for the next few days and examine the wound daily. Apply antibiotics according to the approved animal protocol if wound infection occurs. Remove wound clips ten days after surgery when the wound has fully closed.</li>
</ol>

<table><tbody><tr><td>28 gauge needle (hypodermic syringe with attached needle)&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Kendall</td><td>30339</td><td>Part Number: 8881500014</td></tr><tr><td>Basement membrane matrix&lt;br/&gt; (Geltrex)</td><td>Invitrogen</td><td>&amp;nbsp;A1413202</td><td></td></tr><tr><td>Ethanol 200 proof</td><td>Koptec&amp;nbsp;</td><td>V1001&amp;nbsp;</td><td>To prepare 70%&amp;nbsp;</td></tr><tr><td>Filter tip 0.1-10/20 &amp;micro;l XL&amp;nbsp;</td><td>USA Scientific&amp;nbsp;</td><td>1120-3810</td><td></td></tr><tr><td>Isoflurane, 250 ml&amp;nbsp;</td><td>Santa Cruz Animal Health&amp;nbsp;</td><td>sc-363629Rx</td><td></td></tr><tr><td>Ketoprofen</td><td>Zoetis</td><td>&amp;nbsp;4396H</td><td></td></tr><tr><td>Microscope, stereo</td><td>Nikon</td><td>&amp;nbsp;SMZ800</td><td></td></tr><tr><td>Nod/SCID/gamma (NSG) mice</td><td>The Jackson Laboratory</td><td>5557</td><td>NOD.Cg-Prkdc&lt;sup&gt;scid&lt;/sup&gt;Il2rg&lt;sup&gt;tm1Wjl&lt;/sup&gt;/SzJ</td></tr><tr><td>severe combined&lt;br/&gt; immunodeficiency (SCID/NCr)&lt;br/&gt; mice, BALB/C background)</td><td>NCI-Frederick Animal Production Program</td><td>01S11</td><td></td></tr></tbody></table>

ovarian fat pad transplantation assay: an in vivo technique to introduce cells into ovarian fat pad in mouse models

Source: Flesken-Nikitin, A. et al. <a href="https://www.jove.com/t/54444">Transplantation Into the Mouse Ovarian Fat Pad.</a> J. Vis. Exp. (2016)
In this video, we demonstrate an assay for transplantation of experimental cells and tissue fragments into the mouse ovarian fat pad. This method can be used to monitor cell regeneration and tumor transformation via non-invasive techniques.

Ovarian Fat Pad Transplantation Assay: An In Vivo Technique to Introduce Cells into Ovarian Fat Pad in Mouse Models

Source: Flesken-Nikitin, A. et al. Transplantation Into the Mouse Ovarian Fat Pad. J. Vis. Exp. (2016)
In this video, we demonstrate an assay for ...

Transplantation allows the implantation of cells or tissue fragments into specific body sites in a recipient animal. It helps us study the role of the microenvironment in tumorigenesis and metastasis.
To begin, prep an anesthetized immunocompromised female mouse in the prone position. Make a surgical incision in the dorsomedial position to expose its reproductive tract. Locate the ovarian fat pads situated close to the ovary and oviduct. Using a sharp-tipped needle, make an incision puncturing halfway through the fat pad without compromising the ovary.
Next, inject a chilled suspension of experimental cells mixed with a suitable basement membrane matrix into the fat pad incision. Allow the matrix to solidify and secure the cells within. Place the ovarian fat pad back into the peritoneum and suture the incision. Allow the mouse to recover.
On successful transplantation, the fat pad provides a suitable microenvironment, facilitating the expansion of the injected cells. Check the mouse weekly to observe outgrowth of transplanted cells on the ovarian fat pad.

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1.0K Views. Source: Flesken-Nikitin, A. et al. Transplantation Into the Mouse Ovarian Fat Pad. J. Vis. Exp. (2016) In this video, we demonstrate an assay for transplantation of experimental cells and tissue fragments into the mouse ovarian fat pad. This method can be used to monitor cell regeneration and tumor transformation via non-invasive techniques. 

Video: Ovarian Fat Pad Transplantation Assay: An In Vivo Technique to Introduce Cells into Ovarian Fat Pad in Mouse Models - Concept

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Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

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Developmental Biology

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 ...

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

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Ovarian Fat Pad Transplantation Assay: An In Vivo Technique to Introduce Cells into Ovarian Fat Pad in Mouse Models

Transcript

Ovarian Fat Pad Transplantation Assay: An In Vivo Technique to Introduce Cells into Ovarian Fat Pad in Mouse Models

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

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

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model