intraperitoneal injection of cells: a method of delivering cancer cells into the peritoneal adipose tissue of murine model

Intraperitoneal Injection of Cells: A Method of Delivering Cancer Cells into the Peritoneal Adipose Tissue of Murine Model

Load 500 microliters of the fluorescently tagged single cell suspension into the syringe. Then, put the syringe into a sterile capped 25 gauge needle to reduce cell shearing prior to injection. Restraining the mouse with the other hand, pick the mouse up by the scruff of the neck and hold the tail using the palm and forefinger. Then, fix the left hind leg between the ring and little finger. To avoid traumatizing abdominal organs, restrain the mouse well so that it cannot move during the injection.
Imagine a line across the abdomen, and locate a point on the animal&#39;s right side and close to the midline. The point of entry is cranial to and slightly medial of the last nipple. Prepare to insert the needle on the mouse&#39;s right side to avoid the cecum and reduce the risk of puncturing the intestines. Insert the needle at the lower lateral region of the mouse&#39;s abdomen to a depth of approximately 0.5 centimeters. Pull back on the plunger to confirm that the needle has not penetrated a blood vessel or other peritoneal organs.
If no fluid is aspirated, inject the sample using slow steady pressure. Then, withdraw the needle and return the mouse to its cage. Do not recap syringe before disposal in the sharps container.

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All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Intraperitoneal Injection of Cells
NOTE: In our experiments, mice are handled in a laminar airflow or biosafety cabinet within our barrier facility in order to limit the risk of pathogen exposure. In order to demonstrate this technique clearly, the procedures in the accompanying video were conducted in a laboratory approved for animal work.&#160;This particular technique does not require the animals to be under anesthesia. Under approved protocols, perform this technique on live animals. Use appropriate strains of mice for this study. For example: use immunocompromised, athymic nude mice for the study of human ovarian cancer cell (SKOV3ip.1 and HeyA8) colonization; use immunocompetent C57BL/6 mice for study of mouse ovarian cancer cell (ID8) colonization.
<ol>
	<li>Load 500 &#181;l of single cell suspension of ovarian cancer cells into a syringe and put in the sterile capped 25 G needle. This reduces cell shearing prior to injection.</li>
	<li>Pick the mouse up by the scruff of the neck and hold the tail using the palm and forefinger and fix the left hind leg between the ring and little finger (when the mouse is restrained with the left hand). 
	NOTE: To avoid traumatizing abdominal organs, restrain the mouse well so that it cannot move during the injection.</li>
	<li>Imagine a line across the abdomen just above the knees, and locate a point on the animal&#39;s right side and close to the midline. The point of entry is cranial to and slightly medial of the last nipple. 
	NOTE: Inserting the needle on the mouse&#39;s right side avoids the cecum and reduces the risk of puncturing the intestines.</li>
	<li>Insert the needle at the lower lateral region of the mice&#39;s abdomen to a depth of approximately 0.5 cm. Pull back on the plunger to confirm that the needle has not penetrated a blood vessel or other peritoneal organs.
	<ol>
		<li>If any fluid is aspirated discard the syringe (and sample). A greenish-brown or yellow aspirate indicates needle penetration into the intestines or bladder, respectively.</li>
	</ol>
	</li>
	<li>Inject the sample using slow, steady pressure. Withdraw the needle and return the mouse to its cage. Do not recap the syringe before disposal in a sharp container.</li>
	<li>Sacrifice mice via CO2&#160;asphyxiation and vital organ removal at a specific time point post injection.</li>
</ol>

<table><tbody><tr><td>DMEM</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CV</td><td>Without calcium and Magnesium</td></tr><tr><td>26 gauge needle</td><td>BD</td><td>329652</td><td></td></tr></tbody></table>

Source: Krishnan, V. et al. <a href="https://www.jove.com/t/52721">Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose.</a> J. Vis. Exp. (2015)
In this video, we perform an intraperitoneal injection of ovarian cancer cell suspension in a mouse model to study the colonization of ovarian cancer cells in peritoneal adipose tissues and their subsequent metastasis.&#160;

Source: Krishnan, V. et al. Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose. J. Vis. Exp. 
...

The peritoneal cavity is the space between the abdominal wall and the lining of the internal organs. For intraperitoneal injection, begin by restraining a live mouse by its neck region such that the mouse&#8217;s head tilts downward with its ventral abdomen exposed.
Restraining the mouse restricts its movement and shifts the abdominal organs towards the diaphragm, preventing accidental damage to the organs during needle insertion.
Adjacent to the midline across the mouse&#8217;s abdomen, in the lower right quadrant, locate a point for needle insertion. This site prevents the risk of puncturing the caecum.
Next, take a syringe containing a single cell suspension of ovarian cancer cells. Insert the needle at the chosen point to a depth of approximately half a centimeter to penetrate just the outer abdominal wall. Finally, inject the cancer cell suspension slowly. This ensures efficient distribution of cells within the peritoneal cavity.
Return the mouse to its cage and monitor it for tumor growth. The tissues within the peritoneal cavity may release specific chemotactic molecules that attract the cancer cells leading to their migration and colonization, eventually contributing to tumor development.

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Intraperitoneal Injection of Cells: A Method of Delivering Cancer Cells into the Peritoneal Adipose Tissue of Murine Model

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Intraperitoneal Injection of Cells: A Method of Delivering Cancer Cells into the Peritoneal Adipose Tissue of Murine Model