Name: a method for islet transplantation to the omentum in mouse
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about A Method for Islet Transplantation to the Omentum in Mouse at JoVE.com

Some of the authors of this work were supported in part by grants from National Key R&#38;D Program of China (2017YFC1103704), Sanming Project of Medicine in Shenzhen (SZSM201412020), Fund for High Level Medical Discipline Construction of Shenzhen (2016031638), Shenzhen Foundation of Science and Technology (JCJY20160229204849975, GJHZ20170314171357556, JCYJ20160425110110658), Shenzhen Foundation of Health and Family Planning Commission (SZXJ2017021).

All mice used in this study were obtained from the Medical Animal Center of Guangdong Province. The use of animals was approved by the Ethics Review Committee of Shenzhen Second People&#39;s Hospital, in accordance with the principles of animal welfare.
1. Islet Transplantation to the Omentum
NOTE: This protocol requires 2 persons to accomplish.
<ol>
	<li>Assemble surgical materials which are listed in Table 1. Prepare aseptic field in surgical ar...

<ol>
	<li>Guariguata, 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=Global+estimates+of+diabetes+prevalence+for+2013+and+projections+for+2035.">Global estimates of diabetes prevalence for 2013 and projections for 2035.</a> Diabetes research and clinical practice. 103 (2), 137-149 (2014).</li><li>Bellin, M. 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=Potent+Induction+Immunotherapy+Promotes+Long-Term+Insulin+Independence+After+Islet+Transplantation+in+Type+1+Diabetes.">Potent Induction Immunotherapy Promotes Long-Term Insulin Independence After Islet Transplantation in Type 1 Diabetes.</a> American Journal of Transplantation. 12 (6), 1576-1583 (2012).</li><li>Moberg, 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=Production+of+tissue+factor+by+pancreatic+islet+cells+as+a+trigger+of+detrimental+thrombotic+reactions+in+clinical+islet+transplantation.">Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation.</a> The lancet. 360 (9350), 2039-2045 (2002).</li><li>Nilsson, B., Ekdahl, K. N., Korsgren, O. <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+instant+blood-mediated+inflammatory+reaction+to+improve+islets+of+Langerhans+engraftment.">Control of instant blood-mediated inflammatory reaction to improve islets of Langerhans engraftment.</a> Current Opinion in Organ Transplantation. 16 (6), 620-626 (2011).</li><li>Bellin, M. 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=Similar+islet+function+in+islet+allotransplant+and+autotransplant+recipients,+despite+lower+islet+mass+in+autotransplants.">Similar islet function in islet allotransplant and autotransplant recipients, despite lower islet mass in autotransplants.</a> Transplantation. 91 (3), 367-372 (2011).</li><li>Bruni, A., Gala-Lopez, B., Pepper, A. R., Abualhassan, N. S., Shapiro, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+cell+transplantation+for+the+treatment+of+type+1+diabetes:+recent+advances+and+future+challenges.">Islet cell transplantation for the treatment of type 1 diabetes: recent advances and future challenges.</a> Diabetes, metabolic syndrome and obesity: targets and therapy. 7, 211 (2014).</li><li>Shapiro, A. J., Pokrywczynska, M., Ricordi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+pancreatic+islet+transplantation.">Clinical pancreatic islet transplantation.</a> Nature reviews Endocrinology. 13 (5), 268-277 (2017).</li><li>Cantarelli, E., Piemonti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+transplantation+sites+for+pancreatic+islet+grafts.">Alternative transplantation sites for pancreatic islet grafts.</a> Current diabetes reports. 11 (5), 364 (2011).</li><li>Cantarelli, E., 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=Murine+animal+models+for+preclinical+islet+transplantation:+no+model+fits+all+(research+purposes).">Murine animal models for preclinical islet transplantation: no model fits all (research purposes).</a> Islets. 5 (2), 79-86 (2013).</li><li>Beli, E., 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=Erratum+to:+CX3CR1+deficiency+accelerates+the+development+of+retinopathy+in+a+rodent+model+of+type+1+diabetes.">Erratum to: CX3CR1 deficiency accelerates the development of retinopathy in a rodent model of type 1 diabetes.</a> J Mol Med (Berl). 95 (5), 565-566 (2017).</li><li>Hafner, 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=Regional+Patterns+of+Retinal+Oxygen+Saturation+and+Microvascular+Hemodynamic+Parameters+Preceding+Retinopathy+in+Patients+With+Type+II+Diabetes.">Regional Patterns of Retinal Oxygen Saturation and Microvascular Hemodynamic Parameters Preceding Retinopathy in Patients With Type II Diabetes.</a> Invest Ophthalmol Vis Sci. 58 (12), 5541-5547 (2017).</li><li>Schmidt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+islets+find+a+new+transplant+home+in+the+omentum.">Pancreatic islets find a new transplant home in the omentum.</a> Nature Biotechnology. 35 (1), 82017 (2017).</li><li>Voigt, 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=Prevalence+and+Progression+Rate+of+Diabetic+Retinopathy+in+Type+2+Diabetes+Patients+in+Correlation+with+the+Duration+of+Diabetes.">Prevalence and Progression Rate of Diabetic Retinopathy in Type 2 Diabetes Patients in Correlation with the Duration of Diabetes.</a> Exp Clin Endocrinol Diabetes. , (2017).</li><li>Baidal, D. 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=Bioengineering+of+an+Intraabdominal+Endocrine+Pancreas.">Bioengineering of an Intraabdominal Endocrine Pancreas.</a> New England Journal of Medicine. 376 (19), 1887-1889 (2017).</li><li>Espes, 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=Rapid+restoration+of+vascularity+and+oxygenation+in+mouse+and+human+islets+transplanted+to+omentum+may+contribute+to+their+superior+function+compared+to+intraportally+transplanted+islets.">Rapid restoration of vascularity and oxygenation in mouse and human islets transplanted to omentum may contribute to their superior function compared to intraportally transplanted islets.</a> American Journal of Transplantation. , (2016).</li><li>Berman, D. 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=Bioengineering+the+endocrine+pancreas:+intraomental+islet+transplantation+within+a+biologic+resorbable+scaffold.">Bioengineering the endocrine pancreas: intraomental islet transplantation within a biologic resorbable scaffold.</a> Diabetes. 65 (5), 1350-1361 (2016).</li><li>Delaune, V., Berney, T., Lacotte, S., Toso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraportal+islet+transplantation:+the+impact+of+the+liver+micro-environment.">Intraportal islet transplantation: the impact of the liver micro-environment.</a> Transplant International. 30 (3), 227-238 (2017).</li><li>Espes, 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=Rapid+restoration+of+vascularity+and+oxygenation+in+mouse+and+human+islets+transplanted+to+omentum+may+contribute+to+their+superior+function+compared+to+intraportally+transplanted+islets.">Rapid restoration of vascularity and oxygenation in mouse and human islets transplanted to omentum may contribute to their superior function compared to intraportally transplanted islets.</a> American Journal of Transplantation. 16 (11), 3246-3254 (2016).</li><li>Hajizadeh-Saffar, E., 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=Inducible+VEGF+expression+by+human+embryonic+stem+cell-derived+mesenchymal+stromal+cells+reduces+the+minimal+islet+mass+required+to+reverse+diabetes.">Inducible VEGF expression by human embryonic stem cell-derived mesenchymal stromal cells reduces the minimal islet mass required to reverse diabetes.</a> Scientific Reports. 5, (2015).</li></ol>

Islet transplantation to the liver via the portal vein is the most commonly used method of islet transplantation in humans, but there are still efficiency and safety concerns such as portal vein thrombosis and liver steatosis17. Recent studies show that the omentum may be a suitable alternative to the liver, but more research needs to be conducted prior to clinical translation12,14,18,<sup clas...

According to the International Diabetes Federation (IDF), diabetes mellitus currently affects 382 million people, with a projected increase to 592 million people by 20351. In both allogeneic and xenogeneic islet transplantation, systemic immunosuppressive therapy is necessary. Without immunosuppression, immune rejection is a major cause of graft loss2. There is also a significant problem of transplanted islet loss due to the instant blood mediated inflammatory reaction (IBMIR)3,4. However, even in the absence of an immune response such as in syngeneic or auto-transplantation models, islet cells transplanted into the liver via the portal vein are lost due to inflammation and/or to unfavorable environmental conditions, such as poor blood supply with reduced oxygenation and/or nutrients5,6. As a result, in order to ensure long-term metabolic function, higher islet numbers are necessary to compensate for the initial cell loss that reduces engraftment7.
In an attempt to optimize islet engraftment, several alternative anatomical sites have been investigated experimentally as well as clinically, with promising, yet not definitive results8. Whereas some of the alternative sites offer easy and safe access (e.g., skin, kidney capsule, gastric submucosa and anterior chamber of the eye) or a wider surface for larger islet masses (e.g., peritoneal cavity), survival and physiologic metabolic performance of the transplanted islets are still limited and remain a concern9. The search for a more suitable site for islet engraftment is ongoing.
The omentum was among the many anatomical sites that were investigated in the early development of islet transplantation, and proved a successful environment for islets10,11,12,13,14. However, intraportal islet infusion became the clinical choice due in part to the relative simplicity of the procedure and early success in animal models6. Also, in part, the negatives associated with this site, particularly massive early islet loss, were less understood and less constraining in the early days of experimental islet transplantation as the field matured. With more recent compelling evidence indicating that intravascular islet infusion is far from ideal, the omentum is re-emerging as a potentially valuable site for cell transplantation.
The omentum (in the form of an omental pouch) offers relative advantages over the liver15,16. It is well-vascularized and easily accessible. It allows retrieval of the graft (if necessary) and/or biopsy. The ischemic period experienced by the islets is reduced compared to the liver, and the omentum can accept relatively large islet masses which is not possible intraportally, where a rise in portal pressure can cause complications.
A syngeneic mouse model of transplantation was used in the protocol tested in the study, employing C57BL/6 male mice between 6&#8211;8 weeks old with a body weight of 20&#8211;25 g. Islet recipients were rendered diabetic with a single injection of streptozotocin with a dose of 250 mg/kg ip. The induction of diabetes can be considered successful if the blood glucose level of the mouse is greater than 24 mmol/L 48 h after injection and remains above that level for a minimum of 5 days.
Syngeneic islets were isolated from the pancreas of age-matched donors following previously published methods with some modifications. In brief, the collagenase was injected into the gall bladder instead of the bile duct. This was done as an improvement to facilitate the ease of injection. Collagenase infusion was followed by incubation, tissue disruption, density gradient separation and hand-picking to obtain pure islets. Islets were cultured overnight in CMRL-1066 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) in T175 flasks at 37 &#176;C, under 95% air-5% CO2 before transplantation.

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			<td height="19" style="height:19px;width:476px;">Equipment</td>
			<td style="width:385px;"></td>
			<td style="width:135px;"></td>
			<td style="width:237px;"></td>
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			<td height="19" style="height:19px;width:476px;">5 inch (12-13 cm) Scissors</td>
			<td>RWD Life Science&#160;</td>
			<td>S12030-11</td>
			<td></td>
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			<td height="19" style="height:19px;width:476px;">Fine Forceps</td>
			<td style="width:385px;">RWD Life Science</td>
			<td>F11010-13</td>
			<td></td>
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		<tr height="19">
			<td height="19" style="height:19px;width:476px;">Small wound clips</td>
			<td style="width:385px;">RWD Life Science</td>
			<td>R33003-01</td>
			<td></td>
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			<td height="19" style="height:19px;width:476px;">Acutenaculum</td>
			<td style="width:385px;">RWD Life Science</td>
			<td>F31044-13</td>
			<td></td>
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		<tr height="19">
			<td height="19" style="height:19px;width:476px;">2 pair tissue forceps</td>
			<td style="width:385px;">RWD Life Science</td>
			<td>&#160;F13023-10</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:476px;">4-0 Suture with needle</td>
			<td style="width:385px;">Chenghe, China</td>
			<td>17094</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:476px;">200&#956;l Pipette and tips</td>
			<td style="width:385px;">Gilson</td>
			<td>PN11</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:476px;">One Touch ultraeasy Basic blood glucose monitoring system&#160;&#160;</td>
			<td style="width:385px;">Johnson &#38; Johnson</td>
			<td>33391713</td>
			<td></td>
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		<tr height="19">
			<td height="19" style="height:19px;width:476px;">razor blade</td>
			<td style="width:385px;">Philips</td>
			<td>HC1099/15</td>
			<td></td>
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		<tr height="19">
			<td height="19" style="height:19px;width:476px;">Material and animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:476px;">Pentobarbital Sodium</td>
			<td style="width:385px;">Sigmaaldrich.com</td>
			<td>P3761</td>
			<td>For anesthesia&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:476px;">Hydrogel&#160;</td>
			<td style="width:385px;">bdbiosciences.com</td>
			<td>356234</td>
			<td>Basement Membrane Matrix</td>
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		<tr height="19">
			<td height="19" style="height:19px;width:476px;">Fibrin-Thrombin Hydrogel&#160;</td>
			<td style="width:385px;">Baxter.com</td>
			<td>1501250</td>
			<td>Components clot when mixed</td>
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		<tr height="19">
			<td height="19" style="height:19px;width:476px;">70% Ethanol</td>
			<td style="width:385px;">Yingniu medical, Anhui, China</td>
			<td>23170608</td>
			<td></td>
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		<tr height="19">
			<td height="19" style="height:19px;width:476px;">Iodophor</td>
			<td style="width:385px;">Lierkang medical technology, Shangdong, China</td>
			<td>170521</td>
			<td></td>
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			<td height="19" style="height:19px;width:476px;">Normal saline</td>
			<td style="width:385px;">Baxter.com</td>
			<td>2B1324</td>
			<td></td>
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		<tr height="19">
			<td height="19" style="height:19px;width:476px;">Cephalosporin</td>
			<td style="width:385px;">Lukang medical, Shangdong, China</td>
			<td>150303</td>
			<td></td>
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		<tr height="19">
			<td height="19" style="height:19px;width:476px;">Cefazolin</td>
			<td style="width:385px;">Baxter.com</td>
			<td>2G3508</td>
			<td></td>
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		<tr height="19">
			<td height="19" style="height:19px;width:476px;">lubricant eye ointment</td>
			<td>Major Pharmaceuticals</td>
			<td>203964</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:476px;">streptozotocin</td>
			<td>Sigmaaldrich.com</td>
			<td>S0130</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:476px;">collagenase Type V</td>
			<td>Sigmaaldrich.com</td>
			<td>C9262</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:476px;">CMRL-1066 media</td>
			<td>celltrans, Wenzhou, China</td>
			<td>X018D1</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:476px;">histopaque</td>
			<td>Sigmaaldrich.com</td>
			<td>10771</td>
			<td style="width:237px;">density gradient</td>
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		<tr height="19">
			<td height="19" style="height:19px;width:476px;">PE50 tubing</td>
			<td>Braintreesci.com</td>
			<td>PE50 100 FT</td>
			<td>Polyethylene .023&#34; x .038</td>
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		<tr height="19">
			<td height="19" style="height:19px;width:476px;">Calcein AM</td>
			<td>Sigmaaldrich.com</td>
			<td>C1359</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:476px;">Propidium iodide</td>
			<td>Sigmaaldrich.com</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:476px;">optimal cutting temperature compound (OCT)</td>
			<td>Tissue-Tek; Miles, Naperville, IL</td>
			<td>4583</td>
			<td>embedding medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:476px;">insulin antibody</td>
			<td>Cell Signaling Technology, Danvers, MA 01923</td>
			<td>8138S</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:476px;">hematoxylin staining media</td>
			<td>Cell Signaling Technology, Danvers, MA 01923</td>
			<td>14166S</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:476px;">eosin staining media</td>
			<td>Beyotime Biotech, China</td>
			<td>C0109</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:476px;">DAPI</td>
			<td>Thermo Fisher Scientific Inc.&#160;</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:476px;">C57Bl/6 Mice</td>
			<td style="width:385px;">Medical Animal Center of Guangdong Province</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Fetal Bovine Serum</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30084</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">T175 flasks</td>
			<td>Falcon</td>
			<td>353112</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">1.5mL Snap-top tubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

a method for islet transplantation to the omentum in mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

Post-digestive state of the pancreas is shown in Figure 1A. Purified islets are shown in Figure 1B. Dithizone staining and viability testing of the islets are shown in Figure 2. The main steps of the islet transplantation to the omentum are shown in Figure 3. The blood glucose levels of the recipients after omental transplantation are shown in Figure...

Watch this Scientific Journal Video about A Method for Islet Transplantation to the Omentum in Mouse at JoVE.com

A Method for Islet Transplantation to the Omentum in Mouse

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...

This method can help answer key questions in the islet transplantation field such as islet transplantation to the omentum. The implications of this technique extend toward therapy of type I diabetes because the omentum is re-emergent as a potential value site for islet transplantation. The main advantage of this technique is that the isolated islets are mixed with hydrogel and the mixture is placed into the omentum pouch of diabetic mouse.Begin by preparing an aseptic field in the surgical area with sterile material such as drapes and disposables. Place the sterilized surgical instruments in the aseptic field. Pick the donor islets under the stereo-microscope using a 200 microlitre pipette tip.For each animal, collect 450 to 500 islets and transfer to a sterile 1.5 milliliter snap top tube with 100 microlitres of CMRL 10 66 medium. Place the tube on ice until ready to transplant. Once the islets have been harvested, thaw basement membrane matrix hydrogel on ice.Weigh and tag all diabetic recipient mice. After anesthetizing the first mouse according to an institutionally approved protocol, test the depth of anesthesia by administering a toe pinch. If there is no withdrawal reflex, the level of anesthesia is correct for surgery.Administer vet ointment to the eyes to prevent dryness while under anesthesia. Swab the abdomen with 70%ethanol. Then shave the hair from the surgery site.Cover the animal with a sterile drape, and disinfect the area with an iodine scrub. After using ophthalmic scissors to create a 4 to 5 centimeter incision along the midline of the abdomen, move the intestines to the left side and cover with saline soaked gauze to prevent dehydration during the surgery procedure. Then use cotton swabs to fully expose the stomach and locate the omentum below the stomach.At this point, the hydrogel should be completely thawed. Spin the tube containing the islets at 200 times g for 30 seconds. After the spin, remove the supernatant.Then aspirate 50 microliters of the hydrogel add it to the tube containing the islets and mix gently avoiding the formation of bubbles. Next use two pairs of forceps to pick up the edges of the omentum and gently raise it to form a groove between the gastric wall and the intestines. At this point in the surgery, a second person is needed to aspirate the resuspended islet hydrogel mixture with a 200 microliter pipette tip and deliver it into the groove.Ensure that the mixture is well positioned in the groove by gently raising or lowering the edges of the omentum. Complete the positioning of the mixture within three minutes before the hydrogel solidifies. After the hydrogel sets, fold the omentum to cover the graft.The omentum will adhere to the surrounding gastric wall as the hydrogel solidifies. When the hydrogel is completely solidified, use cotton swabs to reposition the intestines in the abdominal cavity taking care not to touch the site of transplantation. Next dispense 200 microliters of an appropriate antibiotic into the abdominal cavity to prevent infection.Then use 4 O suture to close the abdomen. Generally individuals new to this method will struggle because the sites of the omentum in mice is small and tiring to assess. Place the mouse into a warmed recovery cage until it fully regains external recumbency.The next day and every day thereafter for one week, inject antibiotic as post-surgery prophylaxis. Also, measure non-fasting blood glucose level from a tail vein blood sample using a blood glucose meter once a day after the transplant. When transplanting to the omentum, the islet graft may have a delayed function and not reach a completely normal blood glucose level for two to three weeks.The grafted tissue was retrieved from the euthanized mouse 14 days after transplantation. Here the graft is visible as a cluster of nuclei after DAPI staining. The functionality of the grafted tissue is revealed by immunofluorescence staining with anti-insulin antibodies.H&E staining shows the grafted tissues surrounded by the adipocytes of the omentum. Once mastered, this technique can be done in one hour if it is performed properly. While attempting this procedure, it is important to remember too the mouse omentum is fragile.Following this procedure, other methods like used in different high school, MB performance in all that will answer particular questions like the spread of islet in vascuolization and the performance. After this development, this technique paved the way for researchers in their and transplantation to explore treatment for type I diabetes in more than one excerpt. After watching this video, you should have a good understanding of how to transplant the islets to the omentum in the mouse.

Research

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