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.

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Research

JoVE Journal

Biology

Tracheotomy: A Method for Transplantation of Stem Cells to the Lung

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell therapy may alleviate many of the symptoms associated with lung disease and induce alveolar repair in adults. Concurrent with the ongoing search for stem cells applicable for human treatment, precise delivery and homing (to the site of disease) must be reassured for successful therapy. Here, I report that stem cells can safely be instilled via the trachea opening a non-stop route to the lung. This method involves a skin incision, caudal insertion of a cannula into and along the tracheal lumen, and injection of a stem cell vehicle mixture into airways of the lung. A broad range of media solutions and stabilizers can be instilled via tracheotomy, resulting in the ability to deliver a wider range of cell types. With alveolar epithelium confining these cells to the lumen, lung expansion and negative pressure during inhalation may also assist in stem cell integration. Tracheal delivery of stem cells, with a quick uptake and the ability to handle a large range of treatments, could accelerate the development of cell-based therapies, opening new avenues for treatment of lung disease.

Significant breakthroughs in stem cell identification are continuously being made. To translate these discoveries, however, novel methods for cellular delivery must be devised. Here I report that the airways provide a safe route for stem cell transplantation to the lungs.

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell ...

Murine Pancreatic Islet Isolation

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...