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A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination
Published on: June 25, 2014
A rapid qualitative method to assess in vitro immunobarrier competence of pancreatic islets containing
R Calafiore1, G Basta, P Sarchielli
1Department of Internal Medicine and Endocrine and Metabolic Sciences (DIMISEM), University of Perugia, Italy.
Insights
A new method quickly evaluates alginate/poly-L-ornithine (AG/PLO) microcapsules for islet immunoisolation. These microcapsules prevented immune binding and human lymphocyte proliferation in vitro, showing promise for islet transplantation.
Area of Science:
- Biomaterials Science
- Immunology
- Transplantation Biology
Background:
- Alginate/poly-L-ornithine (AG/PLO) microcapsules are investigated for islet transplantation.
- Standardization of immunoisolatory properties is crucial for successful islet allo/xenografts.
Purpose of the Study:
- To describe a rapid method for qualitatively assessing the immunoisolatory properties of AG/PLO microcapsules.
- To evaluate a novel coherent microcapsule (CM) prototype for islet encapsulation.
Main Methods:
- Indirect immunofluorescence was used to detect immunoglobulin (Ig) binding to encapsulated pig islets.
- In vitro co-cultures assessed human lymphocyte proliferation using 3H-thymidine incorporation.
Main Results:
- No Ig binding was observed from islet cell antibody (ICA)-positive human sera to pig islets within AG/PLO CM.
- AG/PLO CM fully inhibited human lymphocyte proliferative capacity in mixed xenogeneic cultures.
Conclusions:
- The described method offers a preliminary approach for standardizing immunoisolatory membranes for islet transplantation.
- AG/PLO CM demonstrate significant potential for islet immunoprotection in allo/xenograft settings.
Abstract:
A quick method for the qualitative evaluation of immunoisolatory properties associated with islet-containing alginate/poly-L-ornithine (AG/PLO) microcapsules is described. In particular, we examined a new AG/PLO coherent microcapsule (CM) prototype that was recently developed in our laboratory, although the procedure could be applicable to other capsule types as well. We observed no binding of immunoglobulins (Ig) contained in islet cell antibody (ICA)-positive human sera (> 60 Juvenile Diabetes Foundation units, JDT U) to pig islets, enveloped within AG/PLO CM, under indirect immunofluorescence examination. Also, CM were shown to inhibit human lymphocyte proliferative capacity fully, as assessed by the 3H-thymidine incorporation rate, in in vitro mixed xenogeneic pig islet/human lymphocyte co-cultures. These results provided us with a preliminary method to attempt standardization of basic physical/chemical properties which should be associated with an immunoisolatory membrane for islet allo/xenograft immunoprotection.
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