Separation of pancreatic islets by fluorescence-activated sorting

Area of Science:

• Endocrinology
• Cell Biology
• Transplantation Immunology

Background:

• Clinical pancreatic islet transplantation requires improved islet yield and purity.
• Neutral red vital staining is a specific, nontoxic method for labeling islets across species.
• Current cell sorting methods may not be ideal for islet isolation.

Purpose of the Study:

• To evaluate the feasibility of using fluorescence-activated cell sorting (FACS) for pancreatic islet isolation.
• To determine if neutral red-stained islets can be accurately identified and sorted using a modified cell sorter.
• To assess the viability and purity of sorted islets for potential transplantation.

Main Methods:

• Intravital staining of rat islets with neutral red.
• Characterization of fluorescence absorbance and emission spectra of stained islets versus unstained exocrine tissue.
• Modification of a PARTEC cell sorter to accommodate larger particles (up to 300 microns).
• High-speed flow system analysis and sorting of dispersed, stained rat pancreatic islets.
• Assessment of islet viability using supravital staining and isogeneic kidney capsule transplantation.

Main Results:

• Neutral red-stained islets exhibit specific fluorescence emission (>610 nm) when stimulated with light (500–560 nm), distinct from unstained exocrine tissue.
• Modified PARTEC cell sorter successfully identified and sorted neutral red-stained islets.
• Achieved islet purity greater than 90% islet tissue.
• Sorted islets remained intact and viable, confirmed by supravital staining and successful transplantation.

Conclusions:

• Fluorescence-activated sorting using neutral red vital staining is a feasible method for isolating intact and viable pancreatic islets.
• This technique significantly improves islet purity, addressing a key limitation for clinical islet transplantation.
• The modified cell sorter offers a potentially cost-effective alternative for high-purity islet isolation.