TITLE: Flash Freezing Unclaimed Islets
AUTHORS: Integrated Islet Distribution Program

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The objective of the Integrated Islet Distribution Program (IIDP) is to develop a standardized procedure for the freezing and storage of unclaimed islet tissue to approved investigators for use in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) sponsored research in the IIDP.  </span><span style = "font-weight:bold;">This SOP replaces the IIDP SOP: SHP-002 Flash Freezing Unclaimed Islets.</span></div><div class = "text-block">This protocol provides an opportunity for the isolating centers to recoup expenses for isolated islets that cannot be placed through the normal distribution system at the time of isolation. It also may provide a less expensive and more advantageous method of islet supply to investigators that need non-viable islet tissue for gene panel research, RNA expression, and other experiments where viable islets are not necessary. This protocol also allows the storage of islet tissue for supplemental experiments that may be realized at a later date, after the original distribution has been completed.</div></div>

[STEPS]
?. [Materials]
Laboratory Supplies:The following supplies are necessary for the preparation of human islets prior to flash freezing for distribution.Islet preparation and distributionSterile pipettes, pipettors, and other appropriate sterile labware for manipulation of islet preparations.Hanks Buffered Salt Solution (HBSS) – no additives.       (Note: Either HBSS with or without Calcium, Magnesium, and phenol red can be used.)15 mL, 50 mL, or 250 mL conical tubes (depending on final volume of islet prep).Thermo Scientific™ Nalgene™ General Long-Term Storage Cryogenic Tubes Self- Standing with Round Bottom (Product #03-337-7D) (This brand and style of cryogenic tubes are recommended but not mandatory)  Cryogenic markers or pencils for tube labeling.Centrifuge with capabilities of spinning the appropriately sized conicals and the 2 mL cryogenic vials at 1000 rpm for 2 minutes.Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of islet preparations.Cryo Storage Log sheetIIDP Frozen Box Labels from IIDP template.Flash Frozen Tissue Shipment Forms.FedEx Shipping Form.Liquid nitrogen and appropriate vessels and equipment for safely freezing the unclaimed islets.Liquid nitrogen storage tanks or   or  freezer Appropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of islet preparations.Hanks Buffered Salt Solution (HBSS) – no additives.       (Note: Either HBSS with or without Calcium, Magnesium, and phenol red can be used.)15 mL, 50 mL, or 250 mL conical tubes (depending on final volume of islet prep).Thermo Scientific™ Nalgene™ General Long-Term Storage Cryogenic Tubes Self- Standing with Round Bottom (Product #03-337-7D) (This brand and style of cryogenic tubes are recommended but not mandatory)  Cryogenic markers or pencils for tube labeling.Centrifuge with capabilities of spinning the appropriately sized conicals and the 2 mL cryogenic vials at 1000 rpm for 2 minutes.Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of islet preparations.Cryo Storage Log sheetIIDP Frozen Box Labels from IIDP template.Flash Frozen Tissue Shipment Forms.FedEx Shipping Form.Liquid nitrogen and appropriate vessels and equipment for safely freezing the unclaimed islets.Liquid nitrogen storage tanks or   or  freezer Appropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Hanks Buffered Salt Solution (HBSS) – no additives.       (Note: Either HBSS with or without Calcium, Magnesium, and phenol red can be used.)15 mL, 50 mL, or 250 mL conical tubes (depending on final volume of islet prep).Thermo Scientific™ Nalgene™ General Long-Term Storage Cryogenic Tubes Self- Standing with Round Bottom (Product #03-337-7D) (This brand and style of cryogenic tubes are recommended but not mandatory)  Cryogenic markers or pencils for tube labeling.Centrifuge with capabilities of spinning the appropriately sized conicals and the 2 mL cryogenic vials at 1000 rpm for 2 minutes.Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of islet preparations.Cryo Storage Log sheetIIDP Frozen Box Labels from IIDP template.Flash Frozen Tissue Shipment Forms.FedEx Shipping Form.Liquid nitrogen and appropriate vessels and equipment for safely freezing the unclaimed islets.Liquid nitrogen storage tanks or   or  freezer Appropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).      (Note: Either HBSS with or without Calcium, Magnesium, and phenol red can be used.)15 mL, 50 mL, or 250 mL conical tubes (depending on final volume of islet prep).Thermo Scientific™ Nalgene™ General Long-Term Storage Cryogenic Tubes Self- Standing with Round Bottom (Product #03-337-7D) (This brand and style of cryogenic tubes are recommended but not mandatory)  Cryogenic markers or pencils for tube labeling.Centrifuge with capabilities of spinning the appropriately sized conicals and the 2 mL cryogenic vials at 1000 rpm for 2 minutes.Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of islet preparations.Cryo Storage Log sheetIIDP Frozen Box Labels from IIDP template.Flash Frozen Tissue Shipment Forms.FedEx Shipping Form.Liquid nitrogen and appropriate vessels and equipment for safely freezing the unclaimed islets.Liquid nitrogen storage tanks or   or  freezer Appropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Thermo Scientific™ Nalgene™ General Long-Term Storage Cryogenic Tubes Self- Standing with Round Bottom (Product #03-337-7D) (This brand and style of cryogenic tubes are recommended but not mandatory)  Cryogenic markers or pencils for tube labeling.Centrifuge with capabilities of spinning the appropriately sized conicals and the 2 mL cryogenic vials at 1000 rpm for 2 minutes.Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of islet preparations.Cryo Storage Log sheetIIDP Frozen Box Labels from IIDP template.Flash Frozen Tissue Shipment Forms.FedEx Shipping Form.Liquid nitrogen and appropriate vessels and equipment for safely freezing the unclaimed islets.Liquid nitrogen storage tanks or   or  freezer Appropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Cryogenic markers or pencils for tube labeling.Centrifuge with capabilities of spinning the appropriately sized conicals and the 2 mL cryogenic vials at 1000 rpm for 2 minutes.Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of islet preparations.Cryo Storage Log sheetIIDP Frozen Box Labels from IIDP template.Flash Frozen Tissue Shipment Forms.FedEx Shipping Form.Liquid nitrogen and appropriate vessels and equipment for safely freezing the unclaimed islets.Liquid nitrogen storage tanks or   or  freezer Appropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Centrifuge with capabilities of spinning the appropriately sized conicals and the 2 mL cryogenic vials at 1000 rpm for 2 minutes.Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of islet preparations.Cryo Storage Log sheetIIDP Frozen Box Labels from IIDP template.Flash Frozen Tissue Shipment Forms.FedEx Shipping Form.Liquid nitrogen and appropriate vessels and equipment for safely freezing the unclaimed islets.Liquid nitrogen storage tanks or   or  freezer Appropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of islet preparations.Cryo Storage Log sheetIIDP Frozen Box Labels from IIDP template.Flash Frozen Tissue Shipment Forms.FedEx Shipping Form.Liquid nitrogen and appropriate vessels and equipment for safely freezing the unclaimed islets.Liquid nitrogen storage tanks or   or  freezer Appropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Cryo Storage Log sheetIIDP Frozen Box Labels from IIDP template.Flash Frozen Tissue Shipment Forms.FedEx Shipping Form.Liquid nitrogen and appropriate vessels and equipment for safely freezing the unclaimed islets.Liquid nitrogen storage tanks or   or  freezer Appropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).IIDP Frozen Box Labels from IIDP template.Flash Frozen Tissue Shipment Forms.FedEx Shipping Form.Liquid nitrogen and appropriate vessels and equipment for safely freezing the unclaimed islets.Liquid nitrogen storage tanks or   or  freezer Appropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Flash Frozen Tissue Shipment Forms.FedEx Shipping Form.Liquid nitrogen and appropriate vessels and equipment for safely freezing the unclaimed islets.Liquid nitrogen storage tanks or   or  freezer Appropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).FedEx Shipping Form.Liquid nitrogen and appropriate vessels and equipment for safely freezing the unclaimed islets.Liquid nitrogen storage tanks or   or  freezer Appropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Liquid nitrogen and appropriate vessels and equipment for safely freezing the unclaimed islets.Liquid nitrogen storage tanks or   or  freezer Appropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Liquid nitrogen storage tanks or  or freezer
-80 °C
-135 °C
?. [Islet Isolation and Distribution]
Perform islet isolation, followed by culture (Islet Culture and Preparation for Cold Shipping SOP) and the distribution of available islets through the IIDP islet allocation system (IA).
?. [Flash Freezing of Unclaimed Islets:]
At the end of the islet distribution final offer deadline, if there are still unclaimed cultured islets of greater than 80% purity, 1 to 10 aliquots of approximately 1,000 IEQ may be flash frozen and stored for future distribution using the following method:
?. [Cataloguing of Frozen Islets:]
Complete the first six columns of the IIDP Cryo Storage Log Sheet. A separate line should be used for each tube frozen.
This Cryo Storage log sheet is made available for in-house use and center convenience (It does not have to be returned to the IIDP)
?. [Cataloguing of Frozen Islets:]
The log sheet should be updated as the islet aliquots are removed.
?. [Broadcast of Frozen Aliquots:]
The flash frozen islets can be broadcasted at any time after the original isolation has been broadcasted and distributed and islets with greater than 80% purity remain. The broadcast is not time dependent and interested investigators can claim these islets at any time after broadcast until all have been distributed.
?. [Broadcast of Frozen Aliquots:]
Log into the IIDP database using the appropriate ID and Password.
?. [Broadcast of Frozen Aliquots:]
Log into the Dashboard, click on “Frozen Islets” on the Navigation Menu on the left hand side of the page. Click on “Broadcast Frozen Islet Offer”.
?. [Broadcast of Frozen Aliquots:]
When the center's list of isolations displays, click on the "Broadcast"  button of the isolation for intended broadcast of the flash frozen islets. A new screen will prompt a few questions about the timing of when the islets were frozen, how many vials are available (the total number of islets will automatically be populated at 1,000 IEQs per vial) and the purity of the frozen samples (the purity must be greater than 80% to be useful as flash frozen samples).
?. [Broadcast of Frozen Aliquots:]
Once the form is completed, click on "Broadcast"  and the information will be entered into the database system. At this point an email will be sent to all possible investigators. A confirmation notice will display that the broadcast has been successful.
?. [Broadcast of Frozen Aliquots:]
The investigators will then enter the system and, if interested, will accept the offers. (This is similar to the cultured islet broadcasts but the flash frozen islets are not time dependent.)
?. [Broadcast of Frozen Aliquots:]
The broadcasting center will be alerted by email when the flash frozen preparations have been accepted. At this point, the center staff should go back into the system to the “All Frozen Islet Offers” and look for “New Request” under the column "Frozen Islets Accepted "  indicating an accepted offer.
Note: Older isolations may have aliquots of 5,000 or 10,000 IEQs.
?. [Broadcast of Frozen Aliquots:]
To ship the Flash Frozen aliquot, the center staff should click on the red “Confirm Shipment” button on the row that has the “New Request” indicator. The “Confirm Recipients” screen will appear and should be completed by center staff. Click Edit  to insert the tracking number and the amount being shipped. After submission, the “Inventory List” will be populated with the appropriate information and emails will be sent to notify acceptance by the center staff with the shipping information to both the investigators and the center staff. Once a recipient is confirmed, click on the “Print Tissue Shipment Forms” and include the appropriate form with each shipment.
Note: The center must click on the Print Tissue Shipment Form (TSF) in order to complete the distribution process and trigger the email distribution.
?. [Flash Freezing of Unclaimed Islets:]
Calculate the necessary volume of the islet suspension needed to provide approximately 1,000 IEQ per appropriately sized conical tube and divide left over islets needing to be frozen into the required number of tubes.
?. [Flash Freezing of Unclaimed Islets:]
Label the appropriate number of cryogenic tubes with Isolation #, RRID, and date using a cryogenic marker or pencil and set aside. All tubes should have a final aliquot of approximately 1,000 IEQ.
?. [Flash Freezing of Unclaimed Islets:]
Centrifuge conical tubes containing the islets at  for 2 minutes at 4°C.
Centrifuge: 1000 33
?. [Flash Freezing of Unclaimed Islets:]
Carefully evacuate supernatant from the islet pellet.
?. [Flash Freezing of Unclaimed Islets:]
Add 1 mL of serum free HBSS to the conical tube in order to dilute any serum from the remaining culture media. Gently resuspend the islet pellet and transfer islet suspension to a 2 mL labeled cryogenic tube. Add another 0.5 mL of HBSS to conical and rinse to capture remaining islets. Transfer to the cryogenic tube. Rinse with 0.5 mL HBSS. Repeat for all tubes to be frozen.
?. [Flash Freezing of Unclaimed Islets:]
Centrifuge cryogenic tube at   for 2 minutes.
Centrifuge: 1000 33
?. [Flash Freezing of Unclaimed Islets:]
Evacuate supernatant, leaving a dry pellet.
?. [Flash Freezing of Unclaimed Islets:]
Carefully place cryogenic tubes in a liquid nitrogen bath taking all safety precautions to protect you from being burned by liquid nitrogen (mask, gloves, tongs, and apron)
?. [Flash Freezing of Unclaimed Islets:]
Safely transfer the vials into a labeled container and store in a   or  freezer or in the vapor phase of a liquid nitrogen storage tank until shipment.
?. [Shipping of Frozen Aliquots:]
A completed Flash Frozen Tissue Shipment Form should accompany each sample shipment. Approximately five pounds of dry ice should fill a small, thick Styrofoam shipping box. Multiple tubes of 1,000–10,000 IEQ can be sent in a single box if necessary to satisfy the number of requested islets. The tubes should be placed in the middle of the dry ice. The IIDP Frozen Sample Labels should be placed on two sides of the box and the dry ice regulation label should be completed before shipping.
Islets should be maintained below   until ready for shipment.
?. [Shipping of Frozen Aliquots:]
Samples should be sent priority overnight shipping by FedEx with appropriate boxes checked on the FedEx form for dry ice shipments. (No materials will be returned by the investigators.)
?. [Shipping of Frozen Aliquots:]
User Feed Back Forms should be completed online by the investigators.