TITLE: Sentinel Flask Preparation for Cold Shipping Protocol of Human Islets
AUTHORS: Integrated Islet Distribution Program

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">To establish a standardized method for the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) sponsored research in the Integrated Islet Distribution Program (IIDP) for preparing a sentinel flask of human islets in order to monitor sterility and islet integrity at the production center.  This is a representative sample of the islet preparation that has been distributed to investigators and is held at the distributing center as a quality control sample. The islets are cultured in the same shipping media used for islet shipment, for 18 hours at 4-8°C, simulating the cold shipping conditions followed by 2 days at 37°C culture. The flask of islets are verification in the event of a dispute over sterility or islet quality.</div></div>

[STEPS]
?. [SUPPLEMENTAL MATERIALS]
The following supplies are necessary for the preparation of Sentinel Test Flasks for human islet distribution.Islet preparation for distributionWide mouth pipettes and pipettor30 mL shipping bottles - 1 per islet purity batch broadcasted. Shipping media that is to be used for distribution as decribed below.
?. [MEDIA PREPARATION]
Shipping media that is to be used for distribution is prepared as described below.The Prodo Labs PIM(T) should be stored between 2° and 8°C upon receipt but is stable at room temperature.The (heat inactivated) Gemini AB serum and the PIM(G) vials should be stored at -5° to -20°C.
?. [MEDIA PREPARATION]
The  Ciprofloxacin powder can be stored on the shelf but filter sterilized suspension aliquots should be stored at -5° to -20°C.
?. [MEDIA PREPARATION]
Prepare 1 bottle of PIM(T) media prior to the collection by adding the following:Thaw and add 5 mL of PIM(G).Add 12.5 mL of AB serum (2.5%v/v).Add 0.5 mL of prepared sterile ciprofloxacin aliquot. Once all additives have been added to the bottle of PIM(T), it is now referred to as PIM(T) complete.
Note: If a prepared media bottle is to be used from a previous isolation, it must have been filter sterilized at the end of the previous use. The media will expire within 30 days, once it has been fully supplemented.
?. [SENTINEL FLASK PREPARATION ]
Assemble all items described in the Material and Media Preparation Sections.
?. [SENTINEL FLASK PREPARATION ]
At the time of preparation for islet distribution, the cultured islets are consolidated and counted.  Calculate the volume of suspension that is needed from the islet preparation to provide at least 100 IEQs.The formula will be:Total   IEQ/(Total   mL * 1000 μL)= x  IEQ/μL then 100 IEQ/x IEQ/μL = y μL
?. [SENTINEL FLASK PREPARATION ]
Thoroughly resuspend the preparation with a large mouth pipet and remove the calculated aliquot. Place in the 30mL shipping flask. Top with appropriate amount of media using the same batch of PIM(T) complete that will be used for distribution. Repeat for each purity batch that is being distributed.
?. [SENTINEL FLASK PREPARATION ]
Store this/these Sentinel QA Flask(s) between 4°C and 8°C for approximately 18 hours after which the flask(s) should be moved to a 37°C incubator for an additional 2 days.
Note: This culture procedure should simulate the average shipping time followed by culture by an investigator. A lab refrigerator should be appropriate for the first 18 hours of cold temperature culture.
?. [SENTINEL FLASK PREPARATION ]
Observe under a microscope daily for visual islet integrity, quality, and sterility. Record results on Attachment 2 - Sentinel Flask Documentation Sheet.
?. [SENTINEL FLASK PREPARATION ]
Alert the IIDP and the recipients of the distributed islets if there is a problem with the sterility of the preparation. In which case, send a copy of the Attachment 1-Sentinel Flask Documentation Sheet  to IIDP.
The sentinel flask is mainly for the documentation of the preparation by the distribution center in circumstances where islet sterility, quality or integrity of the distributed islets is questioned by a recipient. Photographs may be required to document the visual state of the sentinel flask if there is a challenge.