TITLE: Packaging and Cold Shipping of Human Islets
AUTHORS: Integrated Islet Distribution Program

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This SOP defines a standardized method for packaging and cold shipping of research quality islets to approved investigators of human isolated islet preparations, for use in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) sponsored research in the Integrated Islet Distribution Program (IIDP).  This protocol is written to assist the participating islet isolation centers and investigators who are part of this program.</div><div class = "text-block"><span style = "font-style:italic;">Note: This SOP was developed based on the Prodo Labs, Inc. shipping protocol and results from preliminary studies conducted by the IIDP and commissioned by the original IIDP Project Officer, and External Evaluation Committee (EEC).</span></div></div>

[STEPS]
?. [Materials]
The IIDP will provide each center with the supplies necessary for shipments as listed in the materials section.  Standard lab supplies will be provided by the IIDP Centers as listed below.
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Supplies provided by the IIDP Centers:Islets for distributionRoutine lab supplies for transferring islets Completed FedEx Shipping LabelsPreprinted Fresh Tissue Box LabelsIslet Allocation Tissue Shipment Forms for appropriate recipientsMarkers for labeling vesselsRoutine lab supplies for transferring islets Completed FedEx Shipping LabelsPreprinted Fresh Tissue Box LabelsIslet Allocation Tissue Shipment Forms for appropriate recipientsMarkers for labeling vesselsCompleted FedEx Shipping LabelsPreprinted Fresh Tissue Box LabelsIslet Allocation Tissue Shipment Forms for appropriate recipientsMarkers for labeling vesselsPreprinted Fresh Tissue Box LabelsIslet Allocation Tissue Shipment Forms for appropriate recipientsMarkers for labeling vesselsIslet Allocation Tissue Shipment Forms for appropriate recipientsMarkers for labeling vessels
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Supplies provided by the IIDP:The majority of supplies should be stored in appropriate dry, temperature-controlled environments (room temperature 16°-28°C).The Prodo Labs PIM(T)® should be stored, in the dark, between 2° and 8°C upon receipt but is stable at room temperature.The Gemini AB serum and the PIM(G)® vials should be stored at -5° to -20°C in the dark.The Ciprofloxacin can be stored on the shelf but filter sterilized suspension aliquots should be stored at -5° to -20°C.  Aliquots:                                  The Cold Chain Complete Card with the WarmMark indicator on the card and the separate ColdMark indicator can both be stored at room temperature or between 12°C to 32°C to ensure against accidental triggering of either indicator.Ice Packs and CryoPaks.The Gemini AB serum and the PIM(G)® vials should be stored at -5° to -20°C in the dark.The Ciprofloxacin can be stored on the shelf but filter sterilized suspension aliquots should be stored at -5° to -20°C.  Aliquots:                                  The Cold Chain Complete Card with the WarmMark indicator on the card and the separate ColdMark indicator can both be stored at room temperature or between 12°C to 32°C to ensure against accidental triggering of either indicator.Ice Packs and CryoPaks.The Ciprofloxacin can be stored on the shelf but filter sterilized suspension aliquots should be stored at -5° to -20°C.  Aliquots:                                  The Cold Chain Complete Card with the WarmMark indicator on the card and the separate ColdMark indicator can both be stored at room temperature or between 12°C to 32°C to ensure against accidental triggering of either indicator.Ice Packs and CryoPaks.The Cold Chain Complete Card with the WarmMark indicator on the card and the separate ColdMark indicator can both be stored at room temperature or between 12°C to 32°C to ensure against accidental triggering of either indicator.Ice Packs and CryoPaks.
Record media preparation on Attachment 1:  Solutions Preparation Sheet :
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After the islets have been allowed to recover from the insult of the isolation process at 37°C for approximately 18-48 hours, the quality of the cultured islets should be assessed by inspecting the islets in several flasks. If the islets have rounded up to greater than 80% of all size islets, the isolation should be ready for shipping.  See attachment: Tips for Determining Islet Quality for Shipment.
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Preparation of PIM(T) Media:Prepare one 500 mL bottle of PIM(T) media prior to the isolationThaw and add 5 mL of PIM(G)Thaw and add 25 mL of AB serum (5% v/v)Thaw and add 0.5 mL of prepared ciprofloxacin sterile aliquotOnce all additives have been added to the bottle of PIM(T), it is now referred to as PIM(T) complete.Thaw and add 5 mL of PIM(G)Thaw and add 25 mL of AB serum (5% v/v)Thaw and add 0.5 mL of prepared ciprofloxacin sterile aliquotOnce all additives have been added to the bottle of PIM(T), it is now referred to as PIM(T) complete.Thaw and add 25 mL of AB serum (5% v/v)Thaw and add 0.5 mL of prepared ciprofloxacin sterile aliquotOnce all additives have been added to the bottle of PIM(T), it is now referred to as PIM(T) complete.Thaw and add 0.5 mL of prepared ciprofloxacin sterile aliquotOnce all additives have been added to the bottle of PIM(T), it is now referred to as PIM(T) complete.
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.
Record media preparation on Attachment 1:  Solutions Preparation Sheet, of this SOP.
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Obtain the flasks containing islets from the incubator and examine each culture flask for contamination and aseptically place inside the hood. Obtain one 250 mL conical for each 6 culture flasks and aseptically place inside hood.
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Pool 6 T-175 flasks into each “settling” 250 mL conical by pipetting with a 25 mL pipette wetted with PIM(T). Repeat pooling for each set of 6 flasks into a new “settling” 250 mL conical. Allow conicals to settle while rinsing and checking the flasks.
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Wash the flasks with PIM(T) to collect any remaining islets. Using a 25 mL pipette, deliver 20 mL of media slowly onto the entire bottom surface of the flask in a sweeping motion to loosen islets into the solution. Let the media run to the bottom of the flask by gravity. Repeat this step 2-3 times collecting the 20 mL from the bottom of the flask and delivering it to the top of the flask in a sweeping motion while rocking gently. The same 20 mL can be used for 6 flasks by transferring to the next flask after washing each flask. Collect the wash from all the flasks in to one “count” 250 mL conical.
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Repeat wash on the next 6 flasks and place into the “count conical”.
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After the first wash, examine the flasks under the microscope for stuck islets. If there are many islets attached to the bottom of flask, deliver 20 mL of PIM(T) to the flask for the second time, gently tap on the side of the flask and rock the flask back and forth to get the released islets in to the solution being careful not to get medium into the cap of the flask. Collect the wash and pool in to the “count conical”.
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After the rinses and the “settling conicals” have rested for approximately 10-15 minutes, pre-wet a 25 mL pipette with PIM(T), gently draw the settled islets from the bottom of each of the “settling conicals”, one at a time, by going to the bottom of the conicals containing the settled islets, and pipette with upward movement with only ~ 2-3 mL of media and carefully deliver to the bottom of 250 mL “count conical”.
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Centrifuge the supernatant in the “settling conicals” for 2 minutes @180 g.
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After centrifugation, evacuate supernatant. Gently resuspend the pellets from the “settling conicals” with 2-3 mL of PIM(T) and transfer into the “count conical”.
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Bring count sample up to 200 mL with PIM(T). Take count samples by inverting 250 mL conical 3-4 times, quickly remove cap and have second technician remove exactly 0.5 mL form the “count conical” and place in counting dish. Place the conical containing islets horizontally under the hood while waiting for results from the evaluations.
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According to the islet counts, calculate the concentration of islets/mL and record in the batch record.
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Complete the broadcast records and proceed to broadcast. From this value, calculate how much volume is needed to be distributed into each shipping bottle for each order. Record the value in the batch record.
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Remove 100 IEQ for Viability Test and proceed according to IIDP protocol.
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Remove 400 IEQ for GSIR studies and culture per IIDP protocol.
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As investigator’s orders are determined through the broadcast system, prepare appropriate amounts of islets into proper sized shipping vessel.30 mL bottle for shipments of less than 10,000 IEQ60 mL bottle for shipments of 10,000-50,000 IEQ125 mL bottle for shipments of greater than 50,000IEQ - 100,000 IEQ60 mL bottle for shipments of 10,000-50,000 IEQ125 mL bottle for shipments of greater than 50,000IEQ - 100,000 IEQ
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Distribute appropriate volume into shipping bottles.
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Bring the volume up with PIM(T) stored at 6-10ºC up to the halfway point of the neck of each shipping bottle. Note: Specific volumes for specific bottles sizes are as follows:30 mL bottle – 44 mL of media plus islets260 mL bottle – 75 mL of media plus islets125 mL bottle – 167 mL of media plus islets260 mL bottle – 75 mL of media plus islets125 mL bottle – 167 mL of media plus islets
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Label each bottle with RRID #, Date, #IEQ, and PI Name
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Record appropriate information on the batch record.
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Keep the shipping bottles with islets in the refrigerator set at 6-10º C until ready for packaging.
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Stage all materials needed for packaging the islets and ensure all ice packs and CryoPak pouches are at the proper temperature.
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The Cold Chain Complete Card is shipped with the WarmMark Indicator attached.Store the Card at least 5ºC (9º F) below the activation temperature of the adhered WarmMark of 37ᵒC (therefore less than 32º C). Room temperature is acceptable.The ColdMark is provided separately because it is always active and should be stored at least 10ᵒC (50ºF) above activation temperature of ColdMark of 2ºC (therefore more than 12ºC).Room temperature is acceptable.When ready to use, adhere the ColdMark indicator to the card and activate the WarmMark. ACTIVATE the WarmMark by folding up and pulling out the tab until completely removed.                        Immediately place the Cold Chain Complete Card along with the islet shipping bottles into a Ziploc bag and seal. Include attached Cold Chain Complete FormThe ColdMark is provided separately because it is always active and should be stored at least 10ᵒC (50ºF) above activation temperature of ColdMark of 2ºC (therefore more than 12ºC).Room temperature is acceptable.When ready to use, adhere the ColdMark indicator to the card and activate the WarmMark. ACTIVATE the WarmMark by folding up and pulling out the tab until completely removed.                        Immediately place the Cold Chain Complete Card along with the islet shipping bottles into a Ziploc bag and seal. Include attached Cold Chain Complete FormWhen ready to use, adhere the ColdMark indicator to the card and activate the WarmMark. ACTIVATE the WarmMark by folding up and pulling out the tab until completely removed.                        Immediately place the Cold Chain Complete Card along with the islet shipping bottles into a Ziploc bag and seal. Include attached Cold Chain Complete FormImmediately place the Cold Chain Complete Card along with the islet shipping bottles into a Ziploc bag and seal. Include attached Cold Chain Complete Form
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Line the inner cardboard (S-4434) box with absorbent pad. See attached slide presentation for more information.        Place the bagged shipping bottle(s) containing the islets in the Ziploc bag into the absorbent pad lined, inner cardboard box.Surround bottle with six 8ºC cold packs, one on bottom, one on each of the four sides and one on top.
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Mark the direction for upright bottle. Seal the inner box with shipping tape and stand upright.
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Place one 8°C CryoPak pouch and one 8°C cold pack (stored in the refrigerator) in the bottom of the Styrofoam box.
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Place the inner cardboard box containing the islets on top of the pouch and pack, ensuring that the bottle inside is standing upright and centered in the shipping container.
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Place one 8°C CryoPak pouch and one -20°C frozen ice pack on each long side of the inner box. (Packs/pouches are placed in the larger gap between the inner box and the insulated shipping container.)
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Place one 8°C CryoPak pouch and one 8°C cold pack on the top of the inner box. *NOTE-All packs should be placed in an alternating method, to ensure two of the same packs are not next to each other in the configuration (See Attachment 5-Packaging Islets for Cold Shipping for visual details.)
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Place the Styrofoam lid on top.
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Place Tissue Shipment Form on top of Styrofoam lid.
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Close the flaps of the shipping box and seal with shipping tape.  Add Box label to the Box.
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Attach FedEx label with recipient FedEx number to box and ship priority overnight.
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The shipping center will enter the IEQ to be shipped (Confirm Recipient screen) and will enter FedEx tracking numbers (Islet Tracking) into the Islet Allocation System. In addition, the shipping center should process the shipment by using the on-line FedEx process and click the prompt to alert the recipient of the FedEx tracking number. This will help the recipient investigator follow their shipment through the FedEx system and free up time for the centers and the IIDP.Note: If the investigator has picked up the islets at the distributing center then the center should check “Yes” in  the “Direct Pickup” column and “Yes” under “Islets Shipped?” once they are picked up.
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As soon as the center has entered the number of IEQ shipped and indicated that the investigator will be shipped islets, the investigator’s account will be set automatically to “ineligible”. This will act similar to “on hold” preventing the investigator from receiving additional targeted offers until the User Feedback Form has been completed.
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The investigators will have the FedEx tracking numbers listed in their History of Islet Offers table.