TITLE: Packaging and Shipping of Flash Frozen Non-Islet Pancreatic (Acinar)Tissue
AUTHORS: Integrated Islet Distribution Program

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>To define a standardized method for packaging and cold shipping of research quality non-islet pancreatic (acinar) tissue (NIPT) to approved investigators for use in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) sponsored research in the Integrated Islet Distribution Program (IIDP). This will also include reference to flash frozen NIPT tissue. </span><span style = "font-weight:bold;">This SOP replaces the IIDP SOP:  SHP-007 Cold Shipping of Flash Frozen Non-Islet Pancreatic (Acinar)Tissue</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><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><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block">This protocol provides an opportunity for the isolating centers to recoup expenses for isolated NIPT 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 to investigators that need non-viable islet tissue for gene panel research, RNA expression, and other experiments where viable islets and fresh NIPT are not necessary. This protocol also allows the storage of NIPT for supplemental experiments that may be realized at a later date, after the original distribution has been completed.</div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-style:italic;"> </span></div></div></div>

[STEPS]
?. [Preparation of Supplies and Reagents]
The IIDP will provide each center with the following supplies necessary for islet culture:Gemini Human AB Serum (ABS)-Heat Inactivated (HI)PIM(G)®PIM(T)®Ciprofloxacin Hydrochloride. Either MP Biomedicals™ MP219902005 or Corning™ MT61277RG (61277RF is available in 1 gm bottles and 61277RG in 5 gm bottles. Due to the relatively short shelf life (12-18 months), usually 1 gm bottles will be ordered).Sigma T9128 Trypsin Inhibitor from Glycine max (soybean) – lyophilized powder5 mL Cryogenic tubes for freezingPIM(G)®PIM(T)®Ciprofloxacin Hydrochloride. Either MP Biomedicals™ MP219902005 or Corning™ MT61277RG (61277RF is available in 1 gm bottles and 61277RG in 5 gm bottles. Due to the relatively short shelf life (12-18 months), usually 1 gm bottles will be ordered).Sigma T9128 Trypsin Inhibitor from Glycine max (soybean) – lyophilized powder5 mL Cryogenic tubes for freezingPIM(T)®Ciprofloxacin Hydrochloride. Either MP Biomedicals™ MP219902005 or Corning™ MT61277RG (61277RF is available in 1 gm bottles and 61277RG in 5 gm bottles. Due to the relatively short shelf life (12-18 months), usually 1 gm bottles will be ordered).Sigma T9128 Trypsin Inhibitor from Glycine max (soybean) – lyophilized powder5 mL Cryogenic tubes for freezingCiprofloxacin Hydrochloride. Either MP Biomedicals™ MP219902005 or Corning™ MT61277RG (61277RF is available in 1 gm bottles and 61277RG in 5 gm bottles. Due to the relatively short shelf life (12-18 months), usually 1 gm bottles will be ordered).Sigma T9128 Trypsin Inhibitor from Glycine max (soybean) – lyophilized powder5 mL Cryogenic tubes for freezing
?. [Preparation of Supplies and Reagents]
Laboratory Supplies:The following supplies are necessary for the preparation of NIPT prior to flash freezing for distribution:Islet preparation and distributionHanks 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)Wide mouth pipettes and pipettorSampling wide-bore pipette tip and pipettor (Gilson or Drummond)Routine lab supplies for transferring, media changing and counting isletsThermo Scientific™ Nalgene™ General Long-Term Storage Cryogenic Tubes Self- Standing with Round Bottom (Product #03-337-7H) (This brand and style of cryogenic tubes are recommended but not mandatory) Cryogenic markers or pencils for tube labelingCentrifuge with capabilities of spinning the appropriate sized conicals and the 5 mL cryogenic vials at   Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of tissue preparationsIIDP Frozen Box Labels from IIDP templateFlash Frozen Tissue Shipment FormsFedEx Shipping FormLiquid nitrogen and appropriate vessels and equipment for safely freezing the NIPTLiquid nitrogen storage tanks or   or  freezerAppropriately 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)Wide mouth pipettes and pipettorSampling wide-bore pipette tip and pipettor (Gilson or Drummond)Routine lab supplies for transferring, media changing and counting isletsThermo Scientific™ Nalgene™ General Long-Term Storage Cryogenic Tubes Self- Standing with Round Bottom (Product #03-337-7H) (This brand and style of cryogenic tubes are recommended but not mandatory) Cryogenic markers or pencils for tube labelingCentrifuge with capabilities of spinning the appropriate sized conicals and the 5 mL cryogenic vials at   Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of tissue preparationsIIDP Frozen Box Labels from IIDP templateFlash Frozen Tissue Shipment FormsFedEx Shipping FormLiquid nitrogen and appropriate vessels and equipment for safely freezing the NIPTLiquid nitrogen storage tanks or   or  freezerAppropriately 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)Wide mouth pipettes and pipettorSampling wide-bore pipette tip and pipettor (Gilson or Drummond)Routine lab supplies for transferring, media changing and counting isletsThermo Scientific™ Nalgene™ General Long-Term Storage Cryogenic Tubes Self- Standing with Round Bottom (Product #03-337-7H) (This brand and style of cryogenic tubes are recommended but not mandatory) Cryogenic markers or pencils for tube labelingCentrifuge with capabilities of spinning the appropriate sized conicals and the 5 mL cryogenic vials at   Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of tissue preparationsIIDP Frozen Box Labels from IIDP templateFlash Frozen Tissue Shipment FormsFedEx Shipping FormLiquid nitrogen and appropriate vessels and equipment for safely freezing the NIPTLiquid nitrogen storage tanks or   or  freezerAppropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Wide mouth pipettes and pipettorSampling wide-bore pipette tip and pipettor (Gilson or Drummond)Routine lab supplies for transferring, media changing and counting isletsThermo Scientific™ Nalgene™ General Long-Term Storage Cryogenic Tubes Self- Standing with Round Bottom (Product #03-337-7H) (This brand and style of cryogenic tubes are recommended but not mandatory) Cryogenic markers or pencils for tube labelingCentrifuge with capabilities of spinning the appropriate sized conicals and the 5 mL cryogenic vials at   Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of tissue preparationsIIDP Frozen Box Labels from IIDP templateFlash Frozen Tissue Shipment FormsFedEx Shipping FormLiquid nitrogen and appropriate vessels and equipment for safely freezing the NIPTLiquid nitrogen storage tanks or   or  freezerAppropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Sampling wide-bore pipette tip and pipettor (Gilson or Drummond)Routine lab supplies for transferring, media changing and counting isletsThermo Scientific™ Nalgene™ General Long-Term Storage Cryogenic Tubes Self- Standing with Round Bottom (Product #03-337-7H) (This brand and style of cryogenic tubes are recommended but not mandatory) Cryogenic markers or pencils for tube labelingCentrifuge with capabilities of spinning the appropriate sized conicals and the 5 mL cryogenic vials at   Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of tissue preparationsIIDP Frozen Box Labels from IIDP templateFlash Frozen Tissue Shipment FormsFedEx Shipping FormLiquid nitrogen and appropriate vessels and equipment for safely freezing the NIPTLiquid nitrogen storage tanks or   or  freezerAppropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Routine lab supplies for transferring, media changing and counting isletsThermo Scientific™ Nalgene™ General Long-Term Storage Cryogenic Tubes Self- Standing with Round Bottom (Product #03-337-7H) (This brand and style of cryogenic tubes are recommended but not mandatory) Cryogenic markers or pencils for tube labelingCentrifuge with capabilities of spinning the appropriate sized conicals and the 5 mL cryogenic vials at   Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of tissue preparationsIIDP Frozen Box Labels from IIDP templateFlash Frozen Tissue Shipment FormsFedEx Shipping FormLiquid nitrogen and appropriate vessels and equipment for safely freezing the NIPTLiquid nitrogen storage tanks or   or  freezerAppropriately 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 #03-337-7H) (This brand and style of cryogenic tubes are recommended but not mandatory) Cryogenic markers or pencils for tube labelingCentrifuge with capabilities of spinning the appropriate sized conicals and the 5 mL cryogenic vials at   Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of tissue preparationsIIDP Frozen Box Labels from IIDP templateFlash Frozen Tissue Shipment FormsFedEx Shipping FormLiquid nitrogen and appropriate vessels and equipment for safely freezing the NIPTLiquid nitrogen storage tanks or   or  freezerAppropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Cryogenic markers or pencils for tube labelingCentrifuge with capabilities of spinning the appropriate sized conicals and the 5 mL cryogenic vials at   Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of tissue preparationsIIDP Frozen Box Labels from IIDP templateFlash Frozen Tissue Shipment FormsFedEx Shipping FormLiquid nitrogen and appropriate vessels and equipment for safely freezing the NIPTLiquid nitrogen storage tanks or   or  freezerAppropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Centrifuge with capabilities of spinning the appropriate sized conicals and the 5 mL cryogenic vials at Sterile pipettes, pipettors, and other appropriate sterile labware for manipulation of tissue preparationsIIDP Frozen Box Labels from IIDP templateFlash Frozen Tissue Shipment FormsFedEx Shipping FormLiquid nitrogen and appropriate vessels and equipment for safely freezing the NIPTLiquid nitrogen storage tanks or   or  freezerAppropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).IIDP Frozen Box Labels from IIDP templateFlash Frozen Tissue Shipment FormsFedEx Shipping FormLiquid nitrogen and appropriate vessels and equipment for safely freezing the NIPTLiquid nitrogen storage tanks or   or  freezerAppropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Flash Frozen Tissue Shipment FormsFedEx Shipping FormLiquid nitrogen and appropriate vessels and equipment for safely freezing the NIPTLiquid nitrogen storage tanks or   or  freezerAppropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).FedEx Shipping FormLiquid nitrogen and appropriate vessels and equipment for safely freezing the NIPTLiquid nitrogen storage tanks or   or  freezerAppropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Liquid nitrogen and appropriate vessels and equipment for safely freezing the NIPTLiquid nitrogen storage tanks or   or  freezerAppropriately sized Styrofoam shipping boxes and adequate dry ice (at least 5 lbs).Liquid nitrogen storage tanks or  or freezer
Centrifuge: 1000 33, 2 min
-80 °C
-135 °C
?. [Procedures]
Receipt of Supplies:The majority of supplies should be stored in appropriate dry, temperature-controlled environments (room temperature  to).The Prodo Labs PIM(T) should be stored, in the dark, between  and   upon receipt but is stable at room temperature.The Gemini Human AB serum and the PIM(G) vials should be stored at  to  in the dark.The T9128 Trypsin Inhibitor from Glycine max (soybean) – lyophilized powder should be stored between  and upon receipt.The Ciprofloxacin should be stored as indicated by manufacturer (Corning at room temperature OR MP Biomedicals at 4°C). Aliquots of Ciprofloxin can be prepared prior to the isolation.  Prepare the Ciprofloxacin according to the directions in the table.  Filter sterilized suspension aliquots should be stored at  to
-20 °C
28 °C
2 °C
8 °C
-5 °C
-20 °C
2 °C
8 °C
-5 °C
-20 °C
?. [Begin NIPT (Acinar) Broadcast]
Begin Acinar broadcast on IIDP Secure Access Center Website as soon as the islet isolation is confirmed.
?. [Begin NIPT (Acinar) Broadcast]
Enter donor and isolation information on IIDP secure website under Quick Acinar Broadcast. Complete Donor Information Form and Processing and Distribution of Acinar Tissue Form.
?. [Begin NIPT (Acinar) Broadcast]
Donor Information Form is same as for islets and all entered information will be auto-populated to the subsequent Islet Broadcast.
?. [Begin NIPT (Acinar) Broadcast]
Set your NIPT Broadcast Deadline to the assumed time when the purification will be completed. This will ensure that the NIPT tissue will have minimum holding time prior to freezing the requested NIPT or packaging of the freshly isolated samples. (See Collection, Packaging and Cold Shipping of Fresh Non-Islet Pancreatic (Acinar) Tissue SOP)
?. [Begin NIPT (Acinar) Broadcast]
Relevant information entered on the Processing and Distribution of Acinar Tissue Form will be auto-populated to the Islet Broadcast.
?. [Begin NIPT (Acinar) Broadcast]
When Deadline of Broadcast is met, an automated email will be sent to center asking to View Acinar Offers and to confirm the shipment or cancellation of each request.
?. [Begin NIPT (Acinar) Broadcast]
Complete information regarding NIPT will be available as freshly isolated (Collection, Packaging and Cold Shipping of Fresh Non-Islet Pancreatic (Acinar) Tissue SOP) and/or flash frozen (current SOP).
?. [Preparation of Flash Frozen NIPT]
As investigator’s requests are determined through the broadcast system for Flash Frozen NIPT, prepare appropriate amounts of tissue into proper sized consolidation vessels for reezing from the diluted samples in steps 18 or 19 above. Label each conical for identification during transfer. Label corresponding 5mL cryogenic tube with RRID #, date, amount of requested NIPT tissue and recipient name and hold until ready for final transfer.For 0.5 – 1.0 mL of requested NIPT pelleted tissue, add 5 - 10 mL of NIPT suspension to 15 mL conical.For 1.1 – 2.0 mL of requested NIPT pelleted tissue, add 11-20 mL of NIPT suspension to 50 mL conical.For 2.1 – 3.0 mL of requested NIPT pelleted tissue, add 21-30 mL of NIPT suspension to 50 mL conical.For 3.1 – 4.0 mL of requested NIPT pelleted tissue, add 31-40 mL of NIPT suspension to 50 mL conical.For 1.1 – 2.0 mL of requested NIPT pelleted tissue, add 11-20 mL of NIPT suspension to 50 mL conical.For 2.1 – 3.0 mL of requested NIPT pelleted tissue, add 21-30 mL of NIPT suspension to 50 mL conical.For 3.1 – 4.0 mL of requested NIPT pelleted tissue, add 31-40 mL of NIPT suspension to 50 mL conical.For 2.1 – 3.0 mL of requested NIPT pelleted tissue, add 21-30 mL of NIPT suspension to 50 mL conical.For 3.1 – 4.0 mL of requested NIPT pelleted tissue, add 31-40 mL of NIPT suspension to 50 mL conical.
?. [Begin NIPT (Acinar) Broadcast]
Complete the broadcast records and proceed to Broadcast.
Note: The Broadcast Deadline should be set for the approximate time of when the purification will be completed so that the total amount of accepted tissue will be known prior to when the NIPT tissue is to be prepared. Freshly isolated NIPT should be sent out on the day of the isolation or at the next available FedEx pickup, within 24 hours of isolation (Collection, Packaging and Cold Shipping of Fresh Non-Islet Pancreatic (Acinar) Tissue SOP). This will limit freshly isolated NIPT broadcasts to Sunday evenings through Thursdays (or Fridays if investigators will accept Saturday deliveries).The NIPT Broadcasts are done prior to the Islet Broadcasts.
?. [Collection of NIPT for Transport]
The NIPT, the tissue remaining in the COBE purification bag, should be transferred under sterile conditions, into two 250 mL conical tubes, as soon as feasible during the islet isolation process. Top with Hanks Buffered Salt Solution (HBSS), cap, invert to mix and dilute Ficoll, and centrifuge at
Centrifuge: 180 34, 2 min, 4 10
?. [Preparation of Flash Frozen NIPT]
Centrifuge all samples at to pellet the acinar tissue. Remove the supernatant and wash the tissue with HBSS without serum by topping each conical with appropriate amount of HBSS. This will remove the serum (protein) from the acinar tissue. Resuspend tissue. Repeat the centrifugation
Centrifuge: 180 34, 2 min, 4 10
Centrifuge: 180 34, 2 min, 4 10
?. [Collection of NIPT for Transport]
Obtain PIM-T Complete prepared in Step 5.
?. [Collection of NIPT for Transport]
?. [Collection of NIPT for Transport]
If the deadline has not yet been reached and the total amounts of NIPT accepted is not yet known, suspend up to 20 mL of pellet to 10 times the pellet volume with PIM-T Complete plus inhibitor (200mL). Lay conical of the NIPT suspension on its side at 4°C to avoid pelleting of NIPT while waiting to confirm acinar shipments. Note: As the NIPT program expands more tissue may be held as recipient requests increase.
?. [Collection of NIPT for Transport]
Note: Once inhibitor is added to the PIM-T Complete, it must be used on the day of addition.
?. [Preparation of Flash Frozen NIPT]
Remove supernatant. Wet pipet and transfer each pellet to corresponding labeled 5 mL cryogenic tube, rinsing original tube with HBBS without serum to ensure all tissue is transferred. Centrifuge cryogenic tubes at  to pellet the acinar tissue. Remove all supernatant to ensure a dry, serum-free pellet.
Centrifuge: 180 34, 2 min, 4 10
?. [Preparation of Flash Frozen NIPT]
Freeze pellet by placing tube in liquid nitrogen per your center’s protocol.
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).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.
?. [Completing the Distribution Records]
The shipping center will enter the NIPT to be shipped (Confirm Recipient screen) and will enter FedEx tracking numbers Broadcast 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.  The investigators will have the FedEx tracking numbers listed in their History of Acinar Tissue Offers table.
Note: If the investigator has picked up the NIPT at the distributing center then the center should check “Yes” in the “Direct Pickup” column and “Yes” under “NIPT Shipped?” once they are picked up.
?. [Completing the Distribution Records]
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.
?. [Completing the Distribution Records]
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 NIPT can be sent in a single box as necessary to satisfy the number of requested NIPT. 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.
NIPT should be maintained below   until ready for shipment.
?. [Completing the Distribution Records]
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.)
?. [Completing the Distribution Records]
User Feed Back Forms should be completed online by the investigators.
?. [Collection of NIPT for Transport]
Once deadline has been reached and final amounts of accepted FRESH and FROZEN NIPT is confirmed, calculate the amount of PIM-T needed.  Complete that which is needed for all shipments.  Add 1 mL of Trypsin Inhibitor stock (Attachment 1) to each 100 mL of PIM-T Complete for NIPT shipping (~150mL of media/1mL NIPT total requested). This will give a final working concentration of 100 μg/mL of Trypsin inhibitor in the media. Resuspend only the amount of NIPT tissue needed to complete the requests in 10x the volume of PIM-T Complete plus inhibitor.     Example: If there is one request for 1.5 mL, only resuspend 1.5 mL of NIPT in 15 mL of PIM-T Complete plus                 inhibitor. This will save on the cost of the inhibitor and media.
?. [Preparation of Ciprofloxacin, Media and Trypsin Inhibitor]
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 12.5 mL of AB serum (2.5% 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 12.5 mL of AB serum (2.5% 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 12.5 mL of AB serum (2.5% 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.
Record media preparation on Attachment 1:  Solutions Preparation Sheet, of this SOP.
?. [Preparation of Ciprofloxacin, Media and Trypsin Inhibitor]
Preparation of Ciprofloxacin:Aliquots of Ciprofloxacin can be prepared prior to the isolation.  Prepare the Ciprofloxacin according to the directions in the table.  Filter sterilized suspension aliquots should be stored at     .
-20 °C
?. [Preparation of Ciprofloxacin, Media and Trypsin Inhibitor]
The T9128 Trypsin Inhibitor from Glycine max (soybean) – lyophilized powder should be stored between  and .  Aliquots of Trypsin inhibitor can be prepared prior to the isolation.  Prepare the Trypsin inhibitor according to the directions in the table.  Filter sterilized suspension aliquots should be stored at     .
2 °C
8 °C
-20 °C