TITLE: Collection, Packaging and Cold Shipping of Fresh Non-Islet Pancreatic (Acinar) Tissue
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, fresh Non-Islet Pancreatic (Acinar) Tissue (NIPT) 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 by the IIDP with assistance from Dagmar Klein, PhD, at University of Miami who has been working with acinar since 2011 when it became clear, as far as shipping and culture was concerned, that protease inhibitors must be used in the medium otherwise the tissue will digest itself.</span></div></div>

[STEPS]
?. [Preparation of Supplies and Reagents]
The IIDP will provide each center with the following supplies necessary for NIPT fresh shipping:Gemini Human AB Serum (ABS) -Heat Inactivated (HI)PIM(G)® - 5 mL – Glutamine/GlutathionePIM(T)® - 500 mL – Transport MediaCiprofloxacin Hydrochloride. Either MP Biomedicals™ MP219902005 or Corning™ MT61277RG (61277RF is available in 1 gm bottles and 61277RG in 5 gm bottles, however due to the relatively short shelf life (12-18 months), usually 1 gm bottles are supplied).Sigma T9128 Trypsin inhibitor from Glycine max (soybean) – lyophilized powderThermo Scientific Nalgene 0.2 µm bottle top filter30 mL vessel for shipping NIPT60 mL vessel for shipping NIPT125 mL vessel for shipping NIPT250 mL vessel for shipping NIPTCorrugated, cardboard, inner box for shipping (7x5x5”)Kendall Durasorb Underpads #949Ziplock Bags, 8x10, 2 milCold Chain Complete card with WarmMark ascending indicator adhered to cardColdMark descending indicator Insulated shipping container (8x6x9”) (outer box)Phase 22 pouches-cryopak: small, ambient temperature pouches @ 8° C (four per shipment box)8 oz cold packs @ 8° C (eight per shipment box)8 oz cold packs @ -20° C (two per shipment box)Shipping tape (2 mil) 8 x 11” Labels for outside of boxesCiprofloxacin Hydrochloride. Either MP Biomedicals™ MP219902005 or Corning™ MT61277RG (61277RF is available in 1 gm bottles and 61277RG in 5 gm bottles, however due to the relatively short shelf life (12-18 months), usually 1 gm bottles are supplied).Sigma T9128 Trypsin inhibitor from Glycine max (soybean) – lyophilized powderThermo Scientific Nalgene 0.2 µm bottle top filter30 mL vessel for shipping NIPT60 mL vessel for shipping NIPT125 mL vessel for shipping NIPT250 mL vessel for shipping NIPTCorrugated, cardboard, inner box for shipping (7x5x5”)Kendall Durasorb Underpads #949Ziplock Bags, 8x10, 2 milCold Chain Complete card with WarmMark ascending indicator adhered to cardColdMark descending indicator Insulated shipping container (8x6x9”) (outer box)Phase 22 pouches-cryopak: small, ambient temperature pouches @ 8° C (four per shipment box)8 oz cold packs @ 8° C (eight per shipment box)8 oz cold packs @ -20° C (two per shipment box)Shipping tape (2 mil) 8 x 11” Labels for outside of boxes
?. [Preparation of Supplies and Reagents]
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 AB serum and the PIM(G) vials should be stored at to  in the dark.The ciprofloxacin can be stored on the shelf but filter sterilized suspension aliquots should be stored at  to . 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  to  , to ensure against accidental triggering of either indicator.The T9128 Trypsin inhibitor from Glycine max (soybean) – lyophilized powder should be stored between and upon receipt.
16 °C
28 °C
2 °C
8 °C
-5 °C
-20 °C
-5 °C
-20 °C
12 °C
32 °C
2 °C
8 °C
?. [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]
Preparation of PIM(T) Media:Prepare one 500 mL bottle of PIM(T) media prior to the isolation per Attachment 1.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 aliquot per Attachment 1.Once 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 aliquot per Attachment 1.Once 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 aliquot per Attachment 1.Once 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 aliquot per Attachment 1.Once 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 Trypsin inhibitor:The T9128 Trypsin inhibitor from Glycine max (soybean) – lyophilized powder should be stored between  and  upon receipt.  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
?. [Preparation of Supplies and Reagents]
Supplies provided by the IIDP Centers:NIPT for 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 pipettorRoutine lab supplies for transferring NIPT Markers for labeling vesselsIIDP Box Labels from IIDP templateTissue Shipment FormsCompleted FedEx Shipping Labels Islet Allocation Tissue Shipment Forms for appropriate recipients      (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 pipettorRoutine lab supplies for transferring NIPT Markers for labeling vesselsIIDP Box Labels from IIDP templateTissue Shipment FormsCompleted FedEx Shipping Labels Islet Allocation Tissue Shipment Forms for appropriate recipients
?. [NIPT (Acinar) Broadcast]
Begin "Acinar Tissue Broadcast" on "IIDP Secure Access Center" website as soon as the islet isolation is confirmed.
?. [NIPT (Acinar) Broadcast]
Enter donor and isolation information on IIDP secure website under "Non-Islet (Acinar) Tissue Broadcast". Complete "Donor Information" and "Isolation and Distribution of Non-Islet Pancreatic Tissue” forms.
?. [NIPT (Acinar) Broadcast]
The "Donor Information" form is the same as for islets and all entered information will be auto-populated to the subsequent "Islet Broadcast".
?. [NIPT (Acinar) Broadcast]
Relevant information entered on the “Isolation and Distribution of Non-Islet Pancreatic Tissue” form will be auto-populated to the “Islet Broadcast”.
?. [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 (current SOP)] and/or flash frozen [Packaging and Shipping of Flash Frozen Non-Islet Pancreatic (Acinar)Tissue].
?. [NIPT (Acinar) Broadcast]
Complete the broadcast records and proceed to "Broadcast Now".
Note: Set deadline for approximate time of when the purification step will be completed so that the total amount of accepted NIPT will be known prior to when the tissue is to be prepared for shipment or freezing. this deadline can be set before starting the isolation.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. This will limit freshly isolated NIPT broadcasts to Sunday evenings through Thursdays (or Fridays if investigators will accept Saturday deliveries.)
?. [NIPT (Acinar) Broadcast]
Set the "NIPT Broadcast Deadline" to the assumed time when the purification will be completed. This will ensure that the NIPT will have minimum holding time prior to packaging of the freshly isolated samples or of freezing the requested NIPT. [Packaging and Shipping of Flash Frozen Non-Islet Pancreatic (Acinar)Tissue].
?. [NIPT (Acinar) Broadcast]
When the “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.
?. [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 HBSS, cap, invert to mix and dilute Ficoll, and centrifuge at
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]
Obtain the Trypsin inhibitor from Step 6.  Thaw aliquot.
?. [Collection of NIPT for Transport]
After centrifugation, calculate the pellet size, evacuate supernatant. If 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 (200 mL). Lay conical of the NIPT suspension on its side at  to avoid pelleting of NIPT while waiting to confirm acinar shipments. Note: As interest in the program expands and recipient requests increase, more tissue may need to be held.
4 °C
?. [Preparation of Shipping Vessels]
As investigator’s requests are determined through the broadcast system, prepare appropriate amounts of fresh NIPT into proper sized shipping vessel from the diluted samples in Step 19 above.For 0.5 - 0.75 mL of requested NIPT pellet, add 5 - 7.5 mL of NIPT suspension to 60 mL bottle for shipment.For 0.76 – 1.5 mL of requested NIPT pellet, add 7.6-15 mL of NIPT suspension to125 mL bottle for shipment.For 1.6 – 3.0 mL of requested NIPT pellet, add 16 - 30 mL of NIPT suspension to 250 mL bottle for shipment.For 3.1 – 5.0 mL of requested NIPT pellet, add 31 - 50 mL of NIPT suspension to 500 mL bottle for shipment. Any empty PIM(T) media bottle can be used for shipping these large amounts of NIPT.Distribute appropriate volume of NIPT into shipping bottles.For 0.76 – 1.5 mL of requested NIPT pellet, add 7.6-15 mL of NIPT suspension to125 mL bottle for shipment.For 1.6 – 3.0 mL of requested NIPT pellet, add 16 - 30 mL of NIPT suspension to 250 mL bottle for shipment.For 3.1 – 5.0 mL of requested NIPT pellet, add 31 - 50 mL of NIPT suspension to 500 mL bottle for shipment. Any empty PIM(T) media bottle can be used for shipping these large amounts of NIPT.Distribute appropriate volume of NIPT into shipping bottles.For 1.6 – 3.0 mL of requested NIPT pellet, add 16 - 30 mL of NIPT suspension to 250 mL bottle for shipment.For 3.1 – 5.0 mL of requested NIPT pellet, add 31 - 50 mL of NIPT suspension to 500 mL bottle for shipment. Any empty PIM(T) media bottle can be used for shipping these large amounts of NIPT.Distribute appropriate volume of NIPT into shipping bottles.For 3.1 – 5.0 mL of requested NIPT pellet, add 31 - 50 mL of NIPT suspension to 500 mL bottle for shipment. Any empty PIM(T) media bottle can be used for shipping these large amounts of NIPT.Distribute appropriate volume of NIPT into shipping bottles.
?. [Preparation of Shipping Vessels]
Top off the volume of each shipping vessel with PIM(T) Complete plus inhibitor stored at  to  up to the halfway point of the neck of each shipping bottle60 mL bottle - 75 mL of media plus NIPT125 mL bottle - 167 mL of media plus NIPT250 mL bottle - 300 mL of media plus NIPT500 mL bottle - 550 mL of media plus NIPT (Use sterile PIM Media bottle)125 mL bottle - 167 mL of media plus NIPT250 mL bottle - 300 mL of media plus NIPT500 mL bottle - 550 mL of media plus NIPT (Use sterile PIM Media bottle)250 mL bottle - 300 mL of media plus NIPT500 mL bottle - 550 mL of media plus NIPT (Use sterile PIM Media bottle)
6 °C
10 °C
?. [Packaging of Fresh NIPT]
Stage all materials needed for packaging the NIPT and ensure all ice packs and CryoPak pouches are at the proper temperature.
?. [Packaging of Fresh NIPT]
The Cold Chain Complete card is shipped with the WarmMark indicator attached.  Store the card at least  / ) below the activation temperature of the adhered WarmMark of  (therefore less                       than ). Room temperature is acceptable.
5 °C
9 °F
37 °C
32 °C
?. [Packaging of Fresh NIPT]
The ColdMark is provided separately because it is always active and should be stored at least  /above activation temperature of ColdMark of (therefore more than ). Room temperature is acceptable.
2 °C
50 °F
2 °C
12 °C
?. [Packaging of Fresh NIPT]
When ready to use, adhere the ColdMark indicator to the card and attach the WarmMark. ACTIVATE the WarmMark by folding up and pulling out the tab until completely removed.
?. [Packaging of Fresh NIPT]
Immediately place the Cold Chain Complete Card (TipTemp Indicator) and the acinar shipping bottles together into a Ziploc bag and seal.
Do not place ColdMark indicators on or near  -8 °C  frozen cold packs while preparing to ship to prevent accidental triggering of cold indicator.
?. [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 (~150 mL of media/1 mL 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 needed to complete the requests in 10x the volume of PIM(T) Complete plus inhibitor.  Example: If there is one request for 3 mL and two for 1.5 mL, only resuspend 6 mL of NIPT in 60 mL of  PIM(T) Complete plus inhibitor. This will save on the cost of the inhibitor and media.
?. [Packaging of Fresh NIPT]
Line the inner cardboard (#4434) box with absorbent pad.
?. [Packaging of Fresh NIPT]
Place the bagged shipping bottle(s) containing the NIPT in the Ziploc bag into the absorbent pad lined, inner cardboard box.
?. [Packaging of Fresh NIPT]
Surround bottle(s) with six  cold packs, one on bottom, one on each of the four sides and one on top.
8 °C
?. [Packaging of Fresh NIPT]
Mark the direction for upright bottle. Seal the inner box with shipping tape and stand upright.
?. [Packaging of Fresh NIPT]
Place one  CryoPak pouch and one  frozen ice pack (stored in the freezer) in the bottom of the Styrofoam box.
8 °C
-20 °C
?. [Packaging of Fresh NIPT]
Place the inner cardboard box containing the NIPT on top of the pouch and pack, ensuring that the bottle inside as well as the inner cardboard box, is lying on its side to ensure the maximum surface area for the acinar tissue to settle and centered in the shipping container. Note: the inner box and bottle are opposite of the way islet tissue is shipped.
?. [Packaging of Fresh NIPT]
Place one  CryoPak pouch and one  frozen ice pack on the top of the inner box.
8 °C
-20 °C
?. [Packaging of Fresh NIPT]
Add a second layer of cold/frozen packs on top, one  CryoPak pouch and one  frozen ice pack, on top of the first layer. *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
8 °C
-20 °C
?. [Packaging of Fresh NIPT]
Place the Styrofoam lid on top.
?. [Packaging of Fresh NIPT]
Place Tissue Shipment Form on top of Styrofoam lid.
?. [Packaging of Fresh NIPT]
Close the flaps of the shipping box and seal with shipping tape.  Attach "Cold Live Cells" label to box.
?. [Packaging of Fresh NIPT]
Attach FedEx label with recipient FedEx number to box and ship priority overnight.
?. [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.
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							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]
The investigators will have the FedEx tracking numbers listed in their "History of Acinar Tissue Offers" table.
?. [Preparation of Shipping Vessels]
Label each bottle with RRID #, Date, volume of NIPT, and PI NameRecord appropriate information on the batch record.Keep the shipping bottles with NIPT in the refrigerator 6 °C to 10 °C until ready for packaging.Record appropriate information on the batch record.Keep the shipping bottles with NIPT in the refrigerator 6 °C to 10 °C until ready for packaging.