TITLE: Islet Culture and Preparation for Cold Shipping
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 class = "text-block"><span style = "font-style:italic;">It was commissioned due to problems with acquiring the supplies that were used in IIDP SOP: SHP-001 and with the hope that this method is a better means for transportation of IIDP islets. Preliminary studies proved the islets by the Prodo Labs’ method were statistically as good as the original IIDP method, it was preferred by the test researchers, and was much more cost effective for the IIDP. This new method may be modified as future methods are tested and approved by the IIDP Team, Project Scientist (PS), the Program Official (PO), and EEC.</span><span></span></div></div>

[STEPS]
?. [Preparation of Supplies and Reagents]
Receipt of Supplies:The majority of supplies should be stored in appropriate dry, temperature-controlled environments (room temperature 16°-28°C).The Prodo Labs PIM(R) 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 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    .
2 °C
8 °C
-5 °C
-20 °C
-5 °C
-20 °C
?. [Preparation of Supplies and Reagents]
Preparation of PIM(R) Media:Prepare one 500 mL bottle of PIM(R) 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(R), it is now referred to as PIM(R) complete.Note:  If culture for IIDP distribution is greater than 250,000 IEQ, a second bottle of PIM(R) complete will need to be prepared.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(R), it is now referred to as PIM(R) complete.Note:  If culture for IIDP distribution is greater than 250,000 IEQ, a second bottle of PIM(R) complete will need to be prepared.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(R), it is now referred to as PIM(R) complete.Note:  If culture for IIDP distribution is greater than 250,000 IEQ, a second bottle of PIM(R) complete will need to be prepared.Thaw and add 0.5 mL of prepared ciprofloxacin sterile aliquotOnce all additives have been added to the bottle of PIM(R), it is now referred to as PIM(R) complete.Note:  If culture for IIDP distribution is greater than 250,000 IEQ, a second bottle of PIM(R) complete will need to be prepared.Once all additives have been added to the bottle of PIM(R), it is now referred to as PIM(R) complete.Note:  If culture for IIDP distribution is greater than 250,000 IEQ, a second bottle of PIM(R) complete will need to be prepared.
If PIM(R) complete 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.
?. [Post Purification Culture of Islet with ≥70% Purity]
Post purification, all pooled islets with > 70% purity should be brought up in 200 mL of PIM(R) in a 250 mL conical.Thoroughly mix suspension by either pouring between 2 conicals or inverting one conical at least 3 times.Quickly removing cap and count samples should be taken by a second technician.Take replicate 100-200 µl sample volumes from 100 mL final prep using a sampling wide-bore pipette tip and pipettor (Gilson or Drummond).  Two duplicate counts should be performed by two separate technicians or 2 separate counts by one technician.Add 3 drops (30 μL) of the DTZ solution to the islets sample and allow staining for 1 – 2 minutes at room temperature. Cover the bottom of the counting dish with DPBS to approximately ½ the height of the dish. Count the islets under the microscope as described in the SOP: Qualitative & Quantitative Assessment of Human Islets for Distribution Using Dithizone (DTZ) V.2   https://www.protocols.io/view/qualitative-quantitative-assessment-of-human-islet-bhdjj24n While counting, place the conical that contains the islets on its side at room temperature in the hood so that the islets do not pellet. Quickly removing cap and count samples should be taken by a second technician.Take replicate 100-200 µl sample volumes from 100 mL final prep using a sampling wide-bore pipette tip and pipettor (Gilson or Drummond).  Two duplicate counts should be performed by two separate technicians or 2 separate counts by one technician.Add 3 drops (30 μL) of the DTZ solution to the islets sample and allow staining for 1 – 2 minutes at room temperature. Cover the bottom of the counting dish with DPBS to approximately ½ the height of the dish. Count the islets under the microscope as described in the SOP: Qualitative & Quantitative Assessment of Human Islets for Distribution Using Dithizone (DTZ) V.2   https://www.protocols.io/view/qualitative-quantitative-assessment-of-human-islet-bhdjj24n While counting, place the conical that contains the islets on its side at room temperature in the hood so that the islets do not pellet. Take replicate 100-200 µl sample volumes from 100 mL final prep using a sampling wide-bore pipette tip and pipettor (Gilson or Drummond).  Two duplicate counts should be performed by two separate technicians or 2 separate counts by one technician.Add 3 drops (30 μL) of the DTZ solution to the islets sample and allow staining for 1 – 2 minutes at room temperature. Cover the bottom of the counting dish with DPBS to approximately ½ the height of the dish. Count the islets under the microscope as described in the SOP: Qualitative & Quantitative Assessment of Human Islets for Distribution Using Dithizone (DTZ) V.2   https://www.protocols.io/view/qualitative-quantitative-assessment-of-human-islet-bhdjj24n While counting, place the conical that contains the islets on its side at room temperature in the hood so that the islets do not pellet. Add 3 drops (30 μL) of the DTZ solution to the islets sample and allow staining for 1 – 2 minutes at room temperature. Cover the bottom of the counting dish with DPBS to approximately ½ the height of the dish. Count the islets under the microscope as described in the SOP: Qualitative & Quantitative Assessment of Human Islets for Distribution Using Dithizone (DTZ) V.2   https://www.protocols.io/view/qualitative-quantitative-assessment-of-human-islet-bhdjj24n
Note:  This can be accomplished using center specific method as long as a representative sample is taken from a well-mixed suspension and the sample is suspended in PIM(R)
Record counts on Attachment 2-Islet Tabulation Counting Sheet for Culturing Islets using Attachment 3- Islet Ranking Guide for Ranking, of this SOP.
?. [Preparation of Supplies and Reagents]
Laboratory Supplies:The following supplies are necessary for the preparation of flasks for human islet culture prior to distribution.Islet preparation and distributionWide mouth pipettes and pipettorCulture flasks- T-175 non-coated flasksSampling wide-bore pipette tip and pipettor (Gilson or Drummond)37°C CO2 IncubatorConical tubes, 250 ml, sterileRoutine lab supplies for transferring, media changing and counting islets.
?. [Post Purification Culture of Islet with ≥70% Purity]
Once the average count has been determined from the samples taken and recorded on the count sheets, calculate the total IEQs and the IEQ per mL in the islet suspension.
?. [Post Purification Culture of Islet with ≥70% Purity]
Calculate the amount of suspension needed to aliquot 20,000 IEQ per T-175 flask.
?. [Post Purification Culture of Islet with ≥70% Purity]
Calculate the number of flasks needed to culture the islets for broadcast at 20,000 IEQ per flask.Example:  250,000 IEQ/20,000 IEQ=12.5 flasks (or 200 mL/16 mL=12.5.)  Round up the fractional number of flasks to the next whole number and recalculate the proper aliquot to be taken from the islet suspension.Example: 12.5 → 13 Flasks; 200 mL/13 flasks = 15.4 mL islet suspension/flask.Determine the amount of media needed to prewet each flask. Example: 40 mL - 15.4 mL islet suspension = 24.6 mL fresh PIM(R).Example: 12.5 → 13 Flasks; 200 mL/13 flasks = 15.4 mL islet suspension/flask.Determine the amount of media needed to prewet each flask. Example: 40 mL - 15.4 mL islet suspension = 24.6 mL fresh PIM(R).
?. [Post Purification Culture of Islet with ≥70% Purity]
Aseptically transfer the proper amount of T-175 non-coated flasks into the hood and label per center protocol. Pre-wet all but one flask with predetermined amount of fresh PIM(R).
?. [Post Purification Culture of Islet with ≥70% Purity]
Lay the flasks flat on the hood surface making sure the media covers the entire surface being careful not to wet the neck or cap of the flask.
?. [Post Purification Culture of Islet with ≥70% Purity]
Deliver ~20,000 IEQ to appropriate flasks with a 25 mL pipette properly mixing the islet containing solution between each flask to ensure even distribution.
?. [Post Purification Culture of Islet with ≥70% Purity]
Rinse the conical after the (marked) final flask has been loaded with 3 mL of extra media. Repeat.  Total volume will be 40 mL
?. [Post Purification Culture of Islet with ≥70% Purity]
After dispensing all of the islets, place all the flasks in the incubator and set at  37°C ± 0.5°C and 5% ± 0.5% CO2 and culture for at least 48 hours.
Note:  Possible exceptions for culture time.
?. [Post Purification Culture of Islet with ≥70% Purity]
Between 12 and 18 hours after culture begins, perform a 50% media change on all flasks listed in Step 16-26 under Half Media Change Procedure.
?. [Post Purification Culture of Islet with ]
Follow all steps outlined in steps 5-14, however determine the concentration of the cultured islets and the number of flasks needed in Step 7 (bullet point) by multiplying the total flasks by the percent purity.Example: If part of the prep has a 50% purity and 50,000 IEQ; then 50,000 IEQ/(20,000IEQ * 50%) = 5 flasks.Determine all other values based on this calculation.
Note: Islets with
?. [Half Media Change Procedure]
A media change of half the culture volume should be performed 18 -24 hours after the completion of the isolation, Day 1 post culture. If for some reason the islets cannot be shipped out before or on Day 5, a second media change should be performed. (Note: It is unlikely that this scenario will occur.)
?. [Half Media Change Procedure]
Warm up required amount of PIM(R) to room temperature by taking it out of the refrigerator at least 1 hour before the media change is performed. Aseptically place into the laminar flow hood.
?. [Half Media Change Procedure]
Remove the flask(s) from the incubator, keeping them vertical (with the cap facing upward) while transporting from incubator and aseptically place the flask(s) into laminar flow hood.
?. [Half Media Change Procedure]
Rock the flasks gently back and forth keeping them horizontal, to get any islets that are loosely attached to the bottom of the flasks to go into the solution, being careful avoiding any media getting into the caps of the flasks. Return upright and loosen the caps of the flask(s).
?. [Half Media Change Procedure]
Arrange a 50 mL conical rack supported by a 250 mL conical rack for each set of 10 flasks. Placing 5 in each row, position flask(s) at an angle, tilting it towards the longer edge using 250 mL and 50 mL conical racks for support, resting them on the longer edge of the 50 mL conical rack so all islets can congregate to the lower corner of the flask due to gravity. Start a timer set for 45 minutes and record start time in the batch record.
?. [Half Media Change Procedure]
Leave all flask(s) positioned in this manner for 45 minutes to allow islets to settle in the bottom corner surface.
?. [Half Media Change Procedure]
Go to the middle point of the media in one flask and take a 1 mL sample, place in a Petri dish, and examine under the microscope. If islets are less than 50 micron in size proceed to the next step. If larger islets are visible allow more settling time until the media sample has no islets present.
?. [Half Media Change Procedure]
While maintaining the position of the flask (as in step 19) aspirate 50% of the used media (20 mL) from the surface of the liquid layer equidistant from the sides of the flask, without disturbing the islets that are settled in the bottom corner.
?. [Half Media Change Procedure]
Pipette the aspirated used media into an empty sterile container. (This can be checked when the media change is completed for viable islets that can then be returned to a flask.) Repeat steps for all remaining flasks.
?. [Half Media Change Procedure]
Pipette 20 mL of fresh PIM(R) into each flask after aspiration and tighten caps.
?. [Half Media Change Procedure]
Once media change is completed for all the flask(s), check aspirate for any greater than 50 μm islets and if any are found, transfer to a flask. Confirm all ventilated flask caps are tight and return to incubator set to 37°± 0.5°C.
?. [Half Media Change Procedure]
Using a 0.2 μm filter, filter sterilize any media remaining in the fresh PIM(R) complete bottle.  The media will expire within 30 days once it has been fully supplemented.
?. [Preparation of Supplies and Reagents]
The IIDP will provide each center with the following supplies necessary for islet culture:Human AB Serum (ABS) HIPIM(G)®PIM(R)®Ciprofloxacin Hydrochloride