Jun 11, 2020
Version 2
Sentinel Flask Preparation for Cold Shipping Protocol of Human Islets V.2
- Integrated Islet Distribution Program1
- 1Integrated Islet Distribution Program, City of Hope
- Integrated Islet Distribution ProgramTech. support email: iidp-email@coh.org
External link: https://dx.doi.org/10.17504/protocols.io.bac5iay6; https://iidp.coh.org/Investigators/Policies-Standard-Operating-Procedures
Protocol Citation: Integrated Islet Distribution Program 2020. Sentinel Flask Preparation for Cold Shipping Protocol of Human Islets. protocols.io https://dx.doi.org/10.17504/protocols.io.bhdpj25nVersion created by Integrated Islet Distribution Program
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 10, 2020
Last Modified: June 11, 2020
Protocol Integer ID: 38031
Keywords: Human Islets, culture, sentinel flask, IIDP, QA, Quality Assurance, Islet Equivalent, Prodo Labs,
Abstract
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.
Note
Integrated Islet Distribution Program (IIDP) (RRID:SCR_014387)
Attachments
Guidelines
- Integrated Islet Distribution Program (IIDP) (RRID:SCR_014387): The IIDP is a program commissioned and funded by the NIDDK to provide quality human islets to the diabetes research community to advance scientific discoveries and translational medicine. The IIDP consists of the NIDDK Project Scientist and Program Official, the External Evaluation Committee and the CC at City of Hope (COH). The IIDP CC integrates an interactive group of academic laboratories including the subcontracted IIDP centers.
- IIDP Coordinating Center (CC): Joyce Niland, Ph.D., IIDP Principal Investigator leads CC staff to coordinate the activities of the IIDPand assists the participating centers and investigators in the distribution of human islets.
- Islet Equivalent (IEQ): An IEQ is defined as an islet with a diameter of 150 microns. The number of IEQ in each size class is calculated by multiplying the number of islets by a conversion factor for each micron size.
- Approved Investigators: Researchers who have requested islets from the IIDP for basic science studies and whose research protocols have been reviewed and approved by the IIDP.
- Sentinel Flask: A representative sample of the islet preparation that has been distributed to investigators that is held at the distributing center as a quality assurance sample. This sample should be held in the 30 mL shipping flask at the center and should contain at least 100 IEQ cultured in the same shipping media used for islet shipment. One flask must be prepared for each purity batch of islets distributed.
Materials
MATERIALS
Human AB Serum (ABS) HIGemini BioproductsCatalog #100-512; Heat Inactivated
PIM(G)® (5 mL Glutamine/Glutathione)Prodo Laboratories, IncCatalog #PIM(G)®
PIM(T)®Prodo Laboratories, IncCatalog # PIM(T)®
Corning™ Ciprofloxacin HydrochlorideFisher ScientificCatalog #MT61277RG (Corning™ 61277RG)
Equipment
Invitrogen™ EVOS™ XL Core Imaging System or equivalent
NAME
Inverted Microscope
TYPE
Light Microscope
BRAND
AMEX1000
SKU
LINK
Equipment
Corning 4492 or equivalent
NAME
Sterile Polystyrene Serological Pipets
TYPE
Corning™ Stripette™ Wide-Tip Disposable Polystyren
BRAND
07-200-619
SKU
LINK
Sterile, individually wrapped, and certified nonpyrogenic and DNase-/RNase-free
Accuracy within ±2% at full volume
SPECIFICATIONS
Equipment
Gilson™ PIPETMAN Classic™ Pipets- F123601 or equivalent
NAME
Adjustable pipettor -P200
TYPE
Gilson
BRAND
F123601G
SKU
LINK
50 to 200μL, ±0.5, ±1μL
SPECIFICATIONS
Equipment
Fisherbrand™ Large-Orifice Pipet Tips, 1 to 200µL or equivalent
NAME
Genomic/Wide Orifice Pipet Tips
TYPE
Fisherbrand
BRAND
02-707-134
SKU
LINK
1 to 200μL
SPECIFICATIONS
Equipment
Drummond™ Fixed-Volume Microdispensers or equivalent
NAME
Drummond 3000385
TYPE
Drummond™
BRAND
21176F
SKU
LINK
Volumetric Range 100/200UL with borosilicate glass bores
SPECIFICATIONS
Equipment
Bottle Top Filters with PES Membrane or equivalent
NAME
0.2 μm Bottle Top Filter
TYPE
Thermo Scientific™ Nalgene™ Rapid-Flow™ Sterile Di
BRAND
09-741-09
SKU
LINK
0.2 µm membranes for sterile filtration; PES is the best membrane for cell culture fluids; lowest protein binding to maintain protein balance, lowest extractables to maintain media purity
SPECIFICATIONS
Equipment
Thermo Scientific™ Nalgene™ Square PETG Media Bottles with Closure
NAME
30 mL PETG Media Bottles with Closure
TYPE
Thermo Scientific™ Nalgene™
BRAND
03-311-1V
SKU
LINK
30 mL PETG Media Bottles
SPECIFICATIONS
Safety warnings
Please see attached SDS (Safety Data Sheet) for hazards and safety warnings.
Ciprofloxacin Hydrochloride
Precautionary statements:
- P280 - Wear protective gloves and eye/face protection
- P305 + P351 + P338 - IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.
- P337 + P313 - If eye irritation persists: Get medical advice/attention.
- P273 - Avoid release to the environment.
GemCell™ U.S. Origin Human Serum AB
GemCell™ human serum AB is collected from healthy male donors of the AB serotype at FDA-licensed facilities in the United States.
Hazardous Components:
- Biohazard contains human source material. Handle as though capable of transmitting infectious agents.
- Toxicity: Not Established.
Target Organs/Systems: Product could possibly irritate the skin, eyes and respiratory system. Do not ingest this product.
Before start
Prodo Labs, Inc. Protocols and Website: http://www.prodolabs.com
CITATION
CITATION
SUPPLEMENTAL MATERIALS
SUPPLEMENTAL MATERIALS
The following supplies are necessary for the preparation of Sentinel Test Flasks for human islet distribution.
- Islet preparation for distribution
- Wide mouth pipettes and pipettor
- 30 mL shipping bottles - 1 per islet purity batch broadcasted.
- Shipping media that is to be used for distribution as decribed below.
MEDIA PREPARATION
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.
The Ciprofloxacin powder can be stored on the shelf but filter sterilized suspension aliquots should be stored at -5° to -20°C.
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
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
SENTINEL FLASK PREPARATION
Assemble all items described in the Material and Media Preparation Sections.
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:
then
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.
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
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.
Observe under a microscope daily for visual islet integrity, quality, and sterility. Record results on Attachment 2 - Sentinel Flask Documentation Sheet.
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.
Note
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.
Citations
Ricordi, C.. Pancreatic Islet Cell Transplantation.
Ricordi, C. (Ed).. Methods in Cell Transplantation.