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Beta-Pro Islet Cell Isolation Process Tour
Background: Islets of Langerhans, United Network for Organ Sharing (UNOS)
Islets of Langerhans for use in research are isolated from human pancreata procured from
cadaveric donors. Procurement and allocation of cadaver organs is governed by United Network for Organ Sharing (UNOS) guidelines and managed by local organ procurement organizations (OPOs). The organs used for research processes were not able, for
whatever reason, to be placed for transplantation.
As part of the UNOS allocation process, all organ donors are screened for potentially
transmissible infectious diseases. Only donors found to be serologically negative
for Hepatitis B and C, HIV 1 and 2, HTLV 1 and 2, and syphilis are accepted for processing.
Pancreas donors are also tested for cytomegalovirus (CMV); pancreata from donors
testing positive for CMV are used for research purposes only.
Donor pancreata are harvested using surgical techniques by experienced surgical professionals
in a manner similar to that used for procurement of a pancreas for whole
organ transplantation. Pancreata are placed in sterile preservation solution, either
HTK Custodiol solution (HTK) or Viaspan solution (University of Wisconsin, UW),
packaged on ice, and transported to UVA’s cGMP Processing Facility. Shipping of
pancreata to our islet facility is done using either commercial airlines or courier
service.
Following receipt of the pancreas at the cGMP islet isolation facility, islets are
prepared by a semi-automated process. All steps described below are performed under
strict, sterile conditions in the cGMP facility. All materials, including the reagents
used in the various steps of the islet isolation process, are sterile.
Beta-Pro's Islet Isolation Process Consists of 10 Steps
Preparation of isolated islets for clinical transplantion consists of the
following steps:
- Cleaning of the pancreas and cannulation of the pancreatic duct
- Distension and perfusion of the pancreas with digesting enzyme
- Digestion and mechanical disruption of the pancreas
- Dilution and collection of the digested pancreatic tissue
- Washing and concentration of the pancreatic digest
- Purification of islets from the digest using gradient centrifugation
- Enumeration of islets and purity assessment
- Culture of islets
- Final bulk islet product release testing
- Packaging of islets for shipment
Cleaning of the Pancreas and Cannulation of the
Pancreatic Duct
 The pancreas is cleaned under a Class 100 Biological Safety Cabinet (BSC). The organ
is placed in a tray containing cold preservation solution (HTK or UW solution, as
appropriate) and kept submerged during the cleaning process. Extraneous tissue attached
to the pancreas such as spleen, duodenum, and fat are carefully removed by blunt
dissection. The pancreas is cannulated by placement of a high flow angiocatheter
of appropriate size into the pancreatic duct. The organ may be cannulated whole,
or may be cut into two segments (head and tail), by transection in the neck region.
The cannula(e) is(are) secured into the pancreatic duct(s) by suturing. The pancreas
is quickly dipped into an antiseptic solution, and subsequently an antibiotic solution,
to decontaminate the organ. The organ is then rinsed in Hank’s Balanced Saline Solution
(HBSS), and placed in a clean tray.
Distension and Perfusion of the Pancreas with
Digesting Enzyme
 Distension (perfusion) of the pancreas with specially formulated collagenase enzyme
solution is performed under cold conditions. Distension is performed manually using
a 60cc syringe, or mechanically using a peristaltic pump, depending on the quality
of the organ. For manual perfusion, a 60cc syringe is filled with enzyme solution,
connected to the cannula, and gently pushed into the organ until the organ swells
to a maximum volume (insuring complete dispersion of enzyme throughout the organ.
For continuous perfusion the pump is used to deliver the enzyme solution into the
organ for 5 minutes at low pressure (80mm Hg) and an additional 5 min at high pressure
(180mm Hg). Any leaks developing during the distension phase are identified and
clamped using hemostats. The pancreas is then cut.
Digestion and Mechanical Disruption of the Pancreas
 The digestion chamber is filled with the remaining enzyme solution, the metal screen
is placed, and the chamber is securely sealed. The chamber is connected by tubing
to a circuit allowing recirculation of enzyme solution through a heating circuit
and into the digestion chamber. The digestion of the pancreas is accomplished by
recirculation of warmed enzyme solution through the chamber, assisted by systematic
mechanical shaking of the chamber (using an automated shaker). The temperature of
the enzyme solution is gradually increased to 37 ºC and maintained for 15-30 minutes.
At regular intervals, a representative sample of the digested tissue is taken out
and examined for the release of intact islets using a vital dye (diphenyl thiocarbzone,
DTZ) which stains the islets a brilliant red. Determining the appropriate time to
discontinue enzymatic digestion is a critical step in the isolation of pancreatic
islets.
Dilution and Collection of the Digested Pancreatic Tissue
Enzymatic digestion is stopped by the introduction of cold buffer solution into
the digestion chamber, and collection (rather than recirculation) of the chamber
effluent. Further, mixing of the initial chamber effluent with Dilution Solution
containing human serum albumin (HSA) helps rapidly quench enzymatic activity.
Washing and Concentration of the Pancreatic Digest
The pancreatic digest is collected, recombined, and washed two times with medium
containing HSA by centrifugation (280xg at 4ºC). The collected tissue is pooled
and assessed during this collection phase.
Purification of Islets from the Digest Using Gradient Centrifugation
 Islets are purified from the acinar tissue present in the digest using density gradient
centrifugation using Ficoll based solutions. This step involves centrifugation of
the crude digest using a COBE 2991 cell processing system, during which the islets
are separated into layers of different purity. At the completion of the centrifugation
process the tissue is collected in different fractions, yielding fractions of purified
islets. Islet purity in individual fractions is assessed by dithizone staining,
and fractions are pooled based on their purity. The combined fractions are washed
separately using medium, and samples are taken for determination of islet number
and purity.
Enumeration of Islets and Islet Purity Assessment
Determination of islet counts and islet purity is based on the islets’
uptake of Dithizone (DTZ), which binds to the zinc-rich insulin granules within
islet beta-cells, staining them a brilliant red. A representative sample of the
islet preparation is stained with DTZ and islets are manually counted and sized
under light microscopy. Islet volume is expressed in islet equivalents (IEQ) (number
standardized islet of 150µm diameter). Counts are done in duplicate. Islet purity
is defined as the percentage of DTZ-positive islet tissue vs. DTZ-negative non-islet
tissue present in the preparation.
Culture of Islets
After purification, different islet layers are cultured in medium (based
on CMRL 1066), supplemented with HSA, glutathione and nicotinamide. The islets may
be cultured for up to 48 hours prior to shipment. Islets are cultured at 37ºC in
the presence of 5% CO2 at an appropriate tissue concentration. Culture media is
changed after 24 hours, and again prior to shipment. On the day of shipment, cultured
islets are collected from the culture vessels, washed in medium, and sampled for
product release testing. The islets may be assessed and shipped immediately, if
not cultured. In this case, they are suspended in culture media, supplemented as
described above.
Final Bulk Islet Product Release Testing
All islet preparations undergo specific quality assessments following isolation.
These are listed below. Note that function/potency results are not generally available
until after shipping of the islet product.
|
Sample |
Test |
Method |
|
Islets suspended in culture medium |
Islet Counts |
Manual count of DTZ-stained aliquot |
|
Islet Purity |
Dithizone staining |
|
Viability |
FDA / PI staining |
|
Potency (Function) |
Glucose-stimulated insulin release |
Islet Counts: The determination of islet counting is based on the ability
of the islets to take up Dithizone (DTZ), which binds to the zinc-rich insulin granule
within the beta-cells staining them a brilliant red. A representative sample of
the islet preparation is stained with DTZ and islets are manually counted under
light microscopy. Islets are categorized by size, and total islet volume is expressed
in islet equivalents (IEQ) (number standardized islet of 150µm diameter). Counts
are done in duplicate.
Islet Purity: Islet purity is defined as the percentage of islet (DTZ-stained)
vs. non-islet (non-DTZ-stained) tissue present in the islet preparation, and is
determined by microscopic examination of a representative sample of the islet preparation.
Islet viability: Islet viability testing is performed using dye uptake
and exclusion, testing cell membrane integrity. A representative sample of the islet
preparation is treated with FDA* and PI**, and the specimen is examined under fluorescence
microscopy. A minimum of 50 islets (from at least 5 fields of view) are evaluated
for viability by qualitative means, estimating the percentage of viable cells in
each islet.
*FDA passes through plasma membranes and is hydrolyzed
to produce free fluorescein within the intact cells, and producing a bright green
fluorescence.
**PI cannot penetrate the membrane of viable cells, entering only nonviable cells,
binding nucleic acids, and producing a bright orange/red fluorescence.
Glucose stimulated insulin release testing: The islet product is assessed
for glucose-responsiveness resulting in increased insulin secretion. After overnight
culture (12-18hrs, 37°C), human islets are exposed to low (2.8mM) and high (25.0mM)
glucose concentrations in static culture for a period of two hours. For reproducibility,
incubations are run in duplicate, and secreted insulin (supernatant samples) from
each incubation is measured in triplicate using a human insulin ELISA assay. The
stimulation index is determined by dividing insulin release at 25.0 mM glucose by
insulin release at 2.8 mM glucose.
Packaging of Islets for Shipment
Islets are placed in a sterile, leak-proof air-permeable culture bag. Islet density
and media volume are determined by the number of islets to be shipped and the size
of culture bag to be used. A maximum volume of 600ml is used for shipping in the
3 liter bag, while a maximum of 100ml is used for shipping in the 1 liter bag. Depending
upon the number of islets to be shipped, multiple bags may be used. The bag(s) containing
the islets are packed into insulated shipping containers lined with room temperature
packs and absorbent packaging, and include a temperature monitor (e.g. a HOBO H8
Datalogger). The shipping container is sealed and appropriate labeling is attached
(“Exempt Human Specimen”, labeled "UN3373"). The islets are shipped using a courier
service, to minimize package handling and environmental variability issues. (Alternate
shipping techniques are available at the discretion of the customer.)
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