Chapter 3 Overview of Primary Metadata

3.1 Donor Characteristics

De-identified donor characteristics, including HLA-typing, collected at the time of organ procurement are provided in a de-identified manner to the ADI IsletCore team by the organ procurement organisations. Users should be aware that some information, such as diabetes diagnosis, may not be clinically validated at the time of organ retrieval. Whenever possible, HbA1c measurements are performed directly by the ADI IsletCore using blood samples sent with the organ, and donors indicated as type 1 diabetes (T1D) are validated as much as possible through genetic testing, autoantibody testing, and immunohistochemical analysis. While the Donor Metadata ‘diabetes diagnosis’ reflects the status indicated by the organ procurement organization, the Donor Metadata ‘corrected diabetes status’ indicates a classification based on the totality of information available to the ADI IsletCore (including ‘pre-diabetes’ defined as HbA1c between 5.7 and 6.5%, and ‘non-diagnosed’ donors with HbA1c>6.5% as type 2 diabetes (T2D)).

Available donor characteristics metadata include: Age, Sex, Donation Type (NDD, DCD, MAID), BMI, HbA1c (%), HLA A2 (Yes/No), Diabetes diagnosis, and Corrected diabetes status.

3.2 Organ Characteristics and Processing

Organ characteristic and processing metadata is recorded during the islet isolation by the ADI IsletCore lead technician. Cold ischemia time is calculated from the cross clamp time during organ procurement until the start of the isolation in Edmonton. Please note that some measures, such as ‘organ consistency’ are subjective.

Available organ characteristics and processing metadata include: Cold Ischemic Time (h), Pancreas weight (g), Fatty infiltration (Yes/No), Organ consistency (Soft, Normal, Fibrotic, Inconsistent), Collagenase supplier (Roche, VitaCyte, Serva, Sigma), Colleganse type (Liberase, Clzyme, Gold, NB1, Sigma, Other, Recombinant), Digestion time (min).

3.3 Isolation Outcomes

Islet purity, islet equivalents (IEQ), % trapped, and islet particle index (IPI) are determined by the ADI IsletCore lead technician. Full details can be found here or in our IsletCore welcome booklet. Details on determining Insulin and DNA samples as part of our quality control can be found here.

Available isolation outcomes metadata include: Total islet equivalents (IEQ), Islet equivalents per pancreas weight (IEQ/g), Purity (%), Trapped (%), Islet particle index, Insulin content (ug), DNA content (ug), Insulin:DNA ratio, Insulin per IEQ (ng/IEQ).

3.4 Cell Type Proportions

Cell proportions are estimated based on deconvolution of bulk islet proteomic data from hand-picked islets using canonical proteins as markers (see below). Estimated cell type proportions are shown as either the ‘non-endocrine cell proportion’ (of all cells) or as the proportion of the endocrine compartment only.

Available cell type proportions metadata include: Non-endocrine cell proportion (computed), beta-cell proportion (of endocrine; computed), alpha-cell proportion (of endocrine; computed), delta-cell proportion (of endocrine; computed), and gamma-cell proportion (of endocrine; computed).

3.5 Cell Culture Outcomes

Following isolation, islets are cultured prior to shipment and sample collection for assay. On the day of shipping/collection, islets are recounted to determine islet equivalents (IEQ) and islet particle index (IPI) after culture. Culture time is determined by the end of the isolation until approximately 9 am on a shipping/collection day. The % recovery is calculated as ’post-isolation IEQ/pre-shipment IEQ*100.’ Post-culture samples are collected for insulin and DNA as described.

Available cell culture outcomes metadata include: Culture time (h), Total islet equivalents after culture (IEQ), Percent IEQ recovery after culture (%), Islet particle index after culture, Insulin content after culture, Insulin:DNA ratio after culture, Insulin per IEQ after culture (ng/IEQ).

3.6 Static Insulin Secretion

On the day of sample collection (see section 2.5, above) islets are handpicked to 100% purity and cultured overnight at 37°C with 5% CO2. In triplicates, 15 hand-picked islets from each donor are pre-incubated at low glucose for 2 x 1 hours consecutively, and then sequentially treated with low glucose for 1 hour followed by high glucose 1 hour. Collected supernatant for low glucose, high glucose or insulin content are measured by ELISA (ALPCO STELLUX Chemiluminescence). For each donor, there are multiple glucose combinations that are used. Experimental details can be found here. Glucose pairs are:

1mM → 10mM glucose
1mM → 16.7mM glucose
2.8mM → 16.7mM glucose

Outliers were handled in two ways. Level 1) A single donor-level measurement was computed as the median of the three replicate measures. Using the median instead of the mean reduced the impact of replicate-level outliers on the data. Level 2) The donor-level values were filtered to remove those that were more than 4 standard deviations away from the mean of all donors on a log10 scale. This removed values that were many orders of magnitude different from the population. In the Data Download tool, the data are provided at the replicate level (before outlier removal), while in-tool Omics Analysis is performed on data subjected to both Level 1 and Level 2 outlier removal.

Available static insulin secretion metadata include: Culture time before experiment (days), Insulin content (pg/ml), Secretion at 1 mM glucose (pg/ml), Secretion at 2.8 mM glucose (pg/ml), Secretion at 10 mM glucose (pg/ml), Secretion at 16.7 mM glucose (pg/ml), Stimulation index (1->10 mM glucose), Stimulation index (1->16.7 mM glucose), Stimulation index (2.8->16.7 mM glucose).

3.7 Islet Oxygen Consumption (Seahorse assay)

At the ADI IsletCore, after an overnight culture, islets are hand-picked, and 70 islets per well are assayed in triplicate for each donor. Islets are placed in the center of the well and islet capture screens are used to keep islets in place. Plate is incubated at 37oC for 1 hour without CO2, after which the plate is placed in the Xfe24 analyzer (Agilent). Basal measurements are at 2.8mM glucose, while stimulation is at 16.7mM glucose. 5uM oligomycin, 3uM FCCP and 5uM of rotenone and antimycin A are used as indicated. All solutions are made using DMEM supplemented with 1% FBS, sodium pyruvate and L-glutamine. After the run is complete, islets are collected and a DNA assay is done to normalize data. A detailed protocol is here.

Available islet oxygen consumption (Seahorse assay) metadata include: Basal respiration, ATP-linked respiration, Proton leak, Max glucose response, Stimulated glucose response, Max respiration, and Spare capacity.

3.8 Dynamic Insulin Responses to Macronutrients

After shipment to the University of British Columbia, islets are hand picked and cultured in RPMI for 24-72hrs before the experiment to allow recovery from shipping. 65 islets are loaded per chamber and pre-incubated for 1 hour. Islets are stimulated with 6 or 15mM glucose, 5mM Leucine or 0.75mM Oleic acid/0.75mM palmitic acid as indicated. Samples are stored at -20oC until analysis with RIA. Full details are described here.

Available dynamic insulin responses to macronutrients metadata include: 3 mM Glucose baseline secretion (AUC), 15 mM Glucose-stimulated peak, 15 mM Glucose-stimulated secretion (AUC), 6 mM Glucose-stimulated secretion (AUC), 30 mM KCl-stimulated secretion, after glucose (AUC), 5 mM Leucine-stimulated peak secretion , 5 mM Leucine-stimulated secretion (AUC), 5 mM Leucine + 6 mM Glucose-stimulated secretion (AUC), 30 mM KCl-stimulated secretion, after leucine (AUC), 1.5 mM Oleate/palmitate-stimulated peak secretion, 1.5 mM Oleate/palmitate-stimulated secretion (AUC), 1.5 mM Oleate/palmitate _ 6 mM glucose-stimulated peak secretion, 30 mM KCl-stimulated secretion, after oleate palmitate (AUC).

3.9 Single-cell Function (electrophysiology)

On the day of collection (see section 3.5 above) islets are hand-picked, dispersed to single cells and plated on 35 mm cell culture dishes as described here. 24-72 hours after plating, electrophysiology is performed by whole-cell patch-clamp using procedures and solutions outlined in publications here and here.

For earlier donors, only ‘total exocytosis’ in response to a series of 10 membrane depolarizations from -70 to 0 mV is reported. For more recent donors (from R305 onward) an expanded panel of electrophysiological measures is reported that include measures of cell size, exocytosis, voltage-dependent Ca2+ channel activity, and voltage-dependent-Na+ channel activity. Cell types are identified either by immunostaining (insulin/glucagon) or by single-cell RNA sequencing after the experiment. In the Omics analysis, analyses can be performed on either alpha- or beta-cells, at 1, 5 or 10 mM glucose.

Available single-cell function metadata include: Size (pF), Total exocytosis (fF/pF), Early (RRP) exocytosis (fF/pF), Late exocytosis (fF/pF), Calcium entry (pC/pF), Early calcium current amplitude (pA/pF), Late calcium current amplitude (pA/pF), Sodium current amplitude (pA/pF), Half inactivation sodium current (mV).