Feb 07, 2024

Public workspaceHuBMAP | VU TMC Kidney | Human Kidney Processing

DOI
dx.doi.org/10.17504/protocols.io.5qpvorwexv4o/v1
  • 1Vanderbilt University
Olof Isberg
Open access
DOI: dx.doi.org/10.17504/protocols.io.5qpvorwexv4o/v1
Protocol Citation: Olof Isberg, haichun.yang, Jamie Allen, Melissa Farrow, Audramjudd, Ray Harris, Agnes Fogo, Marc P. De Caestecker, Jeff Spraggins 2024. HuBMAP | VU TMC Kidney | Human Kidney Processing. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvorwexv4o/v1
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: March 07, 2023
Last Modified: February 07, 2024
Protocol Integer ID: 78288
Keywords: HuBMAP, BIOMIC, Vanderbilt, Multimodal Imaging, IMS, MXIF, Codex, Proteomics, Genomics, Kidney
Disclaimer
Adapted from this standard operating procedure established by the VU TMC Eye/Pancreas team.
Abstract
This protocol describes the workflow for collecting human kidney samples for multimodal downstream assays, in use by the Vanderbilt University HuBMAP Tissue Mapping Center.
Materials
Reagents:
  1. Dry Ice
  2. Isopentane, Fisher O3551-4
  3. Wet Ice
  4. Carboxymethylcellulose, Fisher C9481
  5. Richard-Allan Scientific Neutral Buffered Formalin (10%), Fisher 22-050

Equipment:
  1. Solvent compatible ice buckets
  2. Teflon cutting mold
  3. Safety Scalpel #22, Mercedes Scientific CAN SL0022
  4. Surgical Scissors
  5. 8 inch knife
  6. Teflon kidney mold
  7. Steel pans, Grainger 3LEL9
  8. Aluminum cake pan lids (for moving items out of biosafety hood)
  9. Aluminum cake pans (for moving samples from molds to dry ice)
  10. Forceps, various sizes
  11. Tissue Cassettes
  12. Plastic jar for formalin fixation
  13. Aluminum Foil Squares, 5x5", Fisher 50-192-6854
  14. Thermo Scientific Peel-A-Way Disposable Embedding Molds, 20mm x 30mm, Fisher 18-30
  15. Raymay Easy to See Grid Ruler, JetPens Raymay AJH158
  16. iPad Stand
  17. iPhone or camera


2.6% Carboxymethylcellulose Preparation:
Add 500mL water to 500mL bottle
Microwave for 2 minutes
Place bottle on heated stir plate ~70o
Slowly add 13g to warmed water
Stir on low hotplate overnight, occasionally tightening lid and shaking bottle
Store solution in 4o refrigerator
Before each use, pour solution into 250mL squeeze bottle and sonicate for 10 minutes

Safety warnings
Attention
Health and safety:
1. Safety glasses/face shields, proper gloves, and a lab coat/gown are required. The area should be adequately vented.
2. Isopentane: Harmful by inhalation, in contact with skin and if swallowed. Keep container tightly closed and insure adequate ventilation. Flammable liquid and vapor.
Donor Selection Criteria
Donor Selection Criteria
The International Institute for the Advancement of Medicine will identify patient kidneys for research with the following criteria:
Basic Requirements:
Age: 18-55 years
DCD - WIT < 15 minutes
Any Downtime
CIT < 36 hrs
Will accept CMV, EBV, HSV 1 or 2, TOXO & HBsAb (+) w/ confirmed vaccination only
Will accept DCD
Will accept IVDA (current use/ past use)
Will accept Chagas & Strongyloidiasis
Will accept Donor that previously tested COVID-19 (+) & now COVID-19 (-) on Case-By-Case Basis
Will accept Kidneys on a pump
Will accept Biopsy - Fibrosis on CASE-BY-CASE BASIS

Exclusionary Criteria:
Diabetes Type 1 or 2
Hepatitis B/C positive serology results
HIV positive serology results
Meningitis Bacterial/Viral positive
MRSA - any
RPR/Syphilis positive serology
Sepsis

Contraindications:
Biopsy - Necrosis
Kidney Disease (past/current)
DM Type 1 or 2
Donor on dialysis
Cancer (current/past hx)
Bacterial Viral meningitis
MRSA in any culture
Sepsis
Day Before Preparation
Day Before Preparation
  • Ensure that 1 liter of 2.6% CMC is prepared (requires overnight preparation)
  • Label embedding molds with Date-Anterior/Posterior-Block Location
e.g. 05/26/23
Ant - A1

  • Label 2 paraffin fixation cassettes, one for anterior and one for posterior
  • Clear lab benches and safety hood
  • Transfer 2.6% CMC into 6 squeeze bottles and refrigerate overnight
  • Gather materials and place on appropriate benches:
Dry Ice Pans with Lids iPad Stand
Labeled embedding molds Foil squares
Face shields Lab Gowns
Gloves Styrofoam box
  • Clean biosafety hood items with 70% Ethanol. Place in a cleaned (70% EtOH) plastic container in a 4oC refrigerator to cool overnight.
2 steel pans Large knife
teflon mold styrene divider sheets
disposable scalpel surgical scissors
forceps labeled rulers
Day of Preparation Before Sample Arrives
Day of Preparation Before Sample Arrives
  • Clean biosafety hood with 70% Ethanol
  • Print "Sample Mold Chart" with Kidney ID and side for notetaker and freezing station
  • Set up stations
Picture station: iPad stand, iPhone, aluminum tray lid for transporting samples
Placement station: Dry ice pan with ice, disposable aluminum pan, labeled embedding molds,
forceps, plastic jar with 10% formalin
Freezing Station: 2 dry ice pans with ice, isopentane, chilled 2.6% CMC in squeeze bottles,
foil squares, sharpie markers, freezer boxes with labels (kidney ID & date), timers, dry ice
box for holding frozen/wrapped samples
Note Taker: place chair next to biosafety hood, notebook, pens
  • Label rulers, foil trays, embedding molds, and wrapping foil with block names.
Organ dissection
Organ dissection
Remove kidney from shipping container and place on cold dissection tray. Remove as much outer fat as possible.

Dissect the kidney by carefully cutting it in half and remove any visible fat. Work on one half of the kidney while the other half is left on an ice tray to keep cool.


Once the fat is removed, place the kidney half into the millotome. The kidney is first cut longitudinally, after each cut dividers are put down to hold the kidney in its place. Next, the kidney is cut latitudinally. After each latitudinal cut, that row of tissue block is collected and transferred to labelled ruler. The ruler is transferred at a photo stage where an iPad/iPhone is used to take a quick snapshot before each block is transferred to a labelled foil tray on an isopentane slurry.


Note: if the kidney doesn't fill out in the mold, place dividers into the edge of the mold to make it fit.

Note: remove any extra visible fat from the tissue blocks

Collect 1 sample from each side for FFPE.
Choose block and cut a thin nickel thick section. Place in labeled tissue cassette and then into a container of 10% neutral buffered formalin. Follow Freezing and Formalin Fixation of Tissue V.3 for processing the formalin samples.
Place aluminum pan on top of filled dry ice pan. After taking a picture of the sample ruler, place each tissue into the corresponding labeled mold. After two rows are placed, move entire sample pan to the freezing station.
Flash freezing and CMC embedding (FCMC)
Flash freezing and CMC embedding (FCMC)
Prepare isopentane slurry. Place a layer of dry ice into an insulated, lidded dry ice pan and add enough isopentane to cover the dry ice.
Place each tissue mold into the isopentane slurry. Cover the dry ice pan with lid and allow tissue to completely freeze.
Once the tissue is completely frozen, slowly add CMC to the mold until the tissue is completely covered. Keep the lid closed over the slurry as much as possible. When completely frozen, wrap mold in prelabeled square of aluminum foil.
Note time that the CMC is completely frozen for each sample on the Sample Mold chart.
Place all wrapped samples in prelabeled freezer boxes and store at Temperature-80 °C
Protocol references