TITLE: HuBMAP | VU TMC Kidney | Human Kidney Processing
AUTHORS: Olof Isberg, haichun.yang, Jamie Allen, Melissa Farrow, Audramjudd, Ray Harris, Agnes Fogo, Marc P. De Caestecker, Jeff Spraggins

[DESCRIPTION]
This protocol describes the workflow for collecting human kidney samples for multimodal downstream assays, in use by the Vanderbilt University HuBMAP Tissue Mapping Center.

[STEPS]
SECTION: Organ dissection
4. 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.  

 
SECTION: Flash freezing and CMC embedding (FCMC)
8. 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.
SECTION: Flash freezing and CMC embedding (FCMC)
9. Place each tissue mold into the isopentane slurry. Cover the dry ice pan with lid and allow tissue to completely freeze.
SECTION: Flash freezing and CMC embedding (FCMC)
10. 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.
SECTION: Flash freezing and CMC embedding (FCMC)
12. Place all wrapped samples in prelabeled freezer boxes and store at -80 °C


SECTION: Donor Selection Criteria
1. 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
SECTION: Day Before Preparation
2. 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
SECTION: Day of Preparation Before Sample Arrives
3. 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.
        
SECTION: Organ dissection
5. 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

SECTION: Organ dissection
7. 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.
SECTION: Flash freezing and CMC embedding (FCMC)
11. Note time that the CMC is completely frozen for each sample on the Sample Mold chart.
SECTION: Organ dissection
6. 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.