603.3 & 604.5_URMC_HTC_Whole Lung and Lobe Processing

Gloria Pryhuber; Heidie Huyck

Aug 16, 2020

603.3 & 604.5_URMC_HTC_Whole Lung and Lobe Processing

DOI

dx.doi.org/10.17504/protocols.io.biz7kf9n

Gloria S Pryhuber¹,
Heidie Huyck¹

¹University of Rochester Medical Center

Human Cell Atlas Method Development Community
LungMap2 Consortium

Gloria S Pryhuber

University of Rochester Medical Center

DOI: dx.doi.org/10.17504/protocols.io.biz7kf9n

Protocol Citation: Gloria S Pryhuber, Heidie Huyck 2020. 603.3 & 604.5_URMC_HTC_Whole Lung and Lobe Processing . protocols.io https://dx.doi.org/10.17504/protocols.io.biz7kf9n

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: July 26, 2020

Last Modified: August 16, 2020

Protocol Integer ID: 39711

Abstract

Purpose and Scope of the Procedure
Standardize process for processing lung donations into components for storage and distribution
Scope:       coordination of receipt and gross dissection of tissue

Guidelines

Scientific Principles 
Rapid, standardized and safe processing of human tissue for the HTC requires coordination of a team of staff, materials and attention to protocols
Rapid processing is required to maintain the tissues in a state as close to normal as possible
Ultra-High Resolution CT Scan in an inflated state will provide a high-level comparative assessment of human lung structure across developmental ages

Materials

MATERIALS
Reagent10X PBS; Diluted to 1X with DEPC H2OCatalog #Corning 46-013-CM 
Reagent20% Paraformaldehyde (PFA); Diluted to 4% (m/v) PFA in 1X PBSCatalog #EMS 15713-S 
Reagent10% Buffered FormalinCatalog #VWR 89370-094 
ReagentDiethylpyrocarbonate (DEPC)Catalog #Sigma D5758-100ML 
ReagentEthanolCatalog #Koptec V-1001 
ReagentOCT (inflation); 50% (v/v) OCT in 1X PBSCatalog #Tissue-Tek 4583 
ReagentOCT (mold)Catalog #Tissue-Tek 4583 
ReagentSucrose; 30% (m/v) Sucrose in 1X PBSCatalog #Sigma S9378-5KG 
ReagentCarboxymethylcellulose (CMC); 5% (w/w) CMC in DEPC H2OCatalog #Sigma C5678-1KG 
Worksheet       603.A.2 HTC_Whole_or_Partial_Lung_Processing       Worksheet
a.Grossing Station/ Biosafety Cabinet or Fume hood
b.Gloves, face protection and clothing consistent with blood and body fluid precautions
c.Heavy Duty Scissors and Wire Cutters
d.Biohazards Disposal Bag
e.Red Sharps Container
f.Biosafe surface for manipulation of lung
g.Small and Medium Gauze Pads – multi-pack
h.Standard Balance and Weigh Boats: Medium and Large
i.Airway Cannulas – Selection of sizes
    i.16 and 18 gauge angiocatheters (needles removed and discarded)
    ii.tracheal cannulas 2.0-4.0 OD
    iii.endotracheal tubes 2.5 – 8 mm OD; tubes >/= 4.0 cuffed
j.IV extension set tubing with clamp
k.20 ml syringe barrels for fixative
l.Needle free suture material
m.Zip Ties
n.Dissection Instruments: Same or similar to Sakura Series:
    1.#4791 Scalpel Handle with
    2.#4792 or #4793 Scalpel Blades, #61 (curved tip) or #62 (pointed tip), resp
    3.#4785 Trimming Blades, Short and/or #4789 Trimming Blades, Long
    4.#4786 Trimming Handle, Short and/or #4790 Long Straight
    5.#4794 Blade Scissors with #4796 Blades Sharp/Blunt
    6.#4807  Accu-Edge® Grossing Fork 2.5 mm
    7.#4800 Accu-Edge® Grossing Board Inches (L x W x D): 17 x 11.5 x 1
    8.#4802 Accu-Edge® Grossing Wells  Inches (L x W x D): 12.9 x 3.25 x 2.15
    9.#8031 Slide Printer
        a.#8033 Slide Unload Station
        b.#8035 Ink Cartridge
        c.#8040 Slide Magazine
10.Mopec Product #AB079 ProCUT Forcep Kit
o.Camera – iPad in water proof case works well
p.10% neutral buffered formalin – at least one liter with more on hand if needed for larger lungs
q.1xPhosphate Buffered Sodium (PBS diluted in DEPC treated water)
r.4% paraformaldehyde (prepared from 1:4 dilution of 16% stock non-buffered, no menthol formalin in 1x PBS in DEPC water)
s.Liquid Nitrogen in Dewar flask
t.Ring stand(s) and clamps (2 or more) – Fill syringe to 20 ml mark with fixative
u.Rulers – metal 18 inch (45cm) and 12 inch (30.5cm)

Safety warnings

Personnel will adhere to safe work processes outlined in U.S. Public Health Universal Precautions Guidelines for use of human blood and body fluids. PPE will be used including lab coat, closed shoes and gloves. All activity will be behind shield of biosafety cabinet and/or a grossing station with mask and safety glasses. Biosafety level 2 practices will be followed and the work performed in the designated lab space that is covered by annually updated IBC approved protocol.All institutional biosafety measures are followed in any manipulation of these human tissues.

Assemble Team and Materials as needed for processing, to accomplish:
    i.CT Scan
    ii.Separation of Lobes
    iii.Tissue Dissociation
    iv.Formalin Inflation
    v.OCT /CMC Inflation
    vi.Paraformaldehyde Inflation

Record details of procedures in Worksheets. 

Unpacking

Shipped package is opened on arrival to remove a blood sample, if provided, as it should be kept at room temperature.
 The remainder of the package is to be kept in cold room until processing begins to maintain approximately Temperature4 °C   temperature of tissue.

Determine BRINDL PID to assign to tissues and images by clicking the “arrived” button in BRINDL Screening log

Record Information on Shipping Labels in Database or worksheet for entry ASAP
    i.UNOS #; Referring Company #

    ii.Courier and Tracking Numbers

Remove Shipping Labels from package; place aside to be added to Sample File

Open Shipping Box and Remove Styrofoam Cover

i.Note Condition of Packing in Database (ice sufficient, layered packaging, no leakage, etc.)

6.Open container containing tissue and move lung tissue to a clean plastic bag on ice.

    i.Note Condition of Lung Tissue in Database
        a.Is tissue submerged (usually in plastic bag, inside a hard plastic container)
        b.Are Trachea and both lungs included and intact
        c.Is Trachea Occluded, if so how
        d.Are Thymus, Spleen, Blood Sample, Lymph Nodes included?

CT Scan

See separate protocol for CT Scan of Air Inflated Lung procedures.

Grossing of Tissues

Maintain tissues in transplant buffer on ice. Prevent open Trachea from being submerged , +/- ETT removal

Weigh intact organ as sent; Continue to Record all measurements and procedures in Worksheet or directly in BRINDL database                                              

Determine displacement volume without inflation                                              

Determine plan for organ lobes ie which to be inflated or infused and which to be dissociated                                              

Remove excess tissues around trachea and large airways at hilum  
    Collect lymph nodes, excess large vessels, esophagus and nerves 
        (Best to place these back in storage buffer and Tend to Lung First)  
        a.     Weigh each tissue collected  
        b.     The non-lung samples are sectioned into approximately 0.5-1 cm3 portions  
        c.     Divide these tissues between                                                                                                      
             i.    10% formalin, place in labeled tissue cassette in formalin
                    Store at Temperature4 °C   to fix for 44-48 hrs, 
                    Section at mid-time.  
                    Volume of fixative to tissue should be approximately 10:1.  
            ii.     PFA: place in 4%PFA stored at Temperature4 °C   to fix for 20-28 hours before moving to 30%Sucrose  
        d.     Record these tissues and how processed in BRINDL database  

 Dissect free the bronchial branches, identify pulmonary arteries and veins   

Divide airways and vessels to obtain 5 independent lung lobes  

Weigh each lobe once isolated 

Proceed with processing for each lobe as Outlined in Table 1 using appropriate Protocols

Prepare accompanying tissue such as thymus, spleen, esophagus, blood, heart, in similar fashion with specific as outlined in the appropriate Protocol / SOP.

 
 
  Organ
    Starting Tissue
    Prep
    Process
  BioSpecimen Type Collected from Organ
  Trachea and large
  bronchi
    Intact
    Paraffin and OCT
  Frozen
  Bisect longitudinally; Section into <1 cm long half rings  Formalin Fixed,
  Paraffin embedded
  PFASOCT embedded
  and frozen
  
  Alternate Trachea
  and large bronchi
    Intact
    Cell Isolates
    Enzymatic
  digestion and cell isolation
  Frozen isolated cell aliquots
Lung: Right Upper Lobe +/- Right Middle Lobe  Fresh Tissue Blocks
    Mixed Dissociated
  Cells
    Dissociate cells
  by mechanical and enzymatic digestion procedure
  Cryopreserve mixed cell aliquots
  Sorted Dissociated
  Cells
  Flow Activated Cell Sorting dissociated cellsCryopreserve sorted cell aliquots “ex. Endothelial, Epithelial, Fibroblast, Bone Marrow Derived (CD45+) Separation”
Cryo-preserved QICryo-preserved
  cellsRecover frozen
  cell aliquots, test purity by PCR, culture for viability and proliferative/ differentiation potential
Add’l Right Middle Lobe  Intact - Biopsies
    Flash Frozen
    Stored at -80
  Protein Homogenates, RNA and/or DNA Isolation
Alternate Right Middle Lobe  Intact
    Formalin
  Inflation, Paraffin
  Inflation fixed
  with 10% buffered formalin x 24 hr (Intact Lobe); Sectioned by grid into 0.5-1 cm3 pieces; Dehydrated to 70% Ethanol  Inflation fixed,
  paraffin embedded ready for sectioning
  
Alternate Right Middle Lobe  Fresh Tissue
  Blocks
    Fresh Frozen
  Embedded in OCT/CMC/none
  Vertical 0.1 cm slices, Section each slice by grid into 1 cm2 blocks, alternate slices to embed blocks in OCT or approx. 5% CMC (see protocol) or freeze without embedding material  Non-inflated, non-
  fixed flash frozen tissue blocks, mapped to originating location
  
Right Lower Lobe  Intact
    Formalin
  Inflation, Paraffin
  Inflation fix with
  10% buffered formalin x 24 hr (Intact Lobe)
  Sectioned by grid into 0.5-1 cm3 pieces
  Dehydrated to 70% Ethanol  Inflation fixed,
  paraffin embedded ready for sectioning
  
  Left Upper Lobe
    Intact
    PFA Fixed, Sucrose
  Cryopreserved, OCT embedded
  Inflation fixed with 4% paraformaldehyde x 24 hr (Intact Lobe);  Sectioned by grid into 0.5-1 cm3 pieces;  Cryopreserved in 10% sucrose; Block in OCT  Inflation fixed,
  cryo-preserved and frozen for cryo-sectioning
  
Alternate Left Upper Lobe  Intact
    OCT/PBS
    Inflate with 50:50
  OCT:10%PBS
    Frozen for
  cryo-sectioning
   
  
Alternate Left Upper Lobe  Intact
    Air Inflated,
  Vascular Contrast
    To be determined
    To be determined
  
  Left Lower Lobe
   
    Intact
    PFA Fixed, Sucrose Cryopreserved, OCT embedded
  Inflation fixed with 4% paraformaldehyde x 24 hr (Intact Lobe); Sectioned by grid into 0.5-1 cm3 pieces; Cryopreserved in 10% sucrose; Block in OCT  Inflation fixed, cryo-preserved and frozen for
  cryo-sectioning
  
  Additional Tissue
    Intact
    Similar to Lung, see specific SOPs
  These include thymus, spleen, esophagus, heart, nerve, etc; Block as appropriate for tissueFormalin Fixed,
  Paraffin embedded,
  PFA/OCT embedded-frozen
  Single cell dissociation
  Peripheral Blood
    Whole
    Cryopreserved, see specific SOPs
  PBMC isolation, Plasma and Serum Storage  Cryopreserved
  
Table 1: Examples of LungMAP Human Tissue Core BioSpecimen Collection, Processing and Handling at Collection Site 

 
 

Lobe Sectioning Diagrams

Separation into lobes at bronchi

Left Lower Lobe Tissue Blocking Strategy

Right Lower Lobe Tissue Blocking Strategy

Left Upper Lobe Tissue Blocking Strategy

Right Middle Lobe Biopsy and Dissociation Strategy

Right Middle Lobe Tissue Blocking Strategy - alternating slices flash frozen / FFPE / PFAFrozenOCT

Trachea and Bronchi Tissue Blocking Strategy - image shows one side of bisected trachea only

Spleen Tissue Blocking Strategy

Organ	Starting Tissue	Prep	Process	BioSpecimen Type Collected from Organ
Trachea and large bronchi	Intact	Paraffin and OCT Frozen	Bisect longitudinally; Section into <1 cm long half rings	Formalin Fixed, Paraffin embedded PFASOCT embedded and frozen
Alternate Trachea and large bronchi	Intact	Cell Isolates	Enzymatic digestion and cell isolation	Frozen isolated cell aliquots
Lung: Right Upper Lobe +/- Right Middle Lobe	Fresh Tissue Blocks	Mixed Dissociated Cells	Dissociate cells by mechanical and enzymatic digestion procedure	Cryopreserve mixed cell aliquots
Sorted Dissociated Cells	Flow Activated Cell Sorting dissociated cells	Cryopreserve sorted cell aliquots “ex. Endothelial, Epithelial, Fibroblast, Bone Marrow Derived (CD45+) Separation”
Cryo-preserved QI	Cryo-preserved cells	Recover frozen cell aliquots, test purity by PCR, culture for viability and proliferative/ differentiation potential
Add’l Right Middle Lobe	Intact - Biopsies	Flash Frozen	Stored at -80	Protein Homogenates, RNA and/or DNA Isolation
Alternate Right Middle Lobe	Intact	Formalin Inflation, Paraffin	Inflation fixed with 10% buffered formalin x 24 hr (Intact Lobe); Sectioned by grid into 0.5-1 cm3 pieces; Dehydrated to 70% Ethanol	Inflation fixed, paraffin embedded ready for sectioning
Alternate Right Middle Lobe	Fresh Tissue Blocks	Fresh Frozen Embedded in OCT/CMC/none	Vertical 0.1 cm slices, Section each slice by grid into 1 cm2 blocks, alternate slices to embed blocks in OCT or approx. 5% CMC (see protocol) or freeze without embedding material	Non-inflated, non- fixed flash frozen tissue blocks, mapped to originating location
Right Lower Lobe	Intact	Formalin Inflation, Paraffin	Inflation fix with 10% buffered formalin x 24 hr (Intact Lobe) Sectioned by grid into 0.5-1 cm3 pieces Dehydrated to 70% Ethanol	Inflation fixed, paraffin embedded ready for sectioning
Left Upper Lobe	Intact	PFA Fixed, Sucrose Cryopreserved, OCT embedded	Inflation fixed with 4% paraformaldehyde x 24 hr (Intact Lobe); Sectioned by grid into 0.5-1 cm3 pieces; Cryopreserved in 10% sucrose; Block in OCT	Inflation fixed, cryo-preserved and frozen for cryo-sectioning
Alternate Left Upper Lobe	Intact	OCT/PBS	Inflate with 50:50 OCT:10%PBS	Frozen for cryo-sectioning

Alternate Left Upper Lobe	Intact	Air Inflated, Vascular Contrast	To be determined	To be determined
Left Lower Lobe	Intact	PFA Fixed, Sucrose Cryopreserved, OCT embedded	Inflation fixed with 4% paraformaldehyde x 24 hr (Intact Lobe); Sectioned by grid into 0.5-1 cm3 pieces; Cryopreserved in 10% sucrose; Block in OCT	Inflation fixed, cryo-preserved and frozen for cryo-sectioning
Additional Tissue	Intact	Similar to Lung, see specific SOPs	These include thymus, spleen, esophagus, heart, nerve, etc; Block as appropriate for tissue	Formalin Fixed, Paraffin embedded, PFA/OCT embedded-frozen Single cell dissociation
Peripheral Blood	Whole	Cryopreserved, see specific SOPs	PBMC isolation, Plasma and Serum Storage	Cryopreserved

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603.3 & 604.5_URMC_HTC_Whole Lung and Lobe Processing