Nov 14, 2021
617.1 URMC HTC BSL2+ Non-Inflated Fresh-Frozen Embedded Lung and Associated Tissue
- Gloria Pryhuber1,
- Heidie Huyck2,
- Lisa Rogers1,
- Cory Poole2
- 1University of Rochester Medical Center;
- 2University of Rochester
- Human BioMolecular Atlas Program (HuBMAP) Method Development Community
- LungMap2 Consortium
Protocol Citation: Gloria Pryhuber, Heidie Huyck, Lisa Rogers, Cory Poole 2021. 617.1 URMC HTC BSL2+ Non-Inflated Fresh-Frozen Embedded Lung and Associated Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.bjtsknne
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: August 16, 2020
Last Modified: November 14, 2021
Protocol Integer ID: 40530
Keywords: OCT, CMC, rapid freeze, lung, COVID-19, SARS CoV2, frozen embedded lung, freezing of human lung tissue, human lung tissue, urmc htc bsl2, lung, sars cov2, known sars cov2, rapid freezing, freezing media, infected tissue, freezing, tissue, associated tissue purpose,
Funders Acknowledgements:
National Heart Lung and Blood Institute
Grant ID: U01HL148861-02S1
Abstract
Purpose and Scope of the Procedure
- Safe work with unfixed potentially or known SARS CoV2 / COVID-19+ infected tissue
- Rapid blocking, embedding and freezing of human lung tissue in no freezing media, 100% OCT or 5% CMC
- Rapid freezing of non-lung tissue in no freezing media, 100% OCT or 5% CMC
Guidelines
Scientific Principles:
- CDC. “COVID-19 Personal Protective Equipment (PPE) for Healthcare Personnel.” Centers for Disease Control and Prevention, 1 Jan. 2020, www.cdc.gov/coronavirus/2019-ncov/downloads/COVID-19-PPE.pdf.
- WHO. “Personal Protective Equipment for COVID-19.” World Health Organization, World Health Organization, 5 May 2020, www.who.int/medical_devices/priority/COVID_19_PPE/en/.
- University of Rochester Medical Center, director. COVID-19 Safety Training, University of Rochester Medical Center , 15 June 2020, rochester.csod.com/LMS/LoDetails/DetailsLo.aspx?loid=e8469bef-ae75-4d87-9ec1-f5d33e4f1519&query=%3fq%3dCOVID-19+Safety+Training&isCompletionRedirect=true&loStatus=16®num=1#t=1.
Materials
MATERIALS
Brady FREEZERBONDZ Polyester thermal transfer printer labelsFisher ScientificCatalog #22-500521
OCT (mold)Catalog #Tissue-Tek 4583
Carboxymethylcellulose (CMC); 5% (w/w) CMC in DEPC H2OCatalog #Sigma C5678-1KG
Worksheet 603.A.HTC_Whole_or_Partial_Lung_Processing
Biosafe surface and environment for manipulation of lung
- Biosafety Cabinet or Grossing Station
- N95 masks, face shields, double gloves, shoe covers, hair nets, sleeve covers, and labcoat for PPE, all consistent with recommended PPE for SARS-CoV2 / COVID-19 work
- Biohazards Disposal Bag
- Red Sharps Container
- Small and Medium Gauze Pads – multi-pack
- Plastic Cryomolds
- Scalpels and Trimming Blades with Handles; Forceps; Other Dissection Equip
- Freezerbondz Labels and Printer
- Standard Balance and Weigh Boats: Medium and Large
- Rulers – metal 18 inch (45cm) and 12 inch (30.5cm)
- 100% OCT (Tissue Tek)
- 5% (w/w) Carboxymethylcellulose (CMC) in DEPC treated water
- Flat ice buckets or large Styrofoam boxes partially filled with dry ice pellets
- Flat metal pans
- 200 proof (100%) ethanol or isobutane to create dry ice bath
- Aluminum foil and labeling tape or freezer storage bags
- Cryovials with red cap inserts
- Small biohazard stickers
Safety warnings
Working with potentially or known SARS CoV2/COVID-19+ human tissue: Personnel will adhere to safe work processes all consistent with recommended PPE for COIVD-19 work. PPE will be used including N95 mask, face shield, lab coat, closed shoes and double gloves, shoe covers, hair net, and sleeve covers. All activity will be behind shield of biosafety cabinet and/or with mask and safety glasses. Biosafety level 2 + practices will be followed, and the work performed in the designatelabspace that is covered by annually updated IBC approved protocol. All institutional biosafety measures are followed in any manipulation of these human tissues.
Fresh Tissue Frozen in Cryomolds
Slicing and blocking procedure should be accomplished in a grossing station or fume hood with the operator taking appropriate blood and body fluid precautions
Prepare dry ice-ethanol (or isobutane) bath by covering bottom of flat ice bucket or styrofoam box with dry ice pellets.
Pour in enough 200 proof ethanol to cover the dry ice and allow to boil. Once bubbling is stable, float a flat metal pan on the ethanol.
Add more dry ice and/or ehtanol as needed to maintain rapid freezing of molds set on the metal pan.
Take care that ethanol doesn't splash over onto tissue in cryomolds.
Keep the surface of the lobes moist and cool at all times
Select lobe or partial lobes for fresh embedding and freezing
Place fresh lobe on cutting surface and section tissue into slices and further into blocks.
Our standard is approximately 1cmx 1cm x 0.5 cm thick. See sectioning diagram for lobe in 604. URMC HTC protocol.
Work quickly to avoid warming of lung tissue.
Photograph resulting tissue sections as a map of the stored blocks. Include labels in photos to record work, location of blocks and processing method applied.
Suggest alternating slices of tissue between different embedding media. For example, a slice each to i-iii, then repeat:
i.Fresh Flash Frozen Block [FFb] (not embedded in any media) - note: may not withstand long freezing period
ii.Fresh OCT Frozen Block
iii.Fresh CMC Frozen Block
In the case where only 1 lobe is received, slices are taken for Cell Dissociation, Fresh Frozen Biopsy (in cryovial) and Formalin Fixed Paraffin Embedded Samples and fresh frozen blocks in OCT with potential postfixation in paraformaldehyde.
For OCT or CMC embedding: Fill the cryomolds with a thin layer of the appropriate embedding medium
Place the tissue in their respectively labeled cryomolds and position them to allow for small amount of embedding medium around the tissue
Gently press down on the tissue with forceps in an attempt to remove as much air as possible and make sure the tissue lays flat along the bottom of the cryomold during freezing so easier to face block for sectioning
Place cryomolds containing tissue on metal pan in dry-ice ethanol bath and allow to freeze while filling the cryomold with freezing medium (OCT or CMC) to completely cover the embedded tissue.
While cryomolds are freezing, print out Freezerbondz labels for each block and place label on middle of a piece of aluminum foil along with a small biohazard sticker.
Applying the label to the foil prior to wrapping the cold blocks promotes better adhesion and reduces any warming of block while wrapping.
Once frozen, quickly wrap each corresponding block with the correctly labeled aluminum foil and return to the frozen metal pan to keep frozen until transferred to -80°C freezer
Collect wrapped cryomolds into labeled freezer bags, place in cold labeled freezer boxes and store in -80°C freezer
Flash Frozen Tissue for homogenates / RNA Isolation
These tissue portions of Lung, Thymus, Spleen and Lymph Node are intended for processing for total RNA and for protein homogenates.
The tissue samples are sectioned into 0.25-1.0 cm3 portions and placed in properly Freezerbondz labeled freezer vials.
The weight of the collected tissue is recorded.
The freezer tubes containing the individual tissues are then frozen over liquid nitrogen and placed at -800C for storage.
Recording and Analysis of Results
Complete worksheet and virtual freezer inventory
Correctly store photographs of grossing and blocking in Database