Apr 20, 2023
Version 1
Ovarian tissue processing from organ donor V.1
- Hannah McDowell1,
- Elizabeth L Tsui1,
- Monica M Laronda1,2
- 1Ann & Robert H Lurie Children's Hospital of Chicago;
- 2Feinberg School of Medicine, Northwestern University
Protocol Citation: Hannah McDowell, Elizabeth L Tsui, Monica M Laronda 2023. Ovarian tissue processing from organ donor . protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jp4dl1b/v1Version created by Elizabeth L Tsui
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: April 05, 2023
Last Modified: April 20, 2023
Protocol Integer ID: 80066
Keywords: human ovary, HuBMAP, tissue process, organ donor, ovary
Funders Acknowledgement:
Center for Reproductive Science T32
Grant ID: T32HD094699
Eunice Kennedy Shriver National Institute of Child Health and Human Development
Grant ID: F30HD107966-01
Eunice Kennedy Shriver National Institute of Child Health and Human Development
Grant ID: U01HD110336
Abstract
Purpose: This protocol is intended to be used for human ovarian tissue processing from organ donors in a research setting. This details initial processing of the female reproductive tract, isolation of the ovary, and preparation of tissue sections for fixation, decellularization, freezing (for protein assays), and cryopreservation (by controlled-rate or slow freezing).
Guidelines
**CRITICAL**Personnel who process and handle human specimens must adhere to Office of Research Safety guidelines at their institution. They must be up to date on their original and refresher courses for the following training (or equivalent):
1. Biosafety Certification
2. Bloodborne Pathogens Certification
3. Training in Filling and Maintaining Liquid Nitrogen Tanks (as appropriate for LN2 use)
4. Working with Formaldehyde Certification (as appropriate for fixation)
5. CITI training (highly encouraged)
Materials
Human Ovarian Tissue Processing
All materials are listed as Type (Vendor, Catalog Number)
- OFC Holding Media (Sage/Origio, #ART-8040)
- Alternative: Leibovitz-15, L-15 (Caisson Labs, LVL02-6X500ML or equivalent)
- Stadie-Riggs Tissue Slicer (Thomas Scientific, discontinued)
- Disposable surgical scalpels (Exel International, 29550)
- Carbon Steel Blades (Thomas Scientific, 555TADIEBL)
- 15 mL conical tubes (Fisher Scientific, 14-959-53A or equivalent)
- 0.1% Sodium Dodecyl Sulfate, SDS
- Sodium Dodecyl Sulfate pellets (Sigma-Aldrich, 75746-1KG)
- dilute in MilliQ water (or equivalent)
- Modified Davidson's Fixative, MDF (Electron Microscopy Sciences, 64133-50)
- Histology grade ethanol (Bioworld, 40120790-3)
- 60 mm petri dish (CellTreat, 229663 or equivalent)
- 2.0 mL microcentrifuge tubes (Fisher Scientific, 05-408-138 or equivalent)
- 2.0 mL cryovial (TPP, 89020 or equivalent)
Safety warnings
Personnel will wear appropriate personal protective equipment. Areas are disinfected with the Dymon Do-it-all germicidal foaming cleaner (or equivalent) according to manufacturer's instructions.
Ethics statement
This work should take place under an IRB protocol or IRB exemption. The Laronda Lab operates in partnership with organ procurement programs and under the Ann & Robert H. Lurie Children's Hospital of Chicago IRB (2021-0549) and appropriate Materials Transfer Agreements (MTA) with the Gift of Hope Organ and Tissue Donor Network. MTA and Data Use Agreements (DUA) are also in place for transferring materials or unmodified derivatives and associated data within HuBMAP collaborations.
Ovarian Tissue Processing
Ovarian Tissue Processing
Record donor information. This may include: sample ID, location of procurement, date received, age, cause of death, sex, race, ethnicity, infectious disease serologies, weight, height, and body mass index (BMI).
Tissue must be processed within 24 hours of procurement in order to maintain optimal viability and is kept at 2-4 °C throughout all processing steps unless otherwise indicated. Additionally, tissue must be kept in appropriate holding media buffered for air, such as OFC Holding Media or Leibovitz-15, and at 2-4 °C to prevent the tissue from drying out.
Throughout processing, ensure notation of anatomic location within the ovary consistent with suggested nomenclature as described in these references.
CITATION
CITATION
Safety information
Personnel who process and handle human specimens must adhere to Office of Research Safety guidelines at their institution. They must be up to date on their original and refresher courses for the following training (or equivalent):
1. Biosafety Certification
2. Bloodborne Pathogens Certification
3. Training in Filling and Maintaining Liquid Nitrogen Tanks (as appropriate for LN2 use)
4. Working with Formaldehyde Certification (as appropriate for fixation)
5. CITI training (highly encouraged)
Personnel will wear appropriate personal protective equipment. Processing areas are disinfected with Dymon Do-it-all germicidal foaming cleaner (or equivalent) according to manufacturer's instructions.
Image anterior and posterior of the whole female reproductive tract (FRT) (uterus, fallopian tubes, and ovaries).
Anterior View
Posterior View
Record the weight of the whole reproductive tract.
Remove each ovary from the FRT by cutting along the hilum. Each ovary is placed in a 60 mm dish and labeled as the left or right ovary according to anatomic position.
Image and record the weight of each isolated ovary.
Bisect each ovary through the hilum (along the long axis) using a straight blade, keeping track of which half is located anatomically superior ("Over") vs. anatomically inferior ("Under").
These regions are named in line with the recently published 3-D regional model of the human ovary described in the following citation.
CITATION
Note any defining features of the bisected halves, if relevant (i.e., presence of hemorrhagic cysts, etc...).
Using the Stadie-Riggs slicer, prepare a 0.5 mm slice of the internal portion of a bisected half to generate a cross-sectional slice of the ovary across its short axis.
Equipment
Stadie-Riggs Tissue Slicer/Microtome Blades
NAME
labForce
BRAND
6727C10
SKU
LINK
Repeat this step with the other ovary to generate a total of two 0.5 mm tissue sections for tissue sections.
If you are proceeding with paraffin-embedded analyses, fix with Modified Davidson's Fixative (MDF).
If you are proceeding with analyses that require frozen sections for Vanderbilt collaborations, please follow the following protocol.
Protocol
NAME
Freezing and Formalin Fixation of Tissue
CREATED BY
Jamie Allen
Central 0.5 mm section for fixation
For MDF-fixed sections: after fixation in MDF overnight (< 24 hours), remove fixative and dehydrate tissue by washing in increasing amounts of histology grade ethanol. Begin at 50% ethanol (repeat 2x), then 60% ethanol (repeat 2x), and finally 70% ethanol. Package tissue into histology cassettes and store cassettes submerged in 70% ethanol at 4 °C until processed and embedded in paraffin by histology core.
Return to the bisected halves (over and under portions) and cut along the central pole to generate two quarter spheres (representing lateral and medial regions) per bisected half.
Bisected "Over" portion
Bisected "Under" portion
Generate tissue sections for decellularization.
With the lateral under portion from each ovary, use the Stadie-Riggs slicer to generate 0.5 mm sections.
Place each section into a labeled 15mL conical, noting anatomic location within the ovary. For example, the first 0.5 mm slice (most superficial) taken from the left ovary is labeled: Left Under, Lateral dECM-1.
Add the appropriate volume (10-20 x tissue volume) of 0.1% SDS to each conical and allow to nutate at 4 °C for 48 hours to decellularize. Replace SDS solution within the 48 hour incubation, if cloudy.
Conicals for decellularization (LU 1-4) corresponding to the 0.5mm slice - 2.0mm slice
Following incubation in 0.1% SDS solution, rinse decellularized samples in double distilled water for two minutes. Repeat 3 times total and proceed to downstream applications or store at -80 °C .
Generating banked tissue sections for slow freeze/cryopreservation.
With the medial under portion of the ovary, slice coronally into sections no larger than 5 mm in any dimension to allow for adequate penetration of cryoprotectant. A typical section is usually 4 mm x 2 mm x existing depth.
Note anatomic location as described above and proceed with cryopreservation protocol.
Protocol
NAME
Slow freeze (cryopreservation) protocol for human ovarian tissueCREATED BY
Elizabeth L Tsui
Generating tissue sections for protein analysis (flash freeze).
With the lateral over portion of the ovary, use the Stadie Riggs slicer to generate 0.5mm slices from the ovarian surface epithelium to the medulla.
Place into labeled 1.5 mL eppendorf tubes, noting anatomic position as described above.
When complete, place tubes in liquid nitrogen until frozen. Once frozen, transfer to -80 °C .
Safety information
Use appropriate personal protective equipment when using liquid nitrogen.
Generating slow frozen tissue sections for downstream RNAseq.
Using the medial over portion of the ovary, divide the quarter sphere into mesenteric (closest to hilum) and antimesenteric portions. For both mesenteric and antimesenteric pieces, slice the tissue coronally into sections no larger than 5 mm in any dimension to allow for adequate penetration of cryoprotectant. A typical section is 2 mm x 2 mm x existing depth.
Note anatomic location (make sure to include mesenteric vs. antimesenteric information) and proceed with cryopreservation protocol.
Protocol
NAME
Slow freeze (cryopreservation) protocol for human ovarian tissueCREATED BY
Elizabeth L Tsui
Overall Protocol Schematic
Overall Protocol Schematic
Protocol references
CITATION
CITATION