Aug 16, 2023

Public workspacePreparation and cryopreservation of human liver samples for analysis by flow cytometry (fresh or after cryobanking)

DOI
dx.doi.org/10.17504/protocols.io.8epv5x7e4g1b/v1
  • 1Charité - Universitätsmedizin Berlin
Wiebke Werner
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DOI: dx.doi.org/10.17504/protocols.io.8epv5x7e4g1b/v1
Protocol Citation: Wiebke Werner, Linda Hammerich 2023. Preparation and cryopreservation of human liver samples for analysis by flow cytometry (fresh or after cryobanking). protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5x7e4g1b/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: June 12, 2023
Last Modified: August 16, 2023
Protocol Integer ID: 83205
Keywords: human samples, liver, liver tumor, HCC, Flow Cytometry, Banking, FACS, human, immune monitoring, immune microenvironment
Abstract
This protocol focuses on the preparation of small human liver samples for single-cell analysis by flow cytometry. It provides two options for sample preparation: (1) immediate flow cytometry with fresh samples or (2) cryopreservation of samples at -80°C using the Stable-Lyse/Store V2 system from SmartTube Inc. for flow cytometry at a later timepoint.

Note: This protocol does not provide guidelines on how to create a multicolor flow cytometry panel!
Guidelines
  1. This protocol is validated for immune cell isolation of human liver and tumor specimen (weight 150-1000 mg). It has not yet been tested on biopsy material (< 20 mg).
  2. The retrieval of parenchymal cells is a by-product of this protocol but it has not been optimized for this purpose!
  3. Experiments should be performed on ice at all times!
  4. Work with filter tips!
  5. When working with fluorochrome-conjugated antibodies, avoid bright (sun) light and consider using aluminum foil to protect your samples from light while incubating.
  6. For waste management and time reasons, transfer of e.g. larger liquid volumes is done by pouring it directly from the bottle. If you prefer otherwise, you can always use serological pipets.

Advantages:
  1. Isolation of all major immune cell subtypes including granulocytes.
  2. Nycodenz gradient ensures minimal amount of parenchymal cells which improves the quality of the FACS analysis.
  3. Ready-to-use quality-controlled cryo-preservation buffers.

Limitations:
  1. As mentioned above, this protocol has not been tested on biopsies (< 20 mg tissue).
  2. If you also focus on isolation of hepatocytes, two-step-collagenase perfusion of bigger liver specimen might be a more suitable approach.

Qualifications:
We recommend basic experience in wet-lab work (e.g. how to pipette) to handle this protocol. You also might need a trial run to familiarize yourself with the procedures.
Materials
INSTRUCTIONS FOR STOCK AND BUFFER PREPARATIONS:
  • 25% BSA aliquots
  • Blocking Buffer
  • GBSS
  • Stop Digest Buffer
  • HBSS + 0,1% BSA
  • FACS Buffer
  • Fixaton Buffer
  • 1X Lysis Buffer
Download Buffers for Human Liver and Tumor Immune Cell Isolation.pdfBuffers for Human Liver and Tumor Immune Cell Isolation.pdf455KB


REAGENTS:
ReagentAlbumin bovine-serumMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4503
ReagentCalcium chloride dihydrate ≥99 % p.a. ACSCarl RothCatalog #5239.2
ReagentCollagenase B (Roche)Merck MilliporeSigma (Sigma-Aldrich)Catalog #11088815001
ReagentD( )-Glucose p.a. ACS anhydrousCarl RothCatalog #X997.2
Reagentdi-Sodium hydrogen phosphate dihydrate ≥990 % p.a.Carl RothCatalog #4984.2
ReagentDNase I (Roche)Merck MilliporeSigma (Sigma-Aldrich)Catalog #10104159001
Reagent1X Dulbecco’s Phosphate Buffered Saline (DPBS) Thermo Fisher ScientificCatalog #14190094
ReagentEDTA - Solution pH 8.0Panreac AppliChemCatalog #A3145
ReagentGibco™ HBSS without Calcium without Magnesium ohne PhenolrotThermo Fisher ScientificCatalog #14175053
ReagentGibco™ RPMI 1640 Medium (with L-Glutamine)Thermo Fisher ScientificCatalog #21875034
Reagent4% Paraformaldehyde (PFA) Solution in PBSBoster BioCatalog #AR1068
ReagentNycodenz AG®ProteogenixCatalog #1002424
ReagentMagnesium chloride hexahydrate ≥99 % p.a. ACSCarl RothCatalog #2189.1
ReagentMagnesium sulphate heptahydrate ≥99 % p.a. ACSCarl RothCatalog #P027.1
ReagentPotassium chloride ≥995 % p.a. ACS ISOCarl RothCatalog #6781.3
ReagentPotassium dihydrogen phosphate ≥99 % p.a. ACSCarl RothCatalog #3904.1
ReagentBD Pharm Lyse™BD BiosciencesCatalog #555899
ReagentSodium hydrogen carbonate ≥995 % p.a. ACS ISOCarl RothCatalog #6885.2

REAGENTS ONLY NEEDED FOR CRYOFIXATION:
ReagentStable-Lyse V2SMART TUBE Inc.Catalog #Stable-Lyse V2
ReagentStable-Store V2SMART TUBE Inc.Catalog #Stable-Store V2

REAGENTS ONLY NEEDED FOR FLOW CYTOMETRY:
ReagentSera from humanMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2257-1ml
ReagentSera from mouseMerck MilliporeSigma (Sigma-Aldrich)Catalog #S3509-1ml
ReagentNormal rabbit serum (invitrogen)Thermo Fisher ScientificCatalog #10510
ReagentNormal rabbit serum (invitrogen)Thermo Fisher ScientificCatalog #10710C

+ fluorochrome-conjugated antibodies of your choice
+ fixable viability stain (if desired)

DISPOSABLES OF YOUR CHOICE:
  • Cell Strainer 100 µM (for 50 ml Falcon tubes)
  • Centrifuge tubes (15 ml, 50 ml)
  • Cryotubes (1,8 ml)
  • Microcentrifuge tubes (1,5 ml, 2 ml)
  • Needles (20G)
  • Petri dishs (6 cm)
  • Pipet filter tips (10, 200 and 1000 µl)
  • Scalpel
  • Serological pipets (5 ml, 10 ml, 25 ml)
  • Syringes (10 ml)
  • Urine cups (100 ml)
  • 5 ml FACS tubes with 35 µM cell strainer cap
  • caps for FACS tubes


EQUIPMENT:
  • biosafety cabinet
  • centrifuge
  • (shaking) waterbath
  • ice buckets
  • small scissors
  • fine scale
  • small forceps
  • pipets (0,1-2,5 µl, 0.5-10 µl, 10-100 µl, 100-1000 µl)
  • pipette controler






Safety warnings
If your work with untested human samples (e.g. status of Hepatitis B/C and HIV is unknown), all experiments must be carried out in a Class II biosafety cabinet (with laminar air flow). Liquid waste has to be collected and autoclaved before disposal, contaminated materials have to be collected and disposed of separately from other lab waste as they are potentially infectious.

Use caps/lids to close your FACS tube when removing them from the biosafety cabinet for centrifugation. Use pipet tips with filters.

Depending on your personal safety preferences as well as the specific regulations of your research department/institute/country, there might be other or additional regulations to consider. Contact your biosafety officer!
Ethics statement
Before you work with human samples, you have to acquire an human ethics approval from the local ethics committee of your institution! Every subject/patient has to give informed consent!
Before start
  1. Make sure you read the safety warnings regarding untested human samples!
  2. Make sure an ethics approval has been obtained before working with subjects or patient samples!
  3. Revisit the materials list to make sure all the equipment, materials and reagents are available to you.
Preparations (before samples are aquired from the OR)
Preparations (before samples are aquired from the OR)
30m
30m
Start the biosafety cabinet.

1m
Prepare all the reagents and equipment needed for cell isolation.
Prechill a centrifuge to Temperature4 °C as well as preheat a (shaking) waterbath to Temperature37 °C . Place a 50 ml falcon tube containing HBSS inside the waterbath to warm up.

2m
Place all needed buffers and reagents, including a 50 ml falcon tube containing HBSS, TemperatureOn ice or in the wasterbath.

Note
  • 2x HBSS in 50 ml Falcon (4°C and 37 °C)
  • Stop Digest Buffer (on ice)
  • HBSS +0.1% BSA
  • 1x Lysis Buffer
  • FACS buffer

(when you start FACS right after isolation)
  • Blocking Buffer

3m
Place urin cups filled with around 50 ml Medium (RPMI) for your samples TemperatureOn ice .

3m
Freshly weigh Collagenase B (8.52 mg per sample) and DNase I (1.875 mg per sample) in 2 ml tubes and place them TemperatureOn ice until further use.
Note
You may prefer to predilute your enzymes and freeze them in aliquots. We choose to prepare the enzymes freshly for stable enzyme activity.

10m
Prepare Nycodenz stock solution (14ml per sample) and place TemperatureOn ice .

Note
  • per sample weigh 4g of Nycodenz in a 50 ml Falcon
  • add 14 ml of GBSS to the Falcon
  • close the top tightly and vortex until half of the Nycodenz has dissolved
  • let the falcon rest on the side on the countertop for 5 min
  • vortex again until dissolved completely
  • keep on ice

Set up the biosafety cabinet with all the equipment you'll need and make sure, everything you will need can be reached easily.
Note
Preparing all the equipment you need will save you ample time. Working fast is key in recovering viable liver/tumor immune cells.

5m
Directly before leaving for the OR to pick up the samples, place the Collagenase in the waterbath to preheat.
1m
Digestion (BSL2)
Digestion (BSL2)
50m
50m
Place the liver samples in the urine cups with medium, and transport them back to the lab TemperatureOn ice . Place samples (still on ice) inside the biosafety cabinet.

Note
CAVE - With the exception of the digestion, which takes place at 37 °C, place your samples on ice at all times!

Dissolve the DNase I in an appropriate amount of cold HBSS (Amount7.5 mL per sample) and keep TemperatureOn ice in a 15 ml Falcon.
Note
HOW TO - Using a 1000 µl pipette, dissolve the DNase in 1 ml cold HBSS and transfer to a 15 ml falcon tube. Add remaining amoutn of cold HBSS and mix well.



3m
Dissolve Collagenase B in an appropriate amount of 37 C° warm HBSS (Amount3 mL per sample) in a 15 ml Falcon. Transfer back to Temperature37 °C until needed for digestion.

3m
Weigh the tissue samples and place in petri dishes on a cooled sample holder.
5m
If your samples are very bloody, you may attempt to flush the samples. For this, fill a 10 ml syringe with cold HBSS and attach a needle. Puncture the sample and carefully press the HBSS into the tissue to flush out the blood. Repeat this process several times until the liquid runs mostly clear. Discard the liquid before proceeding.
5m
Optional
Cut sample into very small pieces using first a scapel and then fine scissors until the whole tissue appears pulpous.
3m
Add Amount3 mL Collagenase B to each petri dish, swirl to mix and transfer to a 50 ml falcon by pouring the liquid directly into the tube. Use a small forceps to get all the tissue pieces out of the petri dish.

2m
Add Amount250 µL DNase I to each falcon and close the top tightly. Mix by swirling the falcon
1m
Immediately transfer to a shaking waterbath (Temperature37 °C , Shaker150 rpm ) and digest for Duration00:30:00 . Shake the sample vigorously every 5 min!
Note
CAVE - If you do not have a shaking waterbath, any waterbath will do. However, it is crucial to shake the sample on a regular basis!

30m
Digestion
Critical
After digestion, place falcons TemperatureOn ice and immediately add Amount10 mL cold Digest Stop to end the digestion process.

2m
Filtering and Lysis (BSL2)
Filtering and Lysis (BSL2)
1h 42m
1h 42m
Use a 10 ml serological pipet to pipette the cell suspension up and down multiple times to further detach the cells from their connective tissue. When the cell suspension runs easily through the pipet, proceed to the next step.
2m
Using the same 10 ml serological pipet, filter the sample through a 100 µm cell strainer into a new 50 ml falcon.
1m
Use Amount20 mL cold Digest Stop to rinse the old falcon. Gently press the remaining tissue through the cell strainer with the smooth end of a syringe plunger, repeatingly rinsing the mesh with the Digest Stop from the old falcon.

Note
CAVE - Press the plunger straight down on the cell strainer and release in tapping motions.


5m
Remove the cell strainer and add Amount3 mL DNase I to the cell suspension.

Spin Centrifigation500 x g, 4°C, 00:05:00 .
5m
Centrifigation
Place your falcons back TemperatureOn ice and discard the supernatant by pouring it into the liquid waste bottle without disrupting the pellet.
1m
If you want to perform lysis right away, continue here. Otherwise, continue with step 21.

Note
GOOD TO KNOW - The gradient will also eliminate erythrocytes - if you do not plan to work with the parenchymal cells, you can skip this step. You can also treat parenchymal cells with lysis buffer at a later timepoint or use an erythrocyte depletion kit.


Optional
Resuspend the cells by gently flicking the tube or pipetting up and down. Add Amount2 mL 1X Lysis Buffer , gently pipette up and down to mix and incubate for Duration00:05:00 TemperatureOn ice .
5m
Incubation
Add Amount40 mL cold Digest Stop and Amount4 mL DNase I to each falcon.
2m
Spin Centrifigation50 x g, 4°C, 00:02:00 to pellet the parenchymal cells.
Note
If you set focus on isolating parenchymal cells (hepatocytes), you might extent this step to Duration00:05:00 - keep in mind that you might lose some of your non-parenchymal cells when you do so.

Furthermore, fractions of parenchymal cells will be relatively small, as the tissue section is cut and digested without perfusion.



2m
Centrifigation
Transfer the supernatant to a new falcon and spin Centrifigation500 x g, 4°C, 00:10:00 .



10m
Centrifigation
Discard the supernatant and add cold HBSS + 0,1% BSA with a serological pipet to a final volume of Amount7.4 mL and gently resuspend.

2m
Critical
Add Amount12.6 mL Nycodenz stock solution with a serological pipet and gently mix until homogenous.

1m
Overlay the Nycodenz-Cell Suspension with Amount6 mL HBSS + 0,1% BSA.
Note
HOW TO OVERLAY - Due to the higher density of Nycodenz, HBSS will layer on top if it is applied carefully.

1. The dispension force of your pipette controller has to be deactived.
2. Hold the 50 ml falcon containing the Nycodenz-cell suspension at a 45° angle.
3. Aspirate 6 ml of HBSS into a 10 ml serological pipet and press the tip to the tube wall in a 90° angle (the tip opening should be flat on the tube wall).
4. Very slightly press the dispense button and let the HBSS run down the side of the tube in a constant trickle without any drops forming.

5m
Critical
Transfer the falcon tubes to a centrifuge and spin Centrifigation1400 x g, 4°C, 00:22:00 without break (takes around 45min total).


35m
Centrifigation
Critical
After centrifugation, place the tubes back TemperatureOn ice . Carefully remove the first 2-3 ml from the top layer (debris and dead cells) and discard. Next, use a 10 ml serological pipet to slowly remove the white interphase (middle layer containing the immune cells) and transfer to a new falcon tube.
Note
You can also use a sterile pasteur pipet to remove the middle layer.


5m
Add cold HBSS + 0,1% BSA to a final volume of Amount30 mL and spin Centrifigation500 x g, 4°C, 00:10:00 .

10m
Centrifigation
Discard the supernatant without disrupting the pellet and resuspend the cells in Amount2 mL cold FACS buffer.

1m
Pass the cell suspension through a 35 µm cell strainer cap into FACS tubes placed TemperatureOn ice . Discard the cell strainer cap afterwards.

Safety information
Always close the FACS tubes with a cap before taking them out of the biosafety cabinet!


2m
Spin Centrifigation500 x g, 4°C, 00:05:00

5m
Centrifigation
Discard the supernatant and resuspend the cell pellet in the remaining buffer by flicking the tube.
Note
HOW TO DECANT - Empty the supernatant with momentum and - while keeping the FACS tube in an upside-down position to not disrupt the pellet on the bottom - dip the tube onto a paper towel to get rid of the excess liquid. Turn the FACS tube upright and gently flick to resuspend.

1m
Depending on whether you want to perform flow cytometry on fresh or cryo-preserved samples, you can select different protocol options at this step.
Step case

Proceed to Flow Cytometry right away
18 steps

Use this protocol if you want to perform flow cytometry immediately after immune cell isolation.
Preparations for Stainings (BSL2)
Preparations for Stainings (BSL2)
Label your 5 ml FACS tubes and place them in a cold sample holder or TemperatureOn ice . You might need several tubes per sample, depending on the number of panels and controls you are planning to run.
Note
GOOD TO KNOW - As liver samples are very autofluorescent, recording an unstained control for every sample will give you a far better unmixing/compensation result.

FMO (fluorescence minus one) controls are helpful to discriminate positive and negative (unspecific background fluorescence) signals, especially if the positive cell populations are not distinctly separated from the negative population. In an FMO tube, all fluorochromes in the panel are present except the fluorochrome in question.


Prepare the antibody mix: For each FACS tube, use Amount20 µL blocking buffer and the appropriate amount of each of your antibodies. Always prepare the master mix for one extra sample or add 10% to account for pipetting errors.
Note
EXAMPLE 1
  • 2 samples (one full stain and one control each) + 1 extra for pipetting error = 5x mix
  • 20 antibodies in panel (1µl needed per sample)
  • Blocking Buffer: 5 x 20 µl = 100 µl
  • 5 µl per antibody
  • final volume of antibody master mix: 200 µl
  • mix to add per FACS tube = 20 µl blocking buffer + 20 µl antibodies = 40 µl

EXAMPLE 2
  • 2 samples (one full stain and one control each) = 4x mix +10%
  • 20 antibodies in panel (1µl needed per sample)
  • Blocking Buffer: 4x 20 µl = 80 µl (+ 10%) = 88 µl
  • Each antibody: 4 µl per antibody +10% = 4,4 µl per antibody
  • final volume of antibody master mix: 176 µl
  • mix to add per FACS tube = 20 µl blocking buffer + 20 µl antibodies = 40 µl

Divide the cell suspension between the different FACS tubes. Its important to document the amount of sample going into each tube to allow determination of absolute cell numbers during analysis (eg., calculate cells/ gram tissue).
Bring the volume in each FACS tube to Amount100 µL with FACS buffer. Set aside the unstained control.

Staining (BSL2)
Staining (BSL2)
5m
5m
If you prefer, switch of the light in the biosafety cabinet/lamina flow hood to avoid bleaching the fluorochrome-conjugated antibodies.
Add the appropriate concentration of fixable viability stain to the cell suspension in each of the FACS tubes and mix well by flicking the tube.
Note
HOW TO "FLICK"- Mix well by holding the upper part of the tube between thumb and index finger of one hand and at the same time, gently flick the bottom of the tube repeatedly with the index finger of the other hand.

Incubate for Duration00:05:00 TemperatureOn ice .

5m
Incubation
Add the appropriate amount of the prepared antibody mix to each tube. Gently pipette up and down to mix.

Note
EXPERT TIP - In case you are using FMO control(s), you can prepare the antibody mix for all your tubes without the FMO antibody(ies) and add them later to your full stain tube.

Incubate for Duration00:20:00 TemperatureOn ice .

20m
Incubation
Washing and Fixation (BSL2)
Washing and Fixation (BSL2)
20m
20m
Add Amount2 mL FACS buffer and mix well by pipetting up and down.
Spin Centrifigation500 x g, 4°C, 00:05:00
5m
Centrifigation
Discard the supernatant by decanting and resuspend the cell pellet in the remaining buffer by flicking the tube.

Add Amount1 mL Fixation Buffer and mix well by pipetting up and down.
Incubate for Duration00:10:00 at TemperatureOn ice .

10m
Incubation
Add Amount2 mL FACS buffer and mix well by pipetting up and down.
Note
After fixation, samples can be further processed on the bench, a biosafety cabinet is no longer necessary.

Spin Centrifigation500 x g, 4°C, 00:05:00
5m
Centrifigation
Discard the supernatant and resuspend the cell pellet in the remaining buffer by flicking the tube.
Add Amount200 µL FACS buffer and proceed to sample aquisition.
Note
Fixed samples can be stored at Temperature4 °C overnight and recorded the next day. However, keep in mind that fixatives such as formalin can change the autofluorescence of your cells and destabilize fluorochromes, especially tandem dyes. If possible, record you samples directly after fixation.

Protocol references
https://www.smarttubeinc.com/protocols/stbl/ST_SLSSP1TF-150203.pdf