TITLE: Preparation and cryopreservation of human liver samples  for analysis by flow cytometry (fresh or after cryobanking)
AUTHORS: Wiebke Werner, Linda Hammerich

[DESCRIPTION]
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!

[BEFORE_START]
Make sure you read the safety warnings regarding untested human samples!
Make sure an ethics approval has been obtained before working with subjects or patient samples!
Revisit the materials list to make sure all the equipment, materials and reagents are available to you.

[GUIDELINES]
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).
The retrieval of parenchymal cells is a by-product of this protocol but it has not been optimized for this purpose!
Experiments should be performed on ice at all times!
Work with filter tips!
When working with fluorochrome-conjugated antibodies, avoid bright (sun) light and consider using aluminum foil to protect your samples from light while incubating.
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:
Isolation of all major immune cell subtypes including granulocytes.
Nycodenz gradient ensures minimal amount of parenchymal cells which improves the quality of the FACS analysis.
Ready-to-use quality-controlled cryo-preservation buffers.

Limitations:
As mentioned above, this protocol has not been tested on biopsies (< 20 mg tissue).
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.

[STEPS]
SECTION: Preparations (before samples are aquired from the OR)
1. Start the biosafety cabinet.

SECTION: Preparations (before samples are aquired from the OR)
2. Prepare all the reagents and equipment needed for cell isolation.
SECTION: Preparations (before samples are aquired from the OR)
2.2. Place all needed buffers and reagents, including a 50 ml falcon tube containing HBSS, on ice or in the wasterbath. 

 

SECTION: Preparations (before samples are aquired from the OR)
2.3. Place urin cups filled with around 50 ml Medium (RPMI) for your samples on ice. 

SECTION: Preparations (before samples are aquired from the OR)
2.4. Freshly weigh Collagenase B (8.52 mg per sample) and DNase I (1.875 mg per sample) in 2 ml tubes and place them on ice  until further use.
 

SECTION: Preparations (before samples are aquired from the OR)
2.1. Prechill a centrifuge to 4 °C  as well as preheat a (shaking) waterbath to 37 °C . Place a 50 ml falcon tube containing HBSS inside the waterbath to warm up.

SECTION: Preparations (before samples are aquired from the OR)
2.6. Set up the biosafety cabinet with all the equipment you'll need and make sure, everything you will need can be reached easily.
 

SECTION: Preparations (before samples are aquired from the OR)
3. Directly before leaving for the OR to pick up the samples, place the Collagenase in the waterbath to preheat.
SECTION: Digestion (BSL2)
4. Place the liver samples in the urine cups with medium, and transport them back to the lab on ice. Place samples (still on ice) inside the biosafety cabinet.

 

SECTION: Digestion (BSL2)
5. Dissolve the DNase I in an appropriate amount of cold HBSS  (7.5 mL per sample) and keep on ice in a 15 ml Falcon.
 



SECTION: Digestion (BSL2)
6. Dissolve Collagenase B in an appropriate amount of 37 C° warm HBSS (3 mL per sample) in a 15 ml Falcon. Transfer back to 37 °C until needed for digestion.

SECTION: Digestion (BSL2)
7. Weigh the tissue samples and place in petri dishes on a cooled sample holder.
SECTION: Digestion (BSL2)
9. Cut sample into very small pieces using first a scapel and then fine scissors until the whole tissue appears pulpous.
SECTION: Digestion (BSL2)
10. Add 3 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.

SECTION: Digestion (BSL2)
12. Immediately transfer to a shaking waterbath  (37 °C ,   ) and digest for 30 min . Shake the sample vigorously every 5 min!
 

SECTION: Filtering and Lysis (BSL2)
15. Using the same 10 ml serological pipet, filter the sample through a 100 µm cell strainer into a new 50 ml falcon.
SECTION: Filtering and Lysis (BSL2)
16. Use 20 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.

 


SECTION: Filtering and Lysis (BSL2)
20. If you want to perform lysis right away, continue here. Otherwise, continue with step 21.

 


SECTION: Filtering and Lysis (BSL2)
17. Remove the cell strainer and add 3 mL DNase I to the cell suspension. 

SECTION: Filtering and Lysis (BSL2)
19. Place your falcons back on ice and discard the supernatant by pouring it into the liquid waste bottle without disrupting the pellet.
SECTION: Filtering and Lysis (BSL2)
21. Spin 50 x g, 2 min, 4 °C to pellet the parenchymal cells.
 


SECTION: Filtering and Lysis (BSL2)
22. Transfer the supernatant to a new falcon and spin 500 x g, 10 min, 4 °C .



SECTION: Filtering and Lysis (BSL2)
23. Discard the supernatant and add cold HBSS + 0,1% BSA with a serological pipet to a final volume of 7.4 mL and gently resuspend.

SECTION: Filtering and Lysis (BSL2)
24. Add 12.6 mL Nycodenz stock solution with a serological pipet and gently mix until homogenous. 

SECTION: Filtering and Lysis (BSL2)
25. Overlay the Nycodenz-Cell Suspension with 6 mL HBSS + 0,1% BSA. 
 

SECTION: Filtering and Lysis (BSL2)
26. Transfer the falcon tubes to a centrifuge and spin 1400 x g, 22 min, 4 °C without break (takes around 45min total).


SECTION: Filtering and Lysis (BSL2)
27. After centrifugation, place the tubes back on 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.
 


SECTION: Filtering and Lysis (BSL2)
28. Add cold HBSS + 0,1% BSA to a final volume of 30 mL and spin 500 x g, 10 min, 4 °C .

SECTION: Filtering and Lysis (BSL2)
29. Discard the supernatant without disrupting the pellet and resuspend the cells in 2 mL cold FACS buffer.

SECTION: Filtering and Lysis (BSL2)
30. Pass the cell suspension through a 35 µm cell strainer cap into FACS tubes placed on ice . Discard the cell strainer cap afterwards. 

 


SECTION: Filtering and Lysis (BSL2)
31. Spin 500 x g, 5 min, 4 °C

SECTION: Choose protocol according to desired further sample processing
33. Depending on whether you want to perform flow cytometry on fresh or cryo-preserved samples, you can select different protocol options at this step.
SECTION: Preparations for Stainings (BSL2)
34. Label your 5 ml FACS tubes and place them in a cold sample holder or on ice . You might need several tubes per sample, depending on the number of panels and controls you are planning to run.
 


SECTION: Preparations for Stainings (BSL2)
35. Prepare the antibody mix: For each FACS tube, use 20 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.
 

SECTION: Preparations for Stainings (BSL2)
36. 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).
SECTION: Filtering and Lysis (BSL2)
32. Discard the supernatant and resuspend the cell pellet in the remaining buffer by flicking the tube.
 

SECTION: Preparations for Stainings (BSL2)
37. Bring the volume in each FACS tube to 100 µL with FACS buffer. Set aside the unstained control.

SECTION: Staining (BSL2)
39. 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.
 

SECTION: Staining (BSL2)
40. Incubate for 5 min on ice  .

SECTION: Staining (BSL2)
41. Add the appropriate amount  of the prepared antibody  mix to each tube. Gently pipette up and down to mix.

 

SECTION: Staining (BSL2)
42. Incubate for 20 min on ice .

SECTION: Staining (BSL2)
38. If you prefer, switch of the light in the biosafety cabinet/lamina flow hood to avoid bleaching the fluorochrome-conjugated antibodies.
SECTION: Washing and Fixation (BSL2)
43. Add 2 mL FACS buffer and mix well by pipetting up and down.
SECTION: Washing and Fixation (BSL2)
44. Spin 500 x g, 5 min, 4 °C 
SECTION: Washing and Fixation (BSL2)
45. Discard the supernatant by decanting and resuspend the cell pellet in the remaining buffer by flicking the tube.

SECTION: Washing and Fixation (BSL2)
46. Add 1 mL Fixation Buffer and mix well by pipetting up and down.
SECTION: Washing and Fixation (BSL2)
47. Incubate for 10 min at on ice .

SECTION: Washing and Fixation (BSL2)
48. Add 2 mL FACS buffer and mix well by pipetting up and down.
 

SECTION: Washing and Fixation (BSL2)
49. Spin 500 x g, 5 min, 4 °C 
SECTION: Washing and Fixation (BSL2)
50. Discard the supernatant and resuspend the cell pellet in the remaining buffer by flicking the tube.
SECTION: Washing and Fixation (BSL2)
51. Add 200 µL FACS buffer and proceed to sample aquisition.
 

SECTION: Preparations
34. Label 5 ml FACS tubes and 1,8 ml cryotubes to store your samples. Remember to prepare additional aliquots to allow for staining controls at analysis (such as FMO or unstained control). 
 

SECTION: Fixable Viability Stain (BSL2)
35. Divide the cell suspension between the FACS tubes. Its important to document how you split up the sample because you will need this later to calculate absolute cell numbers (cells gram tissue).
SECTION: Fixable Viability Stain (BSL2)
37. Using a small pipett, add the appropriate concentration of fixable viability stain to the FACS tubes containing full stain and FMO sample and mix well by flicking the tube. Do NOT add fixable viability stain to the unstained control.
SECTION: Fixable Viability Stain (BSL2)
36. Bring the volume in each FACS tube to 250 µL with FACS buffer. Set aside the unstained control.
SECTION: Fixable Viability Stain (BSL2)
38. Incubate for 5 min on ice .
SECTION: Fixable Viability Stain (BSL2)
39. After incubation, add 2 mL FACS buffer to each tube and mix well by pipetting up and down.
 

SECTION: Fixable Viability Stain (BSL2)
40. Spin 500 x g, 5 min, 4 °C .

SECTION: Fixable Viability Stain (BSL2)
41. Using a 1000 µl pipette, carefully pipette off 2 mL of the supernatant without disrupting the cell pellet on the bottom of the tube.
SECTION: Fixable Viability Stain (BSL2)
42. Gently mix the remaining volume (it should be approximately 250 µL ) and transfer to the corresponding 1,8 ml cryotube.
SECTION: Banking tissue immune cells (BSL2)
43. Add 350 µL Stable-Lyse Buffer to each cryotube, mix well by pipetting up and down.
SECTION: Banking tissue immune cells (BSL2)
44. Incubate 15 min at Room temperature .
SECTION: Banking tissue immune cells (BSL2)
45. Add 1000 µL Stable-Store Buffer to each cryotube, mix well by pipetting up and down. 
SECTION: Banking tissue immune cells (BSL2)
46. Close the cryotubes and incubate 15 min at Room temperature .
 


SECTION: Banking tissue immune cells (BSL2)
47. Immediately transfer to -80 °C for long-term storage. Samples can be stored for several months.
SECTION: Prepare samples for staining
48. Transfer your samples from -80 °C  to an open rack to the fridge (4 °C ) to thaw for approximately 60 min .
SECTION: Prepare samples for staining
49. In the meantime, prepare the antibody mix: For each FACS tube, use 20 µL  blocking buffer and the appropriate amount of each of your antibodies. Always prepare the master mix for one extra sample or add 10% for pipetting errors.
 

SECTION: Prepare samples for staining
51. Add 2 mL FACS buffer to each tube and mix well by pipetting up and down.
SECTION: Prepare samples for staining
53. Discard the supernatant and resuspend the cell pellet in the remaining buffer by flicking the tube.
SECTION: Prepare samples for staining
52. Spin 500 x g, 5 min
SECTION: Prepare samples for staining
54.   and repeat wash-spin-discard supernatant. Afterwards follow-up with step 55.

SECTION: Staining
55. Bring each sample to a final volume of 100 µL by filling up the missing amount with FACS buffer.
SECTION: Staining
56. Add the appropriate amount  of the prepared antibody  mix to each tube. Gently pipette up and down to mix.
 

SECTION: Staining
57. Incubate 20 min at Room temperature .
SECTION: Staining
58. Add 2 mL FACS buffer to each tube.
SECTION: Staining
59. Spin 500 x g, 5 min 
SECTION: Staining
60. Discard the supernatant and resuspend the cell pellet in the remaining buffer by flicking the tube.
SECTION: Staining
61. Add 200 µL FACS buffer and proceed to sample aquisition.



SECTION: Digestion (BSL2)
8. 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.
SECTION: Digestion (BSL2)
11. Add 250 µL DNase I to each falcon and close the top tightly. Mix by swirling the falcon
SECTION: Digestion (BSL2)
13. After digestion, place falcons on ice and immediately add 10 mL cold Digest Stop to end the digestion process.

SECTION: Filtering and Lysis (BSL2)
14. 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.
SECTION: Filtering and Lysis (BSL2)
20.2. Add 40 mL cold Digest Stop  and 4 mL DNase I to each falcon.
SECTION: Filtering and Lysis (BSL2)
20.1. Resuspend the cells by gently flicking the tube or pipetting up and down. Add 2 mL 1X Lysis Buffer , gently pipette up and down to mix and incubate for 5 min on ice .
SECTION: Filtering and Lysis (BSL2)
18. Spin 500 x g, 5 min, 4 °C .
SECTION: Preparations (before samples are aquired from the OR)
2.5. Prepare Nycodenz stock solution (14ml per sample) and place on ice .

 

SECTION: Prepare samples for staining
50. Transfer the thawed samples to their corresponding 5 ml FACS tubes.