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Isolation of tubule fragments from organoids (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Leif Oxburgh

Keywords

[‘3D culture’, ‘3D imaging’, ‘Cell dissociation’, ‘culture’, ‘fetal mouse’, ‘isolation’, ‘kidney’, ‘kidney development’, ‘organoid’, ‘tissue dissociation’]

Subjects

[‘Isolation, purification and separation’, ‘Developmental biology’, ‘Cell culture’, ‘Cell biology’]

Release Date

2022-03-21

Abstract

The diffusion of dyes into whole organoids is variable and for many dye-based assays, using fragments of tubules isolated from organoids can simplify analysis. This protocol details a method to enzymatically liberate tubule fragments from mouse kidney organoids.

Introduction

For this protocol, we prepared organoids from nephrogenic zone cells from P0 mouse kidneys by aggregating 100,000 cells in 2 ul droplets on 0.1 um pore size polycarbonate filters suspended at the air-liquid interface over APEL 2 medium (Stem Cell Technologies 05270) supplemented with 200 ng/ml FGF9. Aggregates were subjected to a 1 hour pulse of 8 uM CHIR (Reprocell Stemolecule CHIR99021) in the medium immediately after aggregation. Medium was changed to APEL2 alone 3 days after aggregation, again at 5 days, and the organoids were harvested for tubule digestion at 7 days.

Reagents

HGSC solution 8 ml Hanks balanced salt solution 0.1 ml Glycine 0.5 M 2 ml Soybean trypsin inhibitor 30 mg/ml (yes, this is a lot) 0.1 ml Collagenase type 4 25 mg/ml Mix carefully and sterile filter. Freeze in 0.5 ml aliquots and store for up to 6 months.

Stop solution 9 ml Hanks balanced salt solution 0.05 ml EDTA 0.5 M 1 ml BSA 1% Mix & sterile filter. Freeze in 0.5 ml aliquots

HBSS/BSA 9 ml Hanks balanced salt solution 1 ml BSA 1% Mix & sterile filter. Freeze in 0.5 ml aliquots

Trypan blue 0.2 % 0.5 ml Trypan blue solution 0.4 % for cell culture, sterile 0.5 ml HBSS/BSA

Equipment

Humidified incubator set to 37 degrees
Watchmakers forceps, sterilized
P20 & P200 pipettes
Stereomicroscope

Procedure

Generate organoids at the air-liquid interface and culture for 7 days.
On the day of the experiment, prepare all of the reagents.
Immediately prior to digestion, pipette a 0.1 ml bubble of Hanks balanced salt solution (HBSS) in the base of a Petri dish.
Using forceps, pluck 5 organoids from the filter(s) and place in the HBSS bubble to rinse.
Using a P200, drain the HBSS from the bubble and replace it with prewarmed 0.1 ml HGSC.
Cover the Petri dish and place it in the humidified incubator for 30 minutes.
Add 0.05 ml Stop solution to the bubble, place the dish on ice and drain all solution from the bubble with a P200.
Replace solution in the bubble with 0.1 ml HBSS/BSA.
Drain and replace the solution in the bubble with 0.1 ml HBSS/BSA.
Using the P200 set to 0.075 ml, triturate the solution with organoids through the pipette tip orifice 5 times.
After allowing the liberated fragments to settle on the Petri dish surface, push the undigested organoid rests to one side and use the P20 to vacuum intact tubule fragments and place these in a separate 0.1 ml bubble of HBSS/BSA.
Repeat the trituration step with the remnants of undigested organoids to liberate more fragments. After one or two repeats no more intact tubule fragments will be liberated.
Pool the bubbles with intact tubule fragments and if you want to clean up the preparation to remove single cells and debris, then vacuum the fragments again with the P20 and transfer to a fresh bubble of 0.1 ml HBSS/BSA. Transfer into fresh bubbles of HBSS/BSA until you have a clean preparation.
To check viability, transfer the fragments into a bubble of trypan blue - drain immediately and replace with HBSS/BSA. Damaged fragments will contain deep blue cells, remove those & use the remainder for the experiment.

Timing

The collagenase digestion time may need to be increased for larger organoids.

Critical_Steps

Because the organoids are small, it is important to keep them in a format in which they can be easily visualized throughout the digestion. For this reason, all steps are done in a bubble of liquid in a Petri dish. A 0.1 ml bubble of HBSS in a Petri dish

The organoids have a tendency to float on the surface of the bubble and they do not digest well this way. If they do float when you change solutions, then gently force them into the liquid using the pipette tip. Once they are immersed they sink to the bottom.

It is important to avoid mechanically damaging the organoids before the trituration step - the goal is to keep the organoids intact and to arrest enzyme activity so that the tubule fragments that are liberated do not continue to be digested.

Trouble_Shooting

Frothing during the trituration step can be a problem - deal with this by avoiding introducing any air bubbles in the steps prior to the organoid trituration and make sure that you set the pipette volume to less than the total volume in the bubble for the trituration step. Poor disruption of the organoids results in low tubule yield - make sure every organoid enters and exits the pipette tip orifice in each trituration cycle.

Anticipated_Results

From 5 organoids, expect 20 intact tubule fragments of a length around 0.2 mm. Example of organoid and tubule fragments liberated from it using this protocol

Consortium

(Re)Building a Kidney (RBK) Consortium