Collagenase D digest

[Sebastian Ramisch]

Hi Abs,

I've started using Collagenase D for the BMO digest on day 20 and was wondering how long this process usually takes for you.

When I used Collagenase II, the digestion was very quick; within 30 minutes, there were no clumps, and I had only single cells. However, after switching to 2.5 mg of Collagenase D, as you recommended in your post, it takes over 90 minutes to achieve a single cell suspension (even when I get up to 5mg). Is this normal, or might there be an issue with our enzyme?

Thanks for your help!

Best wishes,
Sebastian

[Yuqi]

Hi Sebastian,

 

We did notice a slightly longer dissociation duration for Collagenase D compared to Collagenase II, but the difference is not that significant as you experienced. The duration can depend on a few factors though, like the number of organoids being dissociated (with fewer organoids requiring less time) and the frequency of agitation or pipetting of the suspension. For dissociating 8-24 organoids, 2.5mg/mL Col D typically takes us 25-40 minutes (Col II was taking a shorter time of 10-20min). We normally do P200 pipetting every 5-10 mins to facilitate the dissociation and I found this critical to speeding up the process.

Also, please note that reconstituted Collagenase D should be stored at -20°C for no longer than one week as recommended by Roche. Although we have used it for up to two weeks post reconstitution without issues. If you kept it for longer, the col D activity may decrease and this may lead to longer dissociation.

Hope this is helpful. If this doesn't solve your problem, please feel free to post your detailed dissociation procedures and we can check if anything is different from what we normally do.

Best wishes,

Yuqi

[Zora Baumann]

Dear all,

I'm currently troubleshooting the enzymatic digestion of the comBOs.

Using Col D + DNase I at the indicated concentrations from the preprint for me leads to a viability of 30-40% only after a 50-60min digestion (before the organoids are not dissociating fully).

Also lowering the DNase concentration did not improve viability. 

I've additionally tried the following:

- Liberase TM (which contains Col II) + Dispase II + DNase I (as Frenz Wiessner did): this works like a charm to get a live single-cell suspension within 25 min, but it cleaves a lot of surface receptors.

- Miltenyi digestion kits (Tumor and Multi-Tissue) are a bit better in terms of viability, but I've noticed the same in terms of cleavage of a few surface receptors (e.g., CD56, and CD7).

I generally pooled 16-32 organoids and used 2-2.5 mL of digestion mix per tube. 

Thus, I was wondering what other people in the community are using for the enzymatic digestion? 

Any cocktails that result in a good viability and don't cleave surface receptors? Or is Col D working for everyone?

Kind regards,

Zora

[Abdullah Khan]

Hi Zora, 

Thanks for sharing - we've had similar experiences with the methods you've described and have stuck to ColD + DNAse I as it seems to be the most consistent way to retain surface markers and viability etc. We've stuck to it routinely for the last year or two. A couple of questions and suggestions: 

1. What buffer are you using to constitute the col D? And do you use a stop buffer with EDTA? 

2. Are you manually pipetting at intervals? We find this is pretty important to get the suspension ready in a reasonable time. I usually set a timer and at 8-10 minute intervals titutrate successively with P1000, P200, and P20s. Typically I'll get a nice suspension within 30-45 minutes and about 90% viability. 

3. If you're doing 16-32 organoids you may need a larger volume or to have some form of continuous agitation to break them up. We usually do about 8 per tube and 600uL of 2.5mg/mL solution. 

Hope this helps - thanks for TOOTing ! 

Abs

[Zora Baumann]

Dear Abs,

Thanks for your detailed answer.

1. I reconstituted the Col D freshly in HBSS (and stored it max. for 1 week at -20°C).  Stopping buffer is PBS +1% FBS +5mM EDTA (which I generally use as flow cytometry buffer).

2. I wanted to standardize the mechanical dissociation and used the gentleMacs c-tubes (on the OctoDissociator machine). The program I used was described by Miltenyi to mimic most closely the intermittent pipetting from your protocol (however, shear forces might be less than using pipette tips). I will try to dissociate manually with p1000 and 200 next, maybe that speeds it up more. 

3. I will incubate them shaking at 37°C to have constant agitation; and in case adapt the total volume of digestion mix.

Thank you so much for all the tips! 

[Abdullah Khan]

Thanks for sharing Zora. 

1. We usually prep the Col D in HBSS @ 5mg/mL and then dilute it 1:1 with an IMDM + 2% FBS solution (keeps cells happy). Stopping buffer we use is PBS + 1% FBS + 20mM EDTA. I don't think these will make a huge difference... but just letting you know!

2. Thanks for sharing this - if you're happy to, do let us know how it goes with the tips compared to the OctoDissociator machine. We've tried the TYGR system with decent results, but are curious if there is a method of mechanical dissociation that works better! 

Good luck!