Hydrogel Preparation

[Karolin Stumpf]

Can aliquots of the hydrogel diluent simply be thawed at 4 degrees ON or do they have to be thawed on ice at 4 degrees ON?

Which is the exact 10xDMEM you are using for preparation of hydrogel diluent?

Is the 1% Sodium Acetate solution just prepared in sterile water or pH-adjusted?

Thanks in advance to the TooT!:)

[Abdullah Khan]

Hi Karo ! Thanks for posting. Answers below: 

Can aliquots of the hydrogel diluent simply be thawed at 4 degrees ON or do they have to be thawed on ice at 4 degrees ON?

The hydrogel diluent can be thawed at RT, in a fridge or on ice. It is a mixture of different media/sources of ions so in and of itself is not temp sensitive, but you want it to be chilled once it's added to your other gel components. You can store it at 4 degC for a long time but again I err on the side of caution and keep small aliquots (filtered and frozen) thawed for use. 

Which is the exact 10xDMEM you are using for preparation of hydrogel diluent?

I have used this in the past:
https://www.sigmaaldrich.com/GB/en/product/sigma/d5796?gclid=CjwKCAiA9dGqBhAqEiwAmRpTC7GFGKfud7nmRgb6tv3MZnd5esw_ZuqTtPIPfJo3DhTsUG_iTui2vxoC_joQAvD_BwE
But it looks like it's out of stock. When I ordered this before COVID it never turned up, hence the switch to 10x PBS (which is what we use routinely).

Is the 1% Sodium Acetate solution just prepared in sterile water or pH-adjusted?

The buffered 1% Sodium Acetate Acetic Acid is prepared in sterile water and then filtered ( I know some of the filters are meant to be incompatible but I am paranoid and feel like a mechnical filter of some kind is useful here). No pH adjustment until after the gel is made up (as per paper).

Thanks for TooTing!

Abs

[Salim Atakhanov]

If If I want to use only a mix of Collagen I and IV (not a single collagen), should I then prepare 40% of Matrigel and 60% of Col I:Col IV mix (reagent prepared as described in the protocol, page 15)? I don’t understand why is it written 30% Col I:IV mix and another "30% of Collagen I (Vitrocol) diluted to 2mg/ml with hydrogel diluent"? 

Thanks in advance :)

[Abdullah Khan]

Hi Salim, 

It is a bit confusing, but hopefully the diagram in supplement clears it up a little bit. 

The final gel composition is: 

40% Matrigel: 60% Collagens. 

The 60% collagen mix is split into 2 components: 

- Half of this (so 30% of total volume) is the prepared mixture of Collagen IV and Collagen I. 

- The other half (30% total volume) is VitroCol (Collagen I preparation) diluted to 2mg/Ml in hydrogel diluent. 

You can use ColIV + ColI mix for the whole collagen component here, but it ends up being VERY expensive. This halves your collagen IV usage which is a substantial saving in terms of material. 

Does this make sense?

Abs

[Karolin Stumpf]

Hi everyone,

When reconstituting collagen I and IV, did you usually end up with full 5 mL of the mixture or did you lose some due to evaporation (although parafilm-sealed) or change in viscosity? And if so, would you further dilute the collagen mix as it might be too concentrated then?

Thanks to the TooT for all the help!

[Abdullah Khan]

Hi ! For me it's usually just a little bit shy of the full 5mL - but I've found it hard to account for the loss so just worked on the assumption it's the full 5. It hasn't caused any issues thus far and seemed a minor loss. 

Don't know what the others think! 

[aude-anais.olijnik]

I agree with Abs about the collagen prep: obviously the viscosity of the collagens means you may be ever so slightly out of the exact 5mL (pipetting loss, leftover on the wall of the tube...) but that should not be by much. I don't think evaporation is an issue but more the viscosity. You can just go ahead that way!

[Salim Atakhanov]

Hi all,

happy new year! I hope 2024 will bring us loads of new data in the BM organoid world!

I guess my brain isn"t in the working modus yet, but could you please explain at which stage and at what ratio we add hydrogel diluent (since it is not exactly written in the protocol)? Because as I understood, we use 40% Matrigel, 30% Collagen I and 30% of Col I and IV mix. So, if I need 10ml (for example) of the total volume of the hydrogel: you will add 4ml of matrigel, 3ml of Col I and 3ml of Collagen I and IV and then neutralize it? 

But I also know that to visualize the neutralization, we need reagents such as 10xDMEM and F12... So I am a bit confused. 

Best,

Salim

Abdullah Khan schrieb am Freitag, 24. November 2023 um 12:20:25 UTC+1:

[Abdullah Khan]

Hi Salim, 

Happy new year - we can hope eh ;) I would settle for lots of people happily growing organoids. 

The way I would think of it is this way: 

40% Matrigel - matrigel neat 

30% Collagen I - this solution isn't neat Collagen I. It is Collagen I diluted down to 2mg/mL (and the diluent is the hydrogel diluent). 

30% Collagen I and IV mix - this solution also isn't a neat mix, it is diluted in the diluent. 

So although we say, for example 30% Collagen I what we mean is 30% Collagen I diluted down to 2mg/mL in hydrogel diluent. This diluent has the 10x DMEM/PBS and F12 so all you need to do is work out your dilution volume to get the Col I to 2mg/mL, make up the difference with diluent (and 1M NaOH)

Does that make sense? 

Abs

[Salim Atakhanov]

Hi Abs,

thanks a lot! I think I got it, will have to try. For Matrigel, what the protein concentration do you use? Because I saw on data sheet that we need to dilute it first for a certain protein concentration. And then from that diluted matrigel, you take 40% for the hydrogel, correct? Or you take 40% of Matrigel for the hydrogel directly from the bottle that arrives from Corning without any pre-dilution? 

Best,

Salim

[aude-anais.olijnik]

Hi Salim,

Thanks for your question. We use Matrigel directly from the bottle without any further pre-dilution. We aliquot Matrigel in small volume if we know we won't be using the whole amount to limit freeze/thaw cycles.

We are aware that lots of Matrigel can have different concentrations of protein (certificates of analysis for the latest lots range from 8.6 to 10.9mg/mL). I have mentioned this issue to a Corning representative so that we can get a more consistent product. This will have an impact on how the hydrogel behaves during the week long embedding (can either maintain a nice and uniform gel or lose some structure).

Thanks,

Aude.

[Salim Atakhanov]

Hi everyone,

I yesterday had to embed in the hydrogel my aggregates. All 5 days, I think they looked nice. However, when I prepared the first layer of the hydrogel, the gel looked a bit weird. 

Do you have any idea why this happened? Attached is the pic of the first layer. Somehow my gut feeling tells me that this is the collagen I and IV mix... I did everything on ice and quick, however the collagen mix was in the end 3ml and not 5 (I don't know where the rest 2ml were gone lol)

Best,

Salim

[Abdullah Khan]

Hey Salim, 

That does look a bit strange. Those big fibres look like Col IV , but they should be better dispersed. I also can't see the rest of the gel volume (there should be lots of little fibres everywhere). Can you send us your maths for everything if you have it? 

The col mix usually ends up around 4-4.5 mL not so dramatic as 3mL. 

I'm doing some gels tomorrow if I remember I will pop some images on here. 

Abs

[Salim Atakhanov]

Hi Abs,

I never worked with hydrogels, so I don't know how they should look like. Sending your images would be very helpful.

So I wanted to prepare 6ml in total of the first layer of the hydrogel for 12 wells (500µL/well):

700µL Hydrogel diluent

1.5ml Col I+IV mix (I prepared it as in the protocol with the collagen diluent over three days)

1.4ml Col I Vitrocol (stock 3mg/ml > we need 2mg/ml: so I calculated 2.1ml (30% of the total 6ml volume) as 700µL Hydrogel diluent + 1.4ml Col I Vitrocol)

2.4ml Matrigel 

then added dropwise 1M NaOH using Pasteur pipette.

the order of adding the components of the hydrogel to the falcon was the order I wrote it. Let me know if there are any mistakes.

Best,

Salim

[Abdullah Khan]

Hi Salim - it is a bit of a chaotic week but I will get back to you in more detail probably next week. Quick comments below and some questions. 

I would have done (from memory)

600uL Hydrogel diluent 

1.2mL(ish) VitoCol 

1.8mL Col I + IV Mix

2.4mL Matrigel (correct)

+ 52.2uL NaOH (I would suggest working out a titration volume against your collagen or acetic acid/sodium acetate mix before the experiment rather than add drop wise)

BUT I don't think the differences in protein are so much that it would affect the overall behaviour of your gel. to troubleshoot: 

There are images of what the gel should look like in the well in the protocols paper I shared with you - how did yours compare to this? 

Is your collagen I/IV mix evenly distributed and cloudy? 

Can you send some photos of your gel (maybe set up some cell free test hydrogels first).

Abs

[Salim Atakhanov]

Hi Abs,

sorry for late reply, I am also running between several projects, busy weeks. 

It seems like it worked out, today is day 14 already. I got 50 organoids from the first time and would like to change the medium. Do you add 50µL per well of 96-well plate with the double amount of all the cytokines or only rhTGFb you add double amount? 

Best,

Salim

[Abdullah Khan]

Only rhTGFb is at double amounts - for the others keep at the listed concentrations. 

[Sebastian Ramisch]

Hi Abs,

We came across this post just a day ago and thought “Wow, something doesn’t look quite right here”, only to encounter the same issue with our embedding process the next day. 

Previously, we had used only VitroCol (Collagen I) for our Hydrogels, and didn’t experience any complications. However, this time, upon mixing the Col I + Col IV solution to the VitroCol, we observed the formation of large Collagen aggregates

Collagen aggregates after mixing Col I+IV to VitroCol and Matrigel: 

The Collagen I+IV mixture appeared well dissolved, slightly cloudy and was easy to pipette (although the viscosity was increased compared to the VitroCol type I solution) and was reconstituted exactly as described in the protocol (0.25% acetic acid + sodium acetate buffer). 

Collagen I+IV mixture (in 15ml tube) before addition to VitroCol and Matrigel:

The large aggregates only started forming once the Collagen Mix (150uL) was added to the previously mixed VitroCol (100uL) + Hydrogel diluent (50uL).

I was able to partially disperse these through vigorous pipetting, but couldn’t achieve complete resolution. The collagen aggregates are now dispersed throughout the hydrogel, with larger aggregates accumulating at the outside of the hydrogel layer. 

Hydrogel after embedding the Spheroids:

@Abs: Could you please share an image of how it should optimally look like? 

We have around 2.25ml of the Collagen I+IV mixture remaining and are wondering whether there’s a way to save it. We thought that a slight pH adjustment could perhaps facilitate the complete dissolving of the Collagen IV (Since the manufacturer recommends reconstitution solely in a 0.25% acetic acid solution, without sodium acetate).

Any insights or suggestions on this would be greatly appreciated. Thanks for your help!

Best wishes,

Sebastian

[Abdullah Khan]

HI Sebastian/Salim, 

So I think the main thing to point out here is that Collagen IV is fibrillar - it will form what look like big hairy clumps, it is an unusual ECM in that sense. The main thing is to make a solution, with the buffer we describe, that is evenly distributed so you get a good coverage of Collagen IV across the well. 

This: 

Actually looks really good! I suspect it will form nice big sprouts - would you mind sharing an update next week when you can? 

I've attached a PPT with a couple of example images. I think this should clear things up but essentially the above image looks good. My concern with Salim's image (and some of your earlier ones Sebastian) was just that the col IV is not evenly mixed so you get the clumps. Hope this helps - sounds like you're all doing excellently though so very well done! 

BW + thanks for tooting, 
Abs

[Abdullah Khan]

Images!

[Sebastian Ramisch]

Hi Abs,

Thanks a lot for the feedback and for sharing some images! 

Our organoids are forming nice sprouts and the first HSPCs are starting to appear now at D9:

 

In most of our organoids, particularly the larger ones, we observe a brightening of the outsides and a darkening of the core. 

@Abs Is this something you've observed as well? 

We noticed something similar during our previous attempt where we've only used Matrigel (40%) + VitroCol type I (60%), although there it was only visible until day 9 or so. 

Image from previous attempt (day 7):

Does anyone have an idea what might be happening here?

Best wishes,

Sebastian 

[Abdullah Khan]

Excellent news! Very well done. 

We usually see some cell dense darker bits, but to be honest I haven't seen anything as dramatic as in your first couple of images. Hard to say until you've done some sectioning to see what structure in the middle is like, or flow to see if there is a particular concentration of necrotic or apoptotic cells. 

Otherwise this looks great! Congratulations, 

Abs

[Philipp Sergeev]

Hi Abs, 

Could you please clarify, whether you used 10x DMEM, made from powder for hydrogel diluent, or 1x liquid from manufacturer? And also you added Glutamax on the top of L-Glutamine that is in media already, right? Thank you very much in advance! 

среда, 14 февраля 2024 г. в 17:49:07 UTC+2, Abdullah Khan:

[Abdullah Khan]

WE used pre-made 10X dMEM in the paper, but that's proven difficult to get hold of now so we just use 1x Liquid now. 

We don't add Glutamax if Glutamine is in there. 

Abs