Granular Microgel

[Zora Baumann]

Hi Abs,

Thank you again for this immensely resourceful group!

I have some questions regarding the granular microgel:

1. In the methods section of the BioRxiv it says you prepare a "Layer 2" for the hydrogel mix, where it says the VitroCol is 2 mg/mL (diluted from 3 mg/mL with hydrogel diluent). In the graphical abstract of Supp. Fig. 9b it says to use Vitrocol at 3 mg/mL.

Did you pre-dilute Vitrocol to 2 mg/mL or used it directly at 3 mg/mL?

2. For the fibrinogen, you state to use 33 µL (per 96w plate) "stick @ 20 mg/mL". 

If I divide this either by 450 µL (or also 468 µL, which is the total sum of gel produced by summing up all components in the graphical abstract of Supp. Fig.9b), I result in a higher fibrinogen concentration than 1.25 mg/mL mentioned in the methods section. 
Which concentration or dilution did you exactly use? 

3. Do you resuspend the polymerized gel first with APEL2 medium and then fragmented (as mentioned in Supp. Fig. 9b) or you fragmented it first, and then resuspended in APEL2 medium (as mentioned in the methods)? 

Thank you so much for all your efforts and taking the time. 
I hope your grant submissions will be successful; finger's crossed!

Cheers, 

Zora

[Abdullah Khan]

Morning Zora, 

These are good questions - I will try and clarify them below but do let me know if anything is unclear. 

1. This is correct: VitroCol is 2 mg/mL (diluted from 3 mg/mL with hydrogel diluent). The stock solution is 3mg/mL, and we use it at a final concentration of 2mg/mL in the solution. 

2. For the fibrinogen - we prepare a stock @ 2mg/mL, and use a final concentration after dilution of 1.25mg/mL.

3. You can do this either way, I have found it easier to fragment with some volume of APEL2 medium. What I will do if I am preparing say 4 plates and need a final volume of 20mL APEL2 + fragmented gel:

- I take 20mL of APEL2 in a falcon tube

- SPlit it into 2x 10mL volumes in 50mL Falcon tubes. 

- I fragment the gel using 10mL of APEL 2 (easier to pass through the high gauge needles) in a 6-well plate. 

- Then dilute in the remaining gel volume. 

Doing the fragmentation in too much media winds up being quite messy so this is a good middle ground. In reality, you can do this any which way you prefer really, as long as your gel is moving through the guages a few time it will fragment well. The considerations are:

-  'ease' (you don't want to be trying to force the syringes). 

- 'loss' (you don't want to lose a lot of your gel in the process of fragmenting) 

- 'sterility' (you don't want to be splashing lots of liquid around you'll get an infection!) 

By using some media you make it easier to fragment and are less likely to lose gel and keep your quantities consistent across experiments. 

Hope this makes sense - good luck, and do let us know how it goes with the new protoocl. 

BW
Abs

[Salim Atakhanov]

Hi all,

I wonder what will happen if we use Cellulose instead of the hydrogel as it is much cheaper than all the hydrogel components? Do you think it will affect dramatically the BMO composition? I am not a biomaterial guy, so any thoughts  will be appreciated!

Best,

Salim

[Salim Atakhanov]

Hi all,

For fibrinogen, after reconstituting, you will have a lot of leftovers right? Can you freeze it back? In the data sheet of the company it states that storing at 4deg is possible only for 1 week. How do you do this?

BW,

Salim

[Abdullah Khan]

Hey Salim, 

Yes you wind up with quite a lot left over. I usually freeze it and it's good for about 6 months after you freeze aliquots.

Worth checking that it still polymerises well if you're using the same stock for more than a few months. 

Abs

[Abdullah Khan]

Oh sorry I didn't see your cellulose question. 

I think it's always worth a try, but my feeling is that you will see much less vascularisation. A lot of the sprouting will be a mix of the 3D scaffold and integrin activation which the collagens are so good for. If you try it, let me know if it works ! 

[CAMILLA TOMBARI]

Hi Abs,

following up Zora’s questions, I don’t know if I have well understood the fibrinogen/vitrocol dilution in the preparation of granular microgel.

Let me recapitulate what I got:
For a 96well-plate you prepare a hydrogel (total volume of ~540 uL) as follows:
· 200uL Matrigel
· 133uL collagen I-IV mix prepared as in Olijnik et al.
· 100uL vitrocol 3mg/mL diluted adding 50uL hydrogel diluent to have a final concentration of 2mg/mL
· 33uL fibrinogen 2mg/mL diluted adding 20uL hydrogel diluent to have a final concentration of 1.25mg/mL
· 2uL thrombin

Additionally, when you say to fragment the gel using 10mL of APEL 2, do you mean that you add it directly to the syringe where the hydrogel has polymerized and then start passing the solution through the needles?

I also take the opportunity to ask another very practical question: once d5 aggregates are resuspended into the granular microgel, do you use p200 tips to dispense the 50 μL volume per well into the 96 well-plate? Are the aggregates not too large at this point for using these tips?

Thank you very much!

Camilla

[Abdullah Khan]

Hi Camilla, mostly right some clarification here which we will amend in the revised manuscript. 

With this method I usually prepare a few plates, it is a bit easier to do a a larger batch. This is what I did this morning for 7x 96-well plates: 

Prepare hydrogel (total 3mL of hydrogel). 

• 1.2 mL rGF mGel (from stock, no dilution)
• 800 uL Collagen I + IV mix (prepared as in the Nat Protocols paper)
• 600 uL Vitrocol (use @ stock conc. 3mg/mL, no dilution anymore needless complication).
• 200 uL Fibrinogen (@ stock concentration of 20mg/mL)
• 166 uL Hydrogel diluent (as per Nat protocols paper)
• 30 uL 1N NaOH
• 4U Thrombin. 

You can divide this by 7 for your per plate calculation. We stopped diluting the vitrocol and just using a lower volume (though check what your batch concentration is as it can be variable)

Once this has been polymerised, I fragment it and I add it to 32mL APEL 2 (supplemented to be 5% KO Serum and 5U/mL Heparin) so the final solution is 35mL in volume. 

When I say fragment using X volume of APEL2: It is easier to fragment with some diluent, so I usually take 10mL of APEL2 from the 32mL and fragment into that, then combine the whole lot to make a single solution,

This 35mL solution is what you then add cells to and dispense. 

Does that make sense? Please do let me know if not. I will work on the revision to make it clearer. I'm also fleshing out a step by step protocol to put in here, but it will take me a bit of time to get there!

Yes we use p200 tips - they're usually ok ! This dispensation is the bit where you need to be careful - keep mixing as the aggregates like to settle so you get an even distribution, and assume that you will have some blank wells! With some practice you don't have loads... There are some nice robots now that detect single aggregates and dispense them, if you have a very generous PI .... who might be willing to help to buy you one.... 

Let me know if this isn't clear! 

Abs

[Zora Baumann]

Hi Abs,

 

Thank you so so much for all the detailed answers!

I performed my first embedding (of two different lines), which looked pretty ok at first, but then over time I see massive aggregates in the well (see photos).

Can it be that I just didn't mix the hydrogel components well enough before polymerization? I checked the pH and it's fine, and I'm certain I pipetted the components in the right proportions. Also, I observed a few bubbles in the syringe that I couldn't get rid of, which might have resulted in uneven gelation?

We don't think it's a contamination, but might be wrong.

Have you (or anyone in this community reading this message) ever experienced this? (I've previously only worked with pure Matrigel, so I'm new to this :)

 

Thank you so much for your help.

 

Kind regards,

 

Zora

[Abdullah Khan]

Hi Zora - sorry about the delay busy few weeks. This looks like contamination to me - the cloudiness, clumps, and shrinkage of the cell aggregates suggest that to me! It happens unfortunately... 

Abs