MSCs form aggregates when you subject them to chondrogenic differentiation. The biomechanics of the aggregates allows them to differentiate into chondrogenic lineage. One way I would suggest for differentiating MSCs into Chondrocytes on a 2D surface is to try micromass culture, if it suits your needs.

Briefly, you resuspend the cells at the cell density you desire in a micro volume such as 50-100 uL and seed it in the center of the well and very carefully move it into incubator without media and let the droplet incubate for 3-4 hrs at 37oC, 5% CO2. After 3-4 hrs, add your chondrogenic differentiation media onto the walls of the plate very very gently without disturbing the cell mass. change media every 2 days and from Day 5 you will see chondrogenic differentiation.

You will have to work very carefully with the cell mass as it is very easy to detach them with slightest force of media wash. Do not place them on a shaker or orbital mixer.

You can find a brief method written in the following paper.

Article Evaluation of Human adipose-derived stromal cell behaviour f...

You can use you IHC protocols on the cell clusters if you manage not to disturb them from attachment. SEM is a little tricky, if your SEM has a cryo setup, you can gently lift your cell cluster with the tip of a pipette, transfer it to OCT to embed and try to get the images. SEM is easy said than done and may end up a complete disaster this way. Traditional pellet way is ideal for SEM.

Hope this helps.

Ritihaas Surya Challapalli many thanks for the suggestions. These are deffinitely very vital for me.

I understand that I need to be extra careful with the medium exchanges and so on in order not to detach the cells, but I'm a bit concerned about this. I have the 21-days experiment of chondrogenic differentiation, where the MSCs are seeded in a well plate, and in every well there will be a polymeric fabric excreting active substances. There is a concern if the cells will attach to it as it is not a well bottom, but a different material. Another one is that in 21 days of culture there will be e.g. 40-something medium exchanges. Very challenging I guess...

Ritihaas Surya Challapalli I am sorry I just have one more question. I read your paper (thank you!). I would like to ask how you managed PBS washing without detaching the cells?

In my study I need to immunostain 3 collagens, with fixationm, permeabilization and blocking at first, so that means many washings 5x PBS.

Paulina Anna Trzaskowska In the attached paper, I used 24-well plate to seed the cell cluster, incubate and perform all the experiments needed. The cluster forms a 3D mound and starts differentiating. Cells that are attached to the surface (here 24-well plate for tissue culture) will hold the entire mound up to a certain degree of disturbance. If you seed your cluster properly (in the center of the well) and use the walls of you well to gently add and remove contents you will be fine the entire time. I suggest you give a trial run with 4-5 replicates to perform one experiment and you will get a feel of how things work and when you are comfortable, you can perform your actual experiments.

Coming to your previous comment on the polymeric fabric, if the pore size of your material is less than 0.8 um and you do not need to disturb the membrane once it is set, you can try to grow chondrocytes on the fabric with the said method without many problems. The 0.8 um pore size is meant for suspended fabric such as the membrane in a trans-well plate insert. if your membrane has a firm support such a bottom of the well, the pore size does not matter unless you are removing the membrane from the well frequently.

Hope this helps. Please feel free to get back to me and I will try my best to help you sort the situation.

Ritihaas Surya Challapalli Thank you very much for your kind answer.

Dear Ritihaas Surya Challapalli we are starting the micromass culture of MSC soon. If you dont mind, I'll ask a technical question. We will be working on polymer fabrics placed in 48-well plates. Thus, we will use 0.5 ml of medium for each well. In your paper, you used 24-well plates, 1ml of medium for each well and initially you placed 20 ul of the suspension of 8*10^4 cells, so that was 4*10^3 cells/ul (the cells density in the droplet seeded in each well). To keep the ratio and to obtain 8*10^4 cells/ml (same as you) and we should have 10 ul of 4*10 ^4 cells. Do you think that 10 ul instead of 20 ul of cell suspension would work?

Thank you very much in advance.

Hi Paulina Anna Trzaskowska

10 uL in the center of the well should work, in theory. but since the diameter of the wall is less compared to a 24well plate, you just have to be extra careful while working with the micromass mound. Also it is no necessary to reduce the number of cells/total volume. you can still work with 20uL suspension (and cell density according to your experimental needs). The cluster will come closer in a few days, giving you enough room to add or remove contents from the well. In my paper, I had to perform several experiments on one sample between P3-P5, so cell number was a limiting step and I had to use minimum number of cells possible. you may increase or decrease the cell density relatively (according to your experimental conditions)

The crucial step is to seed the cells on the fabric and incubate without adding media for three hours and then add the media for the first time without moving/disturbing the fabric (Since the fabric will not firmly attach to the 48 well plate). Once the micromass incubates overnight, it will be robust enough to handle your experiments as long as you are careful. I will suggest you do a trial run with a small sample if you are still unsure of the experimental setting without risking your valuable samples.

All the best with the experiments.

Dear Ritihaas Surya Challapalli Thank you very much. We also need to perform a couple of analyses on the cells between P3 and P5, and thus we want to keep the minimal possible cell density. Thanks a lot for your suggestions. Definitely we will run a trial round. If you like, I will post how we are doing.

Thank you so much. I am really interested to know the progress. Please keep me posted if you can. I will be looking forward to hearing your success.