Hi Donald!

     to be honest with you, I never prepared pseudoislets from INS-1E, nor MIN6. What I really do is prepare pseudoislets from neuronal stem cells that secretes several neuropeptides, and insulin. In this case I use non-adherent surface dishes (or if prefered because they are expensive, use normal dish and cover the bottom with agarose 1%) and the cells naturally tend to form spheres that are pretty stable. The conditions of working require to treat them very gently. Pipeting must be slow and centrifugation steps need to be very slow (no more than 1000rpm or 800g depending in the centrifudge). What I would recomendo also is to seed the cells in maximum 6 well plate in order to facilitate the encounter of the cells with each other and favor the pseudoislet formation process. Last advice: once seed the cells, move very gentle the dish into the incubator and close door gently; I have seen that when shaken a lot, cells tend to form big clusters with random morphology rather than sphere.

I hope it helps

Best regards

Hi Juan,

Thank you for your response.  I am unclear by what you mean here:

"seed the cells in maximum 6 well plate in order to facilitate the encounter of the cells with each other and favor the pseudoislet formation process"

Do you mean a 12 or 24-well plate would be more beneficial or less beneficial?  I don't whether I am misunderstanding the protocol.  Don't you form the pseudoislets in a non-adherent flask or dish, and then seed them onto a plate or cover slip?  If that's the case, I'm assuming that even gentle trypsin and centrifugation and pipeting will break up some of the pseudoislets.  You would then seed the already formed pseudoislets.

"Facilitate the encounter of the cells with each other and favor the pseudoislet formation process"

It was my impression that when you seeded cells more sparsely, they had a greater tendency to form clusters/pseudoislets.  I have sporadically seen this happen with 832/13 cells, but it seems to happen very readily with INS-1E cells, as I have just noticed with 1:20 split.

Hi Donald,

I use to seed the cells in the ultra low attachment surface plates. The ones I use are:

Corning (ref. 3471) Costar 6 well plate Ultra-low attachment surface.

Again, in my experience using neuronal stem cells to form pseudoislets, seeding in 6 well plates gves me optimum conditions for cell growing and pseudoislet formation. In numbers: if I seed 5x105 cells in these 6 well plates I have seen that the cells form better pseudoislets (neurospheres in this case)rather than the ones formed if I seed the same amount of cells in a bigger dish (i.e. 75mm agarose coated dish).  That's why I believe that in this case, the bigger the volume, sparsely seeding, worst for sphere forming. But I insist, this is my experience with neuronal stem cells, if something of the information may be extrapolated to INS-1E will be nice.

Let me know if had success and if I can help, just send another question.

Best regards

Juan 

Hi Juan,

I've been testing this a bit, and seeding more cells does make better pseudoislets.  2 days after seeding on a petri dish, I'm seeing the majority of PIs starting to adhere.  Do you plate the PIs after they adhere or while they're in suspension?  1 or 2 days after plating on ultra low attachment surface?

Hi Donald! Good to hear is working! Look, I do the palting maximum each 2 days, when they still are in pseudislet format. The ultralow attachment plates I replace each 2 weeks of use...after that I have observed cell attachment.

Best regards

Juan