Celine Gandar

Meeting Summary (Help)

Meeting Date: Jan 26

Summary: I explained everything I did to figure out the "RGD problem". 1) no spreading (3T3 fibroblasts) even at higher RGD concentration 2) no spreading even with RGD coming directly from my swiss lab 3) it works when 2D experiment = 3T3 seeded on top of the hydrogel.

Action Plan:

Do again the EBs-sprouting experiment on my gel but on 2D way this time (as Yu-Shik did it with Matrigel) + I'm waiting for the DTNB reagent to assess free SH concentration in my RGD aliquots, so that I will definively know if nothing's wrong with RGD peptide I have here.

Progress on issues from last meeting: I investigated the possible origins of my "no-spreading" issues, but for the moment I still can't find it...

Meeting Date: Jan 6

Summary: was not present (on holidays)

Action Plan: still same as previous lab meeting

Progress on issues from last meeting: nothing (sorry I had some vacation time)

Meeting Date: Dec 19 2008

Summary: I explained what happen with my encapsulated ES cells. Problems encounted:

1) no spreading, neither with the positive control (fibroblasts) => abnormal !

2) negative control (plain media only) showed MMP presence. Due to FBS?

Action Plan:

1) Do a new fibroblasts encapsulation varing the RGD concentration in order to define if the problem comes from the RGD peptide itself or not. (i.e. make some gels with very high RGD concentration so that fibroblasts are 100% expected to spread)

2) look for information in scientific papers to prove presence of MMP in FBS or not. In the case I won't be able to show much higher MMP secretion in encasulated cells compared to plain media, I would have to make again the experiment but FBS substitute.

3) Do a new live/dead assay of my cells (9days after encapsulation) and see how much they divide and are alive despite this lake of spreading.

Progress on issues from last meeting:

Meeting Date: Dec 11, 2008

Summary: I had just encapsulated ES cells the same day of that meeting. So I briefly explained the 5 encapsulation conditions I did. I also talked about the results of the first zymography test I carried out with supernatant extracted from 2D EBs culture on Matrigel. (see Yu-Shik's previous work)

Action Plan: Monitor the evolution of my cells in the hydrogel over time. Extract the media every other days and analyze it with zymography to demonstrate the presence of MMP...or not. Do a negative control when zymography => media only.

Progress on issues from last meeting: I finally got enough ("healthy") stem cells to start EB culture for 5 days, trypsinization and then encapsulation. Moreover, WooYoung and I have borrowed from another lab two vials of new R1 ES cells. From now on, everyone working with stem cells in the lab would have younger and "healthier" ES cells available. We hope not to have differentiation problems anymore.

Meeting Date: December 2

Summary: I exposed my future project and some of the very first experiments I did.

Action Plan: Starting as soon as possible (depending on when ES cells get "ready") my first important experiment: ES cells encapsulation within MMP-sensitve PEG hydrogel and assess their viability as well as their MMPs secretion.

Meeting Date: October 28

Summary: New to group, first meeting.

Ali's notes: Prior work with MMP-sensitive PEG hydrogels (Hubbell and Lutolf, Switzerland). Advantages: Michael type addition=> no UV required + progressive degradation by the cells themselves + signaling peptides incorporation.

Action Plan:

First try of ES cells encapsulation within this hydrogel.

Work with Yu-shik to better define my project, learn how to culture ES cells, fabricate microwells, do SDS-PAGE and zymography, immunostaining etc.