Tissue Morphdynamics Lab: Week 7-8

Hi, it's Danny again from Dr. Nelson's Tissue Morphodynamics Lab and its been about a week or so since my last update. Most of my time has largely been devoted to working on lung morphogenesis or essentially lung development, but also I got introduced to a new and exciting topic regarding divisional axes in tissue. Although the two topics don't necessary overlap in terms of research, the work I did the previous year at a UCLA Head and Neck Cancer Lab is very similar in terms of techniques but very different in terms of direction.

Sriram's project focuses on the effects of endogenous stress and motility in cell division. In previous experiments, it was shown that when two cells are attaches at opposite poles of another cell, they tend to create a polarity and divide along a parallel axis. Additionally, it is known that if there is no endogenous stress on the walls of a cell, the cell divides in an unpredictable manner. The objective of Sriram's graduate work is to determine how endogenous stress and cell motility affect cells' divisional pattern on 250 to 500 micron squares.

The process in create a small micron square and treat it with the necessary drugs and cells is a rather long but straightforward. Using soft lithography one can etch patterns into a film that can be used as a base for future experiments. Lithography is useful because it can create any pattern or shape but the pattern that is desired for this particular experiment is 250 micron to 500 micron squares. An essential material of the experiment is Polydimethylsiloxane (PDMS) masters, which is made by mixing cross-linkers to PDMS in a 1:10 ratio and vacuuming out an air bubbles. PDMS masters can then be applied to the lithography film which will result in a negative copy of the film. Then the negative PDMS masters stamp is coated with silane, a substance that prevents PDMS from sticking to itself. After the silane has dried, the PDMS masters negative can be used as a stamp and reused multiple times, until the quality of the stamps produced is subpar. PDMS masters is applied to a negative base and cultured in a thermo-regulator at about 60°C for 3 to 4 hours. As the PDMS masters is being cultured, one can spin down cover slips with PDMS masters on it. Once fours hours has passed, the positive copy is removed from the stamp, which is then cut into little squares using a razor. The PDMS masters squares are then treated with fibronectin protein and then stamped onto the PDMS cover slips. These cover slips are then treated with mammalian cells with a variety of drugs and analyzed using a con-focal microscope over 12 hours.

The machine used to spin down PDMS on cover slips
in order to create an even layer.

PDMS and cross-linkers (right)
Vacuum used to remove air bubbles (left)

I was supposed to attempt to make a cover slip treated with cells this week, but the fume hoods in the culture room were replaced due to ventilation issues. Sriram told me that the insides of new fume hoods are usually quite dirty and suggested that we wait some time before culturing and analyzing new cells. The past two weeks have been a mixture of chicken embryo dissections and imaging lungs as well as a lot of Imaris and image analysis.

PDMS masters stamps that will eventually be covered with fibronectin proteins.  

The next week is going to consist of micro-dissecting about 4 lungs in the morning and 4 lungs in the afternoon for day 4, day 5, day 6, and day 7 chicken lungs in order to start preparing for my poster and presentation. I will also hopefully attempt to go through the aforementioned process with the help of Sriram, if the fume hoods are restocked with equipment by then.

In terms of life outside of the lab, I've been working on my senior thesis paper over the summer and started on the dreaded summer homework. Josh has just left his lab, so if he reads this, hopefully he got back home safely. It's been a great 8 weeks so far at the lab and I've learned so much and met so many great people.

On a side note, today is Friday and I just got a new assignment to start going back through a set of data and count the number of cells in the data that has over 110 cells at around 11 hours. I've been counting cells for the past two hours because I would really dislike doing this on a Monday morning, so if you need anyone to count up to 150 in 2's as quickly as possible; I'm your guy.