Chem 8157

Nancy Allbritton from the University of North Carolina at Chapel Hill presented an interesting talk on the development of a microfabricated device for cell sorting. This new technique involves using standard microfabrication techniques to create an array of SU-8 ‘pallets.’ Arrays of these pallets are then used as platforms for cell adhesion. The voids between these individual pallets in the array are filled with either a virtual air wall when the geometry allows or a more complex polyethylene glycol wall to prevent cells from adhering to the glass between the pallets. Once the cells are adhered to the pallets, standard microscopy and fluorescence techniques are used to single out pallets that contain cells that are expressing the desired properties. As these pallets are identified, a laser can fired at the interface between the SU-8 and the glass creating a plasma that ejects the pallet from the surface thus separating the desired cells. The technique was validated using cell viability studies to ensure the adhering cells could survive the ejection process and microscopy images were collected to confirm cell adhesion to the pallets. The talk concluded that this new technique is quite adaptable to the needs of various biological studies and the devices are easily customizable to the needs of individual experiments and gives results on a faster timescale with increased efficiency over previous cell sorting techniques which can be quite harmful to the target cells.

I believe this seminar was one of the best thus far, certainly not only in content but presentation as well. The work she presented on micopatterning arrays so that only a single cell could be isolated was revolutionary. She presented the information very well and had plenty of supporting evidence to back up the results and conclusions she drew. It seemed that she had every angle covered - she had considered multiple objections to her work and then conducted experiments to disprove those objections. The cell pictures and videos were stunning and provided excellent evidence that the work she conducted was successful.
I am curious though about one aspect. She grew a couple of different types of cells on the pallets with no problem, but does this extend to many other types of cells? Can a human cell grow in an environment like this? If so, I would envision that this technology could be used to probe certain types of human cells to see if they are able to grow under certain conditions. For instance, coat the top of the pallet with an new anti-cancer drug and attempt to grow tumor cells on the pallet. If all the pallets are coated with the same drug, multiple cell types could be tested at once with an array like this...this is just an idea I would be interested to see her address.

The seminar today by Nancy Allbritton from UNC was about platformed cell colonies and was one of the most interesting talks I have listened to in to in the past 7 months. With the goal being to identify single cell signaling pathways, a technique was developed to grow small groups of cells upon individual platforms. The platforms allowed for the creation of distinct colonies. Cell viability studies were done to ensure cell health upon growing cells, cells on ejected platforms, and collected cells. Very detailed and close-up movies and photos were taken of the platforms to show that the cells did indeed grow on individual platforms. This development has led to some applications in single cell communication and also in new techniques for adhering cells and applications such as finding better cellular indicators. One thing I would have like to have seen more of were the applications of the technique. The technique was very innovative and the ideas of applying the technique are large. I better description of current applications would have been exciting to show how truly innovative the technique is.

Nancy Allbritton from the University of North Carolina at Chapel Hill presented a seminar entitled Microfabricated Devices for Single Cell Analysis. More specifically, her presentation was focused on the separation of single cells for use in biological and bioanalytical techniques. This research is fueled by the desire to develop a cell separation method that does not kill a significant percentage of the overall population. Current methods mentioned in her talk were cell detachment coupled with flow cytometry and antibiotic resistance methods. Chemical cell detachment and flow cytometry are both relatively harsh methods for handling cells and result in significant cell loss. Antibiotic resistant genes only slow the killing of cell populations.
The technique that she developed and presented in this method was the culturing of cells on plates that have removable squares, referred to as pallets. Using photolithography, she was able to place squares of SU-8 polymer on a glass slide in any array desired. By convergently focusing a 2 microjoule pulse from a ND:YAG laser through the glass and directly at the base of the pallet, they are able to create a small plasma plume under the cell. This plume releases a shockwave of gas that pushes the pallet off the cell. To overcome the tendency of cells to collect at the lowest point, the group needed to modify the glass such that the water would not come into contact with it. This was achieved by two separate methods: fluorination of the glass slide and creating polyethylene glycol (PEG) walls between the pallets. Because SU-8 is not an ideal substrate for growing cells, the group was able to modify the pallets with biological media. This is made possible because the pallets are relatively hydrophobic and therefore adsorb proteins extremely well. Standard microscopy was used to verify that cells were adhering to and growing on the surface of the pallets.
By growing several types of cells on the newly developed pallets, they were able to verify that the new substrate was viable for easy to use cell separation platforms. Both fluorescence and conventional microscopy were used to verify that cells could grow on the top of the pallets and would not jump from one to the next. By releasing the pallets into wells, the group was able to show that the releasing process did not harm the cell and the pallet would not crush cells if it landed upside down. She presented a method for releasing the cells in which the array was turned upside down after the pallets of interest are found. A well of cell culture media is then placed under the pallet and the laser fired to release the pallet.
The method presented shows promise as an excellent method for the identification and separation of cells displaying desired characteristics. This seminar was well presented and seemed to cover all of the obvious questions. The criticism that I have regarding her seminar is that everything seemed to work too well. Either this project was extremely easy to bring this far or she did not convey how much hard work went into the project. If the former case is true, this work would likely have been done long ago. This leads me to believe that she understated the amount of work that went into the project.

When posting opinions about the seminar, let's try to read the other comments first.

Then you can elaborate on the previous comments or disagree.

It would be interesting to hear from those that disagree with some of the main points presented above.

It would also be interesting to read the guiding questions and answer questions such as, "What bioanalytical techniques were used in obtaining the results presented in the seminar?"

I am curious if someone can point to some of the techniques covered in the genomics unit that are directly by the Allbritton's group.

Professor Allbritton do use Human cells. I am sure She use HEK cell which is a cell line derived from embryonic human kidney. I also remember she use Hela cells too. So at least some of human cell can grow at the pallet.

The lecture given by Professor A is about developing a new technique for sorting and analyzing cells. The technique is very promising for bioanalysis. The techniques she used in the lecture include cell screening by fluorescence-labeling, cell culture, imaging by Environmental Scanning Electron Microscope (ESEM), gene transfection, laser assisting pallet release and most important the new type of cell array. ESEM is a common way for imaging aqueous biosystem. Gene transfection is used to introduce the fluorescence-labeling and it shows the transfectant keeps stable in the further generation since they are fluorescent too. The first four are conventional ones. The last two are the ones her group has developed.

The lecture is clear and exciting. I really enjoy it.

Echoing many of my classmates’ comments, Nancy gave a fascinating and exciting talk and did a good job of crafting the story of this area of her research. Every time I had a question, she answered it later in her talk. One area of this work that was interesting was the ability to grow a colony on a single pallet that allows for the study of cell to cell interactions. One limitations of this novel technique, however, is that the colonies can not expand too far. That is, I can imagine that the colony of cells would eventually outgrow the pallet. Therefore there is smaller window of time before the cells need to be sorted.

Thinking about this technique as a more gentle way to remove cells from culture makes me curious if this technique could be used to remove larger sections of cells like in a tissue culture, where it is important to gently lift off the tissue. At what point to the pallets become too large for this technique to be useful?

With regard to Melissa's comment, I am not sure that the pallet limiting the size in which a colony can grow is actually a limitation of the method. The results that she showed suggested that the colonies could grow long enough to express any plasmid DNA implanted in the cells. This expression was shown through several generations of the cell line. Since this is the overall idea of the pallet method, it seems that her results are exactly what she was attempting to achieve.

As non-native English speaker, I think that the talk given by Nancy Allbritton from UNC is quite interesting and easily understanding. Her group proposed a new method to select and sort cells using array of SU-8 pallets. Her group firstly coated glasses with SU-8 and partially exposed them to UV-light. After that, there were arrays of SU-8 pallets on the glass. By treating glass with perfluoroalkyl-trichlorosilane, they built up virtual air wall to confine cells to the pallets. The cells on pallets are viable. By fluorescence-labeling, they found the pallets with cells they interested in. The next step was just to release the pallets and culture the cells for later analysis. Since the pallets are releasable, this technique overcome the main issue of conventional cell selection and sorting, that is, many cells die when they detached from a surface. This method seems very simple but amazing.

One of the most common and mature technique to separate and sort heterogeneous mixture of cells in bio-analyses is Fluorescence Activated Cell Sorting (FACS) Methods. In this method, a rapid flow can carry on the cells in suspension. Cells are separated by difference the light scattering or fluorescent properties, based on which they are charged differently and go to different containers by electrostatic deflection. This method is continuous separation and really fast. Also, it can achieve single cell analysis by making one or none cell in each droplet.
An obvious limitation is that cells can only be sorted by their light scattering or fluorescent properties, while more often, we are concerned about many other properties. Also, the viability of cells after flow cytometry decreases a lot. The microarray of SU-8 pallets presented by Professor Nancy Allbritton can sort cells with multiple concerns at the same time. It is convenient to sort cells with different fluorescent property, different size and shape, different growing speeds, ect. Moreover, individual cell can be traced on the array and addressed by taking off with laser in this method; however, in FACS cells are sorted into several groups. Last but not least, the cells show high viability (> 90%) after firing the pallets. One thing I doubt is that since we must dilute the cells suspension to very low degree before adhere them onto the pallets, it means there is a limited amount of cells that can be analyzed one time on a chip compared to flow cytometry. So I am not sure about the efficiency if we look for some target in tons of cells.

Regarding to Mellisa and Eric’s comments, I think the size of the pallets is very critical. If it is large, the selection will not be efficiency because the chance of having both interested and uninterested cells sit on one pallet would be very large. However, if it is small, there is not enough space for the cells growth. I just wonder how they disperse cells onto the arrays.

I can't add much to the discussion, as many of the major points have been covered. I do agree that the presentation was very informative, and the technology used is not only cutting edge, but very useful in application as well. Bioanalytical techniques and instrumentation used by her group included select and sort flow cytometry, laser capture microdissection, scanning electron confocal, and fluorescence microscopy. Additionally, immunofluorescent arrays were used to select and sort cells based on growth rate. While some of this instrumentation was not covered in this class directly, I'm sure most of us have seen this in past analytical classes.

Oh, I almost forgot, by using a Gaussian Error Function, Nancy's group was able to analyze laser pallet release! So cool!

I must say that I agree with everybody. I thoroughly enjoyed the talk, and it was a good example of having a good idea and following it through to a practical application. The idea of being able to choose which cells you want and then growing them up into a cell you can use is a good one, however, someone does need to screen for the phenotype that you desire. She talked a lot about the use of fluorescent tags, however, what if the cell phenotype you want cannot be labeled using a fluorescent tag? The job then becomes developing a new screen, which may limit the application of this technique. And going back to what Eric and Melissa were discussing, will you have enough cell on the pallet to test for the phenotype, because I could some screens killing the cell in order to figure out if it has the desired properties.

Reply to Josh’s comment, I think Nancy’s method can only apply to adherent cells. Many cells are nonadherent cell. If the human cells won’t attach to the pallet surface, this separation strategy may not work. So I think this may be the biggest limitation of this method.

In Nancy’s talk, I was very impressed by the stem cell sorting. Because there is no cell marker for stem cell sorting, the only method is using the shape and size to distinguish stem cells from other cells. Stem cell function is still a big question, so I think this method may be a breakthrough for stem cell research. But as Jon mentioned, the cell number on the pallet is very low. Even though you can culture the separated cells in another well for proliferation, that must take a lot of time to get enough cell number for experiment.