Electrofusion of cells on chip. From a static, parallel approach to a high-throughput, serial, microdroplet platform.
Promotion date: November 15.
Promotor: Prof.dr. Albert van den Berg
Assistant promotor : Dr. Floor Wolbers
| Electrofusion of cells is an important tool for generating antibody producing hybridomas. These hybridomas are the result of the fusion between a B-cell and a myeloma cell. The conventional method for generating hybridomas has a very low efficiency, typically around 0.001%. In combination with the increasing need for antibodies, this has motivated us to explore a more efficient method for generating hybridomas. Here microfluidic chips offer several advantages, compared to conventional systems. To generate hybridomas, it is of utmost importance to fuse exactly one B-cell with one myeloma cell. In the static approach, cell pairing is achieved using cell traps while the high-throughput droplet based method focuses on deterministic cell encapsulation. Moreover, the fusion process has to be followed at a cellular level, with the use of fluorescent dyes, and the successful fusion products need to be harvested for further analysis The cell traps were optimized for the electrofusion of single human peripheral blood B-cells and mouse myeloma (NS-1) cells. An electrofusion efficiency of paired cells 51 ± 11 % was shown. Furthermore, hybridoma formation was observed after culturing the fused cells for 14 days in a conditioned medium. In the microfluidic droplet platform we tried to obtain high-throughput cell fusion. The basis is the formation of monodisperse droplets using two immiscible fluids. Furthermore, droplets are physically and (bio)chemically isolated that can be generated, merged, and sorted at kHz rates, enabling high-throughput single cell experimentation and analysis. For the first time, successful electrofusion of two HL60 cells in a droplet was shown. For this, six successive pulses of 4 kV/cm with pulse lengths ranging from 50-300 µs were applied These results ascertained the feasibility of electrofusion of cells in droplets, albeit so far with low efficiency (<0.1%).The obtained results provide a basis to realize a platform for efficient high-throughput electrofusion of cells in droplet-based microfluidic devices, which can definitely compete with the existing conventional fusion methods.
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Can you recall some special moments during the PhD project?
Our approach to make use of microfluidic chips in this field of research, is quite new, especially by focusing on the microfluidic droplet platform. Droplet formation and electrofusing of the cells herein, are completely novel.
The results obtained, do open up new strategies for high throughput production of hybridomas. Within our BIOS Lab-on-a-chip group I am happy to say a new set of research activities has come to life and is now led on by a successor PhD researcher. Overall, I am very happy my research project did have some impact on our group. The droplet platform theme is a hot item in research worldwide, and our group is attributing to this now.
Also the collaboration with other groups proved to be fruitful, like the Developmental Bioengineering group of professor Marcel Karperien (Mira Institute) and Physics of Fluidics group, led by professor Detlef Lohse. Also the three months collaboration with the Condensed Matter Physics group in Harvard UK was special, leading to a nice article on single cell encapsulation. Here the preparing and creative work of master student Rogier Schoeman was of utmost importance, leading to the first observations of efficient cell-in-droplet encapsulation. This collaboration led to a successful start in Harvard. Here it was possible for me to collect all information and impressions there like a sponge, so to say.
More specific, I was really excited observing the desired fusing mechanisms in the droplets actually taking place. From this moment on I was more secure this novel approach could lead to high throughput devices in the end. A lot of work has to be done in the near future of course. Some calculations however show a success rate of ten percent must be feasible in lab-on-a-chip devices using the droplet platform. This success rate is necessary to make competitive devices. At this moment I am convinced this will be the case in the future.
Did you manage to publish some nice articles?
Three articles were published in: Lab-on-a-Chip, the Analyst Journal and in Electrophoresis. In the last journal I was involved in as a second author. Right now Rogier Schoeman and I work on a final article, hoping to be published shortly.
In what way did you develop personally as a scientist and researcher?
These changes are taking place gradually. Now I am much more of an independent researcher, making my own strategic decisions. One of the features is, I allow myself to make mistakes along the way, asking bold research questions. When things don’t work out as planned beforehand, this doesn’t mean one didn’t obtain any result at all. The euphoria however when things do work out as hoped or expected, is hard to beat. Nothing compares to that.
What are your future plans?
Right now I am trained as a chemical analyst and laboratory specialist. My ambition is to work on new methods and tests to be of consulting relevance in the future. I really like to be of immediate importance to everyday practice in the hospital environment. Hopefully a leading position in the laboratory will be available in future, being of strategic importance for new diagnostic methods. In this position interesting lines of research and personal interests do meet in which techniques and the demand of patients and doctors interact.
What, in your opinion, is important for Mesa+ to stay successful in future?
The social interactions at Mesa+ are very pleasant and altogether leading to progress. I notice in a lot of research topics, biotechnical aspects become more and more important. This theme of research will be of prime importance in future, I believe.
I like the way Mesa+ presents itself as an institute at all layers of promotion, be it at an international level or in the region of Twente, stimulating youngster to learn about nanotechnology.
I also liked the way of inviting famous speakers to the Mesa+ institute by a voting system in which the Mesa+ researchers can speak out their preferences. This way of inviting speakers brings about great involvement. Also I am convinced the technicians play an important role in the success of Mesa+,