Week 9: Synthetic Development Biology

Summary

This week's lecture was about the design and development of synthetic tissue engineering. We saw various examples of bioreactors (i.e. advanced cell culture systems that recapitulate living cellular environment in the laboratory). used to grow different tissue types. Finally, we considered an amusing version of bioreactors: building biotic video games!

In the experimental part (which was actually the first experimental work done at EMW Streetbio!) we had to design a playground for paramecia, i.e. and control their movement with the phenomenon of galvanotaxis! Here is a glimpse of the fun we had designing the experiment:

Designing experiment.
Assesing lasercut.
Assesing lasercut.

Design Assignment

Problem 1: Design considerations for electrical strimulation systems

Stimulus waveform, Tandon et al. 2009.
Fig.1: Stimulus waveform withough (blue) and with (pink) the bioreactor attached.

We were presented with the oscilloscope reading (Fig. 2) for a stimulus waveform from a commercial electrical stimulator.

  1. As noted we can see that when the bioreactor is attached, the waveform gets distorted and is not applied correctly to the load. In detail, upon attachment the stimulus no longer reaches the desired 5V, but maxes out at 3V and drops with a significant slope through the stimulation time. Moreover, when the pulse stops, there is a drift towards a negative voltage before the bioreactor's voltage recovers back to zero.

  2. The most reasonable explanation is due to current limitations of the stimulator. By Ohm's law, for a certain impedance we need a proportional amount of current to reach the desired voltage levels. The maximum voltage of the applied field is Im*Z, where Im is the maximum current that can go through the stimulator and Z the impedance of the bioreactor.

  3. Having many stimulation chambers connected in parallel would degrade even more the electrical field applied to the bioreactor, as the current needed is adding up for each separate bioreactor. \(R_{sum} \ll R_{tot}\)

  4. To have control over the applied field, you can either reduce the amount of current needed to reach the desired amplitude, by making the stimulus amplitude lower, or lessen the resistance of your bioreactor (e.g. shortening the distance of the electrodes) or, lastly, increase the available current by a higher current rating stimulator or an operational amplifier with high current gain.

    Human adiposite stem cells.
    Explanatory figure from the paper of Nina Tandon et. al on cardiac bioreactors.

Problem 2: Morphological changes associated with electrical stimulation

Human adiposite stem cells.
Fig.2: Images from human adiposite stem cells (hASC) during various phases of the experiment.
  1. To use Image J (FIJI), open cell image, change threshold color

    • t=0H :: Cell Length = 215.22um ; Orientation angle = -5.57 degrees

    • t=2H :: Cell Length = 225.01um ; Orientation angle = -4.32 degrees

    • t=4H :: Cell Length = 299.89um; Orientation angle = 3.34 degrees

  2. We took the average of 10 cells in each image (t=0, t=2, t=4) using the Image J software.

    FIJI (ImageJ) software.
    Fig.3: We used FIJI (ImageJ) to measure the length of the cells.

Problem 3: Design considerations for perfusion bioreactors

  1. TBD
  2. TBD
    Flow simulation.
    Fig.4: Medium flow through anatomically precise TMJ bone constructsduring bioreactor cultivation(from Grayson et al 2009).
  3. TBD

Experimental Assignment - Paramecium Playground!

Lasercutting the playground!

We began to build a paramecium playground by modifying the provided vector drawing using CoralDraw and then rastering it out of acrylic using a lasercutter. It took a couple tries.

Modified design for our experiment!
Fig. 5: EMW Streetbio modified paramecia playground designed in coreldraw.
Stimulus waveform, Tandon et al. 2009.
Fig. 6: Playground lasercut on acrylic! It looks neat!

Setting up the experiment

Our little fellas arrived in the mail from Carolina: Paramecia, a genus of unicellular ciliated protozoan, widespread in freshwater and brackish environments. Their magic power; galvanotaxis, the directional movement of motile cells in response to an electric field. (ok- it's not that magical..you've heard of moths having phototaxis).

Tools for the setup.
Fig. 7: The setup includes: Paramecia from Carolina, copper tape, pencil leads, pippeters, arduino and of course the lasercut playground!

The set up required us to create an electrode along each edge of the 'playground', which would control movement in that direction. As Nina described, it is very important to find a electrode material that does not create paramecium-toxic byproduct with electrochemical degradation, when a current is run through. We used pencil lead, connected with copper tape to our arduino-controled power source .

Playground and Arduino.
Fig. 8: Assembled playground with electrodes connected to arduino!

Imaging the paramecia!

Finally, we placed our assembled device under a microscrope and pippetted the paramecia onto the center opening. We had unforeseen trouble visualizing them because the laser-cutting created a rough surface to the bottom of the well. Thus, the acrylic was not optically clear enough for the microscope to see through. Back to the drawing board!

Imaging.
Fig. 9: Positioning the playground under the microscope and pipetting the paramecia.

Yet, we got some nice paramecia footage by imaging them on top of a microscope slide ! Enjoy :D

Gif.
Fig. 10: Paramecia dancing around on a microscope slide.