RECAP:

We chose to use 3D printing to manufacture the LidPlungers because the piece required precise and accurate dimensions to achieve uniform compression.

Our first attempts at printing used the Lulzbot TAZ4 with Kira (software) rendering software. The Lulzbot is a higher-end consumer 3D printer with ~75 micron resolution and can support multiple materials for printing.

UPDATE:

Our specific design turned out to be extremely challenging for the Lulzbot. On our first print attempts, the printer would lay the base structure before plastic built up at the release valve, causing the printer to fail. Conversations with the manufacturer revealed that the Lulzbot might not be able to handle repetitive, small designs (like the plungers) because it requires the plastic flow to start and stop so rapidly.

While a switch from Kira to Slice3 rendering software was able to get the printer to complete the piece, the final product was not structurally sound. Fragments of plastic were breaking off and the piece was extremely flexible.

The team switched to printing on the 3D Systems Projet 3500 HD max, recently set up in the Mount Sinai Prototyping Center. A few features made this the more attractive printer; 1) it could print with much higher resolution, 2) its proprietary plastic would harden after printing, reducing breaking and bending, 3) it simultaneously printed a supporting wax to ensure no structural deformation.

The final product seemed extremely promising. The structure seemed solid and accurate, however, we couldn’t confirm until the wax melted away.

We set one piece in the oven at 60 degrees C to melt, and two others were left at room temperature in the lab. All three pieces warped and shrank, folding the plungers inward. Furthermore, we found the structure to be quite soft when immediately removed from the oven. The warping and shrinking meant that the piece couldn’t properly fit in the well plate with free movement. With Phil Cook’s help, we were able to reduce warping by reheating and cooling, but the shrinking had taken its toll.

Phone calls with the manufacturer revealed that warping is a common occurrence with designs that have a thin, flat sheet (the base of the plungers). 3D systems recommended 1) thickening the base 2) reprint with the piece slightly rotated about the z-axis, and 3) melt the wax without rapidly heating and cooling the piece.

Thankfully, the final product supported free movement through the wells and was sufficient for a proof of concept. It should be noted, however, that the piece still warped and shrank, even with a 5mm base.

Given our experiences with 3D printing and conversations with manufacturing, it seems that this particular design is not right for printing. The materials cannot support the thin base and repetitive column design. Instead, we propose looking into laser cutting or suction molding.

Programming the Arduino microprocessor to control the linear motors was based on (1) the parameters for cycling the actuators (and thus plunger array) up and down, and (2) the capabilities and limits of both the microcontroller and microprocessor.  Specifically, the actuators needed to cycle between 2 positions — the start and final position, or the “compression off” and the “compression on” positions, which were separated by just a few millimeters.  The linear motors each had an on-board feedback systems able to to detect position.  Whereas the microcontroller cannot detect position, it can detect and control the length of time that current is being applied to the motors to cause actuation at a constant rate.  Thus, multiplying the the rate constant by time, the microprocessor is able to detect position.

To control the location, and command the cyclical actuation, a for-loop was implemented. The for-loop is a conditional command that dictates “if condition X is met, then proceed to the next step, and if condition X is not met, repeat a function Y.”  Thus, in such a system, function Y will be repeated until condition X is met.  In this case, the for-loop controlled for position, and increased (or decreased) the length of the motor incrementally.  If the position of the actuator arm was at position 0, and the ultimate goal was to get to position a position, “10” the actuators were commanded to actuate to position 1. This would then be compared to the desired position (10).  Since position 1 is less then position 10, the actuators would then be commande to actuate another 1 unit, and so on, until position 10 was achieved.  At this point, the system would reverse until position zero was achieved. This cycle would repeat 100 times, at which point the actuators would go to “neutral position,” in which they were raised to their maximum length, to allow for access by a fluid handling arm or other implementation.

Whereas the plunger array was fabricated by 3D printing, machining and milling was selected as the method for fabricating the arms, base, and stage of the system.

All parts were milled from PVC.  A large block of PVC was ordered, and rough estimates of each piece’s dimension were cut with a band saw.

Subsequently, a milling machine (shown below!) was used to to attain exact dimensions, accurate to within a few thousandths of an inch, and with angles accurate within a few thousandths of a degree.

Each piece — the base, the stage, and both arms were cut and milled.  The component of the arms that make contact with the plunger plate were rounded out to add tolerance; even if the actuation timing was slightly imperfect amongst the 2 actuators, both would be able to contact the plunger array and actuate directly downward.

The arms were placed perpendicular to — and at the edge of both lengthwise sides of — the base. Holes were drilled and threaded for screws. Screws were placed into secure the arms to the base.  The stage was secured to the base in a similar fashion, with bolts to enable the motion of the arms when actuated by the linear motors.  Holes were drilled to place screws to mount the linear motors.  The linear motors were secured to the base, and bolted to the arms to enable their controlling of the arms.  The motors were integrated with the microcontroller and the fabricated system was tested.

Here’s Benji hard at work!

Agarose and Alginate have been commonly used in the literature to make 3-D culture constructs. The advantage of alginate is that it does not need to be heated up to solidify. You just simply add sodium alginate to a calcium solution and you get your gel! So we thought to take advantage of this property of alginate to make gel constructs without killing our cells by heat.

We mixed 2% sodium alginate with 102 mM calcium salt solution in the well, hoping it will form a nice disc. But nope! We got this.

Try to make alginate gel

An amorphous blob with uneven consistency!! After consulting with a gel expert in the lab, we were told that in order to make an alginate gel with a defined shape, we will need to construct a negative mold made of agarose and pour the alginate solution into the mold. Seal it up and soak the agarose mold with the alginate in it into a pool of calcium solution. The calcium ions will diffuse through the agarose gel and react with the sodium alginate inside. Then we will get nice alginate disc gels.

Considering the complexity of making alginate gels, we decided to use agarose to make our disc constructs. Although we have to heat the agarose solution to a boiling temperature, we can choose to use a low-melting agarose. Like this one!

Low-melting agarose

Its gelling temperature is also pretty low, much lower than 37 degrees celsius. GREAT! So we wouldn’t kills cells by seeding them with hot agarose solution!

Next, we need to figure out how to make round gel discs to be compressed by our bioreactor.

Gel sandwich construct

We initially designed the the gel constructs to be consisting of two layers agarose gels with a thin layer of cells in the middle. Ideally this could facilitate the confocal imaging process as all cells can be captured in one layer and cells are more accessible to the surrounding media. However, our approach to make the sandwich construct failed on multiple levels!

First, we encountered problem of making gels using the tools we designed.

The  GelMolder

The GelPoker

We designed the gel molder (3-D printed) that was supposed to cut 96 gel discs and transfer them to the well plate. And then we would use the poker to push out the gel discs through the holes of the molder. This way we could make the gel constructs in a high-throughput fashion.

However, the gel molder we 3-D printed was not picking up the discs from the gel slab. And the length of the plungers of the poker was too short that it could not reach to the  other end of the hollow wells and thus could not touch the 3 mm height gels discs.

As a result, we had to use a biopsy punch to punch the gel slab 96 times and use a tweezer to pick up the discs and transfer them into the well plate! This step really needs to be automated in the future, otherwise it defeats the whole purpose of making a “high-throughput” bioreactor.

Make a punch

Pick up the gel disc with a tweezer

In the process of transferring gel constructs. BE VERY CAREFUL!

Good luck try putting the gel construct into the center of the wells!

Another problem with making a gel sandwich is the uneven gel surface. We poured a calculated amount of agarose on to a petridish to make the desired height and waited for it to gel before we added a layer of cells. Without any constraints on the top, the top surface can assume any form. It can bulge, distort and disturb. Not cool. It led to the formation of discs of uneven height. Yuk!

What’s more, as we added the second layer of gel and tried to pick out the sandwich construct by the tweezer, the two layers of gels slipped apart!!! NIGHTMARE!!!!

At this point, it is clear that the gel sandwich approach was not the best way to make little gel discs. And we really needed to figure out a way to make flat, nice gel slabs!

As we are smart, smart students, we quickly found another way to make perfectly flat gel slabs!

Make gel slab using Western Blot!

As shown in the picture, we used two pieces of glass separated by a spacer (the black rubber piece) of 2.8 mm thickness to make gel slabs. The thickness of the spacer is the desired height of our gel discs. By putting the glasses onto a stand that is usually used to make gels for Western blot, we were able to seal one end of the glass pieces and pour the agarose solution from the top. After removing the glass slides when the gel solidified, we got a super nice gel slab! Tata….

Perfectly flat gel slab!!

We first heated up a 4% agarose solution to its boiling temperature. After it’s cooled down below body temperature,we mixed it with an equal volume of tissue culture growth medium (pink) with a certain cell concentration to make an agarose solution with a final concentration of 2%. Then we loaded our agarose solution seeded with cells on to the gel making device and wait for it to solidify.  We measured the actual height of the gel disc to be 2.57 mm, a little less than the spacer due to the force applied by the clamps.

Measuring the actual height of our gel construct!

Great! Now we have made gel discs seeded with cells. The next thing would be performing an assay for testing cell viability. We want to make sure that 1) the reagent of the assay can diffuse through the gels and get to the cells; 2) the cells are healthy and alive inside the gel constructs. We chose to use alamarBlue assay because it’s simple to perform and it is not an end-point assay. You can keep testing cell viability over time.  It indicates cell health by using the reducing power of living cells to quantitatively measure the proliferation of various kinds of cells. Resazurin, the active reagent of alamarBlue, is a non-toxic, cell permeable compound that is blue in color and virtually non-fluorescent. Upon entering cells, resazurin is reduced to resorufin, a compound that is red in color and highly fluorescent. Viable cells continuously convert resazurin to resorufin, increasing the overall fluorescence and color of the media surrounding cells.

We incubated the cell-seeded gel constructs with alamarBlue in the medium for overnight. The next day, to our delight, the color of the well turned pink from blue!!

AlamarBlue Assay results

The right rectangular comes from the medium incubated with cells in gels. The pink color indicates that the cells are alive and healthy in gels! High five!

Now that we know that alamarBlue assay can be successfully performed in our specific case, we can go ahead to test if the compression will have an effect on cell viability. According to literature, intervertebral cells subject to physiological compression actually become more healthy than the cells not subject to the compression. On the other end, cells subject to excessive compression will have decreased viability than the cells subject to physiological or no compression. It will be interesting to test these established  observations on our bioreactor to validate the device.