Background

    Aside from space radiation, microgravity is an unignorable property of space. It surely must be taken into consideration since it greatly affects the function of Se coli in space[1]. However, available devices are lacking ideal simulation of microgravity. Even clinostats, the most commonly used machine for microgravity simulations, can only approximate microgravity in a tiny space near their centers by static equilibrium. Knowing that true microgravity is not yet achievable, we chose two of the most significant phenotypes that microbes might encounter in space and simulated them using hanging drop microfluidic chips[2]. After simulating the two phenotypes with hanging drop microfluidic chips, we crafted an acrylic case with UV lamps inside and placed the chips containing bacteria samples in it. By using this device, the UV tolerance of Se coli can be tested.

    We built our own hardware “MerStage”, a combination of microfluidic chip and UV-C stage to simulate microgravity and space radiation simultaneously. Operating MerStage requires no special training and costs only 5.0 USD for a single test, making MerStage more accessible to the public and able to bring real space environments closer to us. Table 1 shows the comparison on the current microgravity simulating devices.

Table 1. Comparison of current microgravity simulating devices
(RPM: Random positioning machine; DL: Diamagnetic levitation)

Device Overview

    MerStage is a user-friendly and affordable device for 3D cell culture and aggregation experiments under radiation. To deal with the problem that microgravity is unachievable, two of the most significant phenotypes that microbes exhibit in space were simulated by using hanging drop microfluidic chips. The chips containing bacteria samples were placed inside an acrylic case with UV lamps inside to test the UV tolerance of Se coli.

Fig. 1. Hanging drop microfluidic chip

Fig. 2. MerStage

Hanging Drop Microfluidic Chip

Goal

    To simulate 3D cell culture and bacteria aggregation.

Background

    The hanging drops formed under the microfluidic chip are caused by gravity. Bacteria will fall into these drops when the bacteria-rich medium flows through the chip. Because of surface tension and gravity, E. coli will be suspended inside these micro drops and form clusters.

Fig. 3. Hanging drop microfluidic chip

Fig. 4. Bacteria aggregation in a single well

Design

    The microfluidic chip has three different layers[3, 4]:

1. Injection layer has two holes with different sizes. The smaller one is for bacteria injection, and the larger one is the medium inlet. Medium in the pressure tank gives pressure to form drops on the underside of the chip.
2. Channel layer has a hollow oblong space in the middle to let bacteria medium flow into all the wells.
3. Hanging drop layer has 126 wells with a diameter of 1.0 mm where the hanging drops form and the bacteria samples aggregate.

    To easily observe bacteria aggregation and hanging drop formation with our naked eyes, the chip should be made of transparent materials. PMMA is chosen because it costs less.

Aggregation Experiment

    Prior to radiation resistance test, bacteria aggregation must be settled first. The time required for it to aggregate was calculated. We first injected bacteria medium with two different concentrations into the chip and used a microscope to observe bacteria aggregation in the hanging drops.

    In high concentration, bacteria clusters inside the chip could not be properly formed due to its fast growth, causing a mix between aggregated and non-aggregated bacteria. In lower concentration, clear aggregation could be observed after 6-8 hours. The experimental observation corresponds to the model simulation results (see Model page). In Fig. 5 and Fig. 6, E. coli aggregation in the chip shares a lot of similarities with that in space. In the following UV tests, we waited for at least 8 hours to let bacteria aggregate before conducting experiments.

Fig. 5. E. coli aggregation in our microfluidic chip (40X)

Fig. 6. E. coli aggregation in space[2]

Well Size Experiment

    The well's diameter is an important factor that must be taken into consideration to form stable hanging drops. Drop formation in wells with diameters of 0.6 mm, 1.0 mm, 1.4 mm, and 1.8 mm were observed under the microscope. In 1.8 mm well diameter, surface tension was not strong enough and the water drop bursted. Contrary to large diameter, in 0.6 mm well diameter, no water drop was formed because the size of the drop was too small. The well with a diameter of 1 mm was found to be the best to fabricate microfluidic chip and form stable hanging drops.

(A)	(B)
(C)	(D)

Fig. 7. Droplets formed by liquid pressure and surface tension.
Well size: 0.6 mm (A), 1.0 mm (B), 1.4 mm (C), and 1.8 mm (D)

Table 2. Well size experiment result.

Growth Curve Experiment

I. Three dimentional aggregation

    Experimental group of E. coli were inside the hanging drop microfluidic chip. For the control group, a hydrophilic membrane was sticked below the chip so it cannot form hanging drops. From Fig. 8, the growth rate of the experimental group is higher than the control group for the first 10 hours. This happens because a cell with 3D structure has a larger surface area compared to a cell with 2D structure. As mentioned above, since the contact area between E. coli and the medium was larger, bacteria consumed more nutrients and grew faster[5].

    After 10 hours of cell culture, the growth curve of the experimental group slowed down, and even lower than that of the control group. According to our previous aggregation experiment result, bacterial aggregation could be easily observed inside our microfluidic chip after 6-8 hours. We concluded that 3D bacteria aggregation may cause the bacteria in the middle of the cluster to have less contact area with nutrients.

Fig. 8. Growth curve (Aggregation/ No Aggregation)

II. Medium exchange

    To extend the UV experiment time, changing the culture medium properly is crucial, for cells may die from nutrient depletion instead of UV light. With the help of Model, we decided to change medium after 8 hours of cell culture. The results showed a higher OD₆₀₀ value than the non-exchanging one.

    For fear that the most bacteria might be sucked out from the chip when changing the medium, we also tested the OD₆₀₀ value of the medium that we sucked out. After some calculation, 90% of the E. coli remained in the chip after medium exchange.

Fig. 9. Growth curve (Changed/ Unchanged)

III. Conclusions

1. E. coli grows faster in 3D cell culture than in 2D (1.8 times faster in the first 2 hr).
2. Changing the medium around 8 hr of culture is preferred.

UV Stage

Goal

    To provide a stable environment where our bacteria samples in microfluidic chips can safely undergo UV-C exposure.

Background

Fig. 10. UV-C stage.

Space radiation consists of radiation with various wavelengths. Short-wavelength radiation can be the most harmful radiation to the living of bacteria in space. Therefore, UV-C (254 nm) was chosen because it has the shortest wavelength among all UV lights.

Design

    MerStage is a device with the ability to adjust UV sources’ height to change radiation intensity. Considering that UV-C radiation may be harmful to humans, we used 5-mm thick black acrylic to craft the outer shell of MerStage. Other than that, UV-C card was also installed to notify if the radiation somehow leaked outside the UV stage. To make it convenient for people to insert the microfluidic chip into the stage and exchange medium, we left a window on top of MerStage. The picture below shows some more detailed designs of MerStage. (Click for more detail, double click to hide.)

Survival Rate in MerStage

Experiment

    First, E. coli was injected into a microfluidic chip, and then the chip was placed inside a sealed box to wait for the aggregation. After more than 8 hr of aggregation, the chip was taken out and put into the MerStage to do the exposure test.

Calculation

    We tested OD₆₀₀ and OD₂₅₄ and used the equations[4] below to calculate the average irradiation intensity in the UV test[5]. Table 2 is the parameter descriptions, Table 3 is the OD value, and Table 4 are the parameters values.

\[{W_{avg}}={W_0(1-e^{-{A_e}L}) \over {A_e}L}\text{ ......(Eq. 1)}\]

\[{A}={alc}\text{ ......(Eq. 2)}\]

\[{OD_{254}}={a \times OD_{600} \times 3 \times 10^8 (cell/ml) \times 10^{-12} (g/cell) \times 10^3 (ml/L) \times l}\text{ ......(Eq. 3)}\]

Table 2. Parameter descriptions

Results

    In Fig. 11, the radiation resistances of E. coli with and without melanin were tested with 9 W UV-C light. According to our calculation, E. coli without melanin receives a higher average irradiation number (0.306 W > 0.020 W) and has a lower survival rate compared to E. coli with melanin protection. In this experiment, E. coli with melanin protection is proven to be more radiation-resistant in our microfluidic chip.

Fig. 11. Survival rate of bacteria with and without melanin (UV light 9 W)
After 3 hours of exposure to UV-C light, the survival rate of melanized bacteria was 40.1%, while non-melanized bacteria was 10.0%.

    In Fig. 12, the survival rate of E. coli without melanin, E. coli with melanin, and our final wet product Se coli were tested with 12 W UV-C light. After calculation, the three average irradiation numbers were 0.041 W (none), 0.026 W (melanin), and 0.030 W (selenomelanin). E. coli without melanin protection received the highest average irradiation number and the lowest survival rate. Compared to E. coli with melanin, Se coli received a higher average irradiation number but had a better survival rate.

Fig. 12. Survival rate of bacteria without melanin, with melanin, and with selenomelanin (UV light 12 W)
After 1 hr of exposure, the survival rate of non-melanized bacteria was 3.8%, melanized bacteria was 14.0%, and selenomelanin was 30.6%.

Conclusions

    MerStage has been proven to successfully provide a low-cost, accurate, and effective way to mimic space experiments. Although it might satisfy the needs of the current period, further improvements are mandatory, given that our experiments are still facing some obstacles. For instance, a simple Geiger counter is needed to detect and measure radiation. Aside from that, UV-C LED may be used to prevent evaporation which leads to the liquid inside the microfluidic chip getting dried out and the bacteria getting killed. Water sprayer may also be placed inside of MerStage to increase humidity.

Hardware Video

Cost Estimation

    Table 4 shows the total cost of initial investment, and Table 5 shows the price for each single chip. The cost of every single experiment, including the cost of tips, medium, and plates, is less than 5 USD.

Table 4. Estimation of the total cost for initial investment

Table 5. Estimation of the total cost for a single microfluidic chip.