Template:Team:XMU-China/cellimmobilization

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Cell Immobilization

1. Overview
After finishing our circuit's construction, it is time to set up the macroscopic device system. Obviously, the foremost thing we should do is to fix the engineering bacteria from suspended batch cultivation to the tubes of our device, namely cell immobilization. Through detailed cmparison of three methods in various aspects, we chose the intra-hollow calcium alginate capsule as matrix for our cell immobilization. And the result proves that it is feasible. After that, we started to establish our digital display device and undertaken the continuous cultivation of our engineering bacteria. Meanwhile, cultivation condition and fluorescence intensity of immobilized cells should be made certain.

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2. Methods of cell immobilization
Cell immobilization is a technique to fix cells in a suitable matrix. In the past, various cells have been immobilized. A number of methods have been founded and developed base on various principles: entrapment, ion exchange adsorption, porous ceramics, and even covalent bonding. After studying literatures and asking senior students for help, we decided to explore three methods to immobilize our engineering bacteria:
2.1 Immobilizing cells into calcium alginate beads
Calcium alginate gel apply to entrapping cells has advantageous properties such as high mechanical strength, inexpensive cost, mild and simple operation conditions and good biocompatibility. Therefore, this method is widely used in cell immobilization. In this project, we chose making calcium alginate beads as our first trial of immobilization experiments.
2.2 Immobilizing cells into intra-hollow Ca-alginate capsules
Embedding cells in intra-hollow Ca-alginate capsules is a technique derives from calcium alginate immobilization system. This method not only covers most of the advantages of calcium alginate entrapment, but also overcomes many difficulties. Substrates and oxygen can easier transfer into intra-hollow capsules than into calcium alginate beads. Moreover, while cells can only grow on the beads' surface and in the gel holes, the grow space of cells largely broaden in capsules since they enwrap liquid interior medium.
2.3 Immobilizing cells into NaCS-PDMDAAC microcapsules
Embedding cells into NaCS-PDMDAAC microcapsules is one kind of new and promising immobilization technique, which has well biocompatibility and excellent light transmission. However, the preparation of NaCS is difficult and time-consuming. Through experiments, we also found that mechanical strength of NaCS-PDMDAAC microcapsules was too low for our project. When the microcapsules were transferred from one culture to another, they broke easily. That indicates if our engineering bacteria were embedded in NaCS-PDMDAAC microcapsules, they cannot be well fixed in some kinds of continuous culture systems, such as our tubes.

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3. Device Design

Figure 1: The simple device that we made for cell immobilization culture.

To construct our display device, we used seven glass tubes (95mm×Φ13mm) to form the shape "日", then filled with our immobilized cells. Each end of the tubes is stuffed up with a silica gel plug, through which leads out a glass connecting jointed with a silica gel pipe to pass through LB medium. The LB medium is parallel and equally pumped into the seven tubes by peristaltic pumps. Inside each tube, we fixed small sieve plates next to the plugs incase the beads /capsules flow out or block the silica gel tubes. Besides, the LB medium is oxygenated before being pumped into the system, which ensures the supply of oxygen to the cells.
In consideration of sterilization, the materials we used can all endure high temperature at least 121 ℃, and the LB medium is also sterilized by an autoclave at 121 ℃ for 20 min, so our culture conditions can be aseptic.

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4. Results and Discussion
At first, we had to make sure which method is the most suitable one to embed our engineering bacteria and to stuff in our device tubes.
4.1 Calcium Alginate Beads
Making calcium alginate beads is a basic method of cell immobilization, and there were many previous research literatures^[1-6]. According to these literatures, we prepared calcium alginate beads embedding engineering bacteria with few challenges. After finishing the preparation, we could easily see the beads turn green while cultivation in LB medium.
But during the cultivation, we found that this method is not perfect for us. The beads are stuffed and opaque, and because some of the bacteria leaked into the medium, the background fluorescence intensity brings a significant influence to the measuring.

Figure 2: The calcium alginate beads emitted fluorescence when embedded PCiGLT. (The bacteria PCiGLT had been cultivated 12 hours.)	Figure 3: Beads in the left flask were shaken at 50 rpm, while beads in the right flask were shaken at 100 rpm and the bacteria had leaked into medium. After cultivation for a few hours, their fluorescence faded.

4.2 NaCS-PDMDAAC Microcapsules
For the relatively new technique—NaCS-PDMDAAC microcapsules, its materials are complex. So we started our exploration with testing if PDMDAAC will restrain the growth of the engineering bacteria. We respectively added 3%, 6%, 9% PDMDAAC solution to isometric bacteria culture and then 37℃ shaker incubated for 12 hours. The result was shown as follows.

Figure 4: Experimentally measured growth curves of E. coli cells with different concentration of PDMDAAC. The figures a, b, c, d respectively added 0%, 3%, 6%, 9% PDMDAAC solution.

As the graph shows, in the first 90 minutes, OD₆₀₀ was relatively high (in a normal value), but then it declined dramatically. That indicated some measure of PDMDAAC will restrain the long-term growth of the engineering bacteria. It was not a very promising outcome, but according to some previous research^[7], NaCS-PDMDAAC microcapsules have relatively well biocompatibility, so we still believed this method is viable at a certain extent, and went on in this way.
With great difficulty, we accomplished the preparation of NaCS-PDMDAAC microcapsules, which looked roundish and transparent, just like what we wanted:

Figure 5: PBADGLT embedded in NaCS-PDMDAAC microcapsules

However, some unavoidable problems have arisen. First, the preparation of NaCS solution is really a complicated and time-consuming process. Another crucial fact is that the microcapsules are extraordinary fragile, namely the mechanical strength is too weak to transfer into our device.

4.3 Intra-hollow Ca-alginate Capsules
The advantage of transparent or semitransparent capsules is very apparent, so we need to search another easier method to prepare capsules. We find that intra-hollow Ca-alginate capsules which can also be prepared easily have a good biocompatibility through referring literatures. Embedding cells in intra-hollow Ca-alginate capsules is an improvement of calcium alginate beads^[8]. Then we prepared intra-hollow Ca-alginate capsules using sodium alginate, CaCl₂ and another composition – CMC (sodium carboxyl methyl cellulose).

Figure 6: Embedding bacteria into intra-hollow Ca-alginate capsules.

4.4 Comparison of Beads and Capsules
Embedding cells in intra-hollow Ca-alginate capsules is an improvement of calcium alginate beads, whereas its preparation process is a little more complicated ^[8]. To make better selection, one of our team members prepared calcium alginate beads and intra-hollow Ca-alginate capsules under the same temperature and air pressure, and compared them in several aspects.

4.4.1 The measurement of diameter of the immobilized beads/ intra-hollow capsules
We randomly selected 25 beads/ intra-hollow capsules from the same batch we prepared. For each bead/ capsule, we measured the diameter 3 times from different positions through the microscope and calculated their average value. Then calculated and wrote down the average of the 25 values.
We can see that the capsules are a bit bigger than the beads from the graph.

Figure 7: Diameter distributions of capsules and beads.[click to data]

4.4.2 The measurement of the thickness of membrane of the intra-hollow Ca-alginate capsules

There is a cavity in each intra-hollow calcium alginate capsule. We slit the capsules by a knife, observed and measured the thickness of membrane from different positions through the microscope. Randomly selected 5 samples and calculated the average value.

Table 1: Membrane thickness of capsules which measured through the microscope.

4.4.3 The measurement of mechanical strength of the beads/ intra-hollow capsules

We used a texture analyzer to measure the mechanical strength of the immobilized beads/ intra-hollow capsules we prepared.
We placed a bead/ intra-hollow capsule on the tray, and pressed from the top of the bead/ intra-hollow capsule at the testing speed of 1.0 mm/min, while observing the changes of the force sensor form a computer screen. For each test, we recorded the force value when the bead/ intra-hollow capsule ruptured and the value reach a maximum (after that the force reduce sharply). Randomly select 20 samples and calculate the average value.

Table 2: The maximum pressure force when a capsule or bead burst. (Testing speed: 1mm/min)

4.4.4 The measurement of fluorescence intensity of the immobilized beads/ intra-hollow capsules

We used a microplate reader to measure the fluorescence. First we randomly selected 9 beads/ intra-hollow capsules from the same batch we prepared and divided into 3 parallel groups. Add 150 µl PBS in each well of a 96 Well Cell Culture Cluster 3599, and then placed each group of microspheres/microcapsules in it. Measured the fluorescence of every group and calculated the average value.
Obviously, the capsules perform better stability of fluorescence and the curve of capsules goes as we expected.

Figure 8: Fluorescence curve of PBADGLT Ca-alginate capsules.[click to data]

Figure 9: Fluorescence curves of PBADGLT in Ca-alginate beads.[click to data]

4.4.5 The measurement of immobilizing capability of the beads/ intra-hollow capsules

If the immobilizing capability of the beads/ intra-hollow capsules is favorable, the bacteria inside rarely leak, and the OD of the medium will be low and steady. We used a microplate reader to measure the OD of the culture medium of the immobilized beads/ intra-hollow capsules.

Figure 10: OD600 Measurement of culture medium of capsules or beads during cultivation.[click to data]

From the graph, we can find the OD₆₀₀ of the culture medium of beads increased faster and higher, indicating that cells immobilized in them leak easier than those embedded in capsules.

Table 3: Comparison of three kinds of immobilization methods.

Even though our experiments showed that the fluorescence intensity of beads is stronger than capsules, the capsules perform better stability of fluorescence, stronger mechanical strength and lower cell leak rate. Compared to a solid bead, a capsule's liquid core can provide a better environment for bacteria to grow because the liquid core is more favorable to the exchange of substance. Additionally, the beads will swell in culture medium but the capsules not. In conclusion, among the three kinds of immobilization methods, intra-hollow calcium alginate capsules should be the best choice of our biological display device.

4.5 Optimization of Culture Conditions
After making the decision of immobilizing our engineering bacteria into intra-hollow Ca-alginate capsules, some of the culture conditions need exploration and optimization.
We designed some different conditions of shaking time before induction and conducted fluorescence measurements. Results pointed out that fluorescence of cells under 3-hour shaking, when cells approximately reached the logarithmic phase, got a higher level than 10-hour shaking.

Figure 11: Fluorescence curves of PBADGLT Ca-alginate capsules induced by arabinose after shaking for 3h.[click to data]

Figure 12: Fluorescence curves of PBADGLT Ca-alginate capsules induced by arabinose after shaking for 10h.[click to data]

4.6 Biological Activity Tests
Also, we must guarantee the activity of cells immobilized inside the intra-hollow capsules. We prepared intra-hollow Ca-alginate capsules, embedding condensed cell suspension. Used a 1 mL syringe to extract a little culture inside a capsule, spread some of the culture inside a capsule on an LB agar plate containing Amp, and incubating overnight at 37℃ .

Figure 13: Cultivation of the liquid extract from the capsules. Picture a is a plate cultivated liquid extracted from capsules embedded bacteria, and picture b is a plate cultivated liquid extracted from capsules embedded aseptic water.

On the next day, we found out that some bacterial colony had grown on the LB agar plate containing Amp, while there was no colony growing on the plate of control group. The result of fluorescence measurement also proved that the cells immobilized into capsules were alive after shaking for a long time. When we refreshed the medium and added inducer again, they can start a new growth cycle.

Figure 14: Fluorescence curves of PBADGLT Ca-alginate capsules.[click to data]

4.7 Digital Display Test
After finishing the experiments above, we began to test our circuits of digital display. We took P_cIGLT and P_BADGLT to conduct fluorescence measurements.

Figure 15: Fluorescence curves of PBADGLT and PcIGLT capsules.[click to data]

As anticipated in circuit construction stage, P_BADGLT should be induced by arabinose and P_cIGLT needs no inducer. So, at the very beginning, there were expressions of green fluorescence proteins in P_cIGLT. But as we know, intra-hollow capsules have limited inner space to provide cells with a certain volume to live. So when first immobilized, cells of P_cIGLT could still reproduce in some free space of capsules, but as amount of cells increase, free space was used up, hence cells reached a critical level where they could not express this fluorescence protein. But the existed proteins were still degraded for its unstable tail. On the other hand, culture P_BADGLT could normally express the protein under induction, as the graph shows.

4.8 Conclusions
In sum, we have designed a biological display device to effectuate our goal. And we chose the method of embedding our engineering bacteria into intra-hollow calcium alginate capsules, due to its better stability of fluorescence, better cultivation environment, stronger mechanical strength and lower cell leak rate. Through experiments, we found that induced cells under 3-hour shaking showed stronger and more stable fluorescence. Moreover, we have proved the cell activity still maintain at a relatively high level when cells are immobilized into the capsules. Therefore cell immobilization is feasible for our device construction. Finally, we conducted the fluorescence measurements of immobilized bacteria P_cIGLT and P_BADGLT to test our circuits of digital displayer and got some promising outcomes.

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References
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[2] A. Blandino, M. Macias, D. Cantero. Immobilization of glucose oxidase within calcium alginate gel capsules[J], Process Biochemistry, 36 (2001) 601-606.
[3] P.H.C. Haroldo Yukio Kawaguti, Joelise Alencar Figueira, and Hélia Harumi Sato. Immobilization of Erwinia sp. D12 Cells in Alginate-Gelatin Matrix and Conversion of Sucrose into Isomaltulose Using Response Surface Methodology[J], Enzyme Res, (2011).
[4] A. Blandino, M. Macias, D. Cantero. Calcium alginate gel as encapsulation matrix for coimmobilized enzyme systems[J], Appl Biochem Biotechnol, 110 (2003) 53-60.
[5] 肖美燕, 徐尔尼, 陈志文. 包埋法固定化细胞技术的研究进展[J], 食品科学, 24 (2003) 158-161.
[6] 李超敏，韩梅，张良，陈锡时. 细胞固定化技术--海藻酸钠包埋法的研究进展[J], 安徽农业科学, 34 (2006).
[7] 章小忠, 梅乐和, 姚善泾. 纤维素硫酸钠的制取反应过程研究及由其制备的微胶囊性能[J], 化学反应工程与工艺, (2003).
[8] 陆悦飞. 中空海藻酸钙微胶囊固定化大肠杆菌表达P450BM-3催化吲哚生成靛蓝的研究[D], 浙江大学, (2006).