View source for Team:Pasteur Paris/Protocols/CellBio2

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        <h1>CELLULAR BIOLOGY PROTOCOLS</h1>
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            <h2 style="order:1;width:1000px">Cortical Cell Culture on Microfluidic Chip </h2>

                


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                    <p>PDMS Chip Coating</p>
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                    <p>Cell Dissociation</p>
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                    <p>Cell counting</p>
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                    <p>Seeding neurons into the microfluidic chip</p>
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                    <p>Fixating and staining neurons</p>
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                <br>
                <h3>Materials</h3>
                <ul>
                    <li> PDMS microchannel chip, see <a href="https://static.igem.org/mediawiki/2018/4/46/T--Pasteur_Paris--Microfluidics-microchannel-chip.pdf"> Microfluidics : microchannel chip </a> </li>
                    <li> Pipettes + tips  </li>
                    <li> Gloves  </li>
                    <li> Falcon tubes  </li>
                    <li> Eppendorf tubes  </li>
                    <li> Automated aspiration system  </li>
                    <li> Incubator </li>
                    <li> UV box  </li>
                    <li> Poly-L-lysine solution (PLL) (Sigma-Aldrich, ref : P8920-100ML)  </li>
                    <li> Laminin form engelbreth-holm-swarm murin (Sigma-Aldrich, ref : L2020-1MG)  </li>
                    <li> Phosphate buffered saline tablet (PBS) (Sigma-Aldrich, ref : P4417-50TAB)  </li>

                </ul>
                <br> 
                <h3>Procedure</h3>
                <br>

                <p> After sterilization under UV, see section 6 of <a href="https://static.igem.org/mediawiki/2018/4/46/T--Pasteur_Paris--Microfluidics-microchannel-chip.pdf"> Microfluidics : microchannel chip </a> , manipulations until the fixations are realized in sterile conditions. </p>

                <p> Drain water with aspiration system (Figure 1). </p>

               <figure>
                    <img src="https://static.igem.org/mediawiki/2018/5/5c/T--Pasteur_Paris--Order-HBHB.png" alt="Figure 1: Order without creation of flow">
                    <figcaption>Figure 1: Order without creation of flow</figcaption>
                </figure>

                <p> Add 50 µL of 10 µg/mL Poly-L-Lysine (PLL) into the top wells and allow the flow through (Figure 2a). After 5 minutes, fill the bottom wells (Figure 2b). Place the device in an incubator overnight at 37◦C. </p>


               <figure>
                    <img src="https://static.igem.org/mediawiki/2018/5/58/T--Pasteur_Paris--Order-HH.png" alt="Figure 2a: Order to create of flow">
                    <img src="https://static.igem.org/mediawiki/2018/e/e1/T--Pasteur_Paris--Order-BB.png" alt="Figure 2b: Order to create a flow">
                    <figcaption>Figure 2: Order to create a flow <br> (2a) left   (2b) right</figcaption>
                </figure>

                <p> Drain the device with aspiration system (Figure 1), and rinse with 30 µL of PBS 1X to wash out the excess fluid of PLL. Add 20 µL of 1mg/mL laminin into the top wells only (Figure 2a). Place the device containing laminin in an incubator at 37◦C for a minimum of 2 hours. </p>

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                <h3>Materials</h3>
                <ul>
                    <li> Bunsen burner  </li>
                    <li> Sterile Eppendorf tubes </li>
                    <li> Sterile 15 mL Falcon tube  </li>
                    <li> Pipettes + tips  </li>
                    <li> Centrifuge  </li>
                    <li> E18 rat cortex (2 pieces of tissue) stored in Hibernate EB (BrainBits)  </li>
                    <li> Sterile papain (BrainBits PAP 6mg)  </li>
                    <li> Hibernate E-Ca (BrainBits HE-Ca 5mL)  </li>
                    <li> Sterilized Pasteur pipette (BrainBits FPP)  </li>
                    <li>  Appropriate cell culture medium. Reagents used for the different cell culture mediums : 
                        <ul>
                            <li> FBS,HI,Qualified, USA origin (Fisher Scientific, ref : 11570516)  </li> 
                            <li> HBSS,W/OCA,MG,PH red 1X (Fisher Scientific, ref : 11530476) </li> 
                            <li> Glutamax 1 100X (Fisher Scientific, ref : 11574466)  </li> 
                            <li> B27 supplement 50X (Fisher Scientific, ref : 11530536) </li> 
                            <li> Neurobasal MED SFM (Fisher Scientific, ref : 11570556) </li> 
                            <li> Dulbeccos modified eagles medium 1X (DMEM) (Fisher Scientific, ref : 21969-035500ML) </li> 
                        </ul>
                    </li>
                </ul>
                <br> 
                <h3>Procedure</h3>
                <br>
                <p> According to <a href="https://static.igem.org/mediawiki/2018/a/a3/T--Pasteur_Paris--Primary-neuron-protocol-brainbits.pdf"> BrainBits protocol </a> . </p>
                <p> Prepare cell dissociation solution by dissolving 6 mg sterile papain in 3mL of Hibernate E-Ca for a final working concentration of 2 mg/mL papain. Incubate for 10 minutes at 30◦C. Then, carefully transfer and save Hibernate EB solution to a sterile tube leaving the tissue with minimal Hibernate EB volume. Add 2 mL of cell dissociation solution to the tissue and incubate for 10 minutes at 30◦C, gently swirl every 5 minutes. Then, carefully remove cell dissociation solution and return the saved Hibernate EB medium. Fire polish the tip of a sterilized Pasteur pipette to an opening of about 5 mm. With this pipette, triturate tissue for about 1 minute and let undispersed pieces settle for 1 minute. Then, transfer supernatant containing dispersed cells to a sterile 15mL, leave about 50 µL of Hibernate EB solution containing debris. Spin 1100 rpm for 1 minute, and discard supernatant leaving 50 µL of Hibernate EB solution containing the pellet. Resuspend pellet in 1mL of appropriate cell culture medium. </p>
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                <br>
                <h3>Materials</h3>
                <ul>
                    <li>  Sterile Eppendorf tubes  </li>
                    <li> Neubauer chamber </li>
                    <li>  Light microscope  </li>
                    <li>  Pipettes + tips  </li>
                    <li>  Trypan blue solution cell culture (Sigma-Aldrich, ref : T8154-20ML) </li>

                </ul>
                <br> 
                <h3>Procedure</h3>
                <br>
                <p> Aliquot 10 µL of cell solution into the tube containing 10 µL of Trypan Blue. Place 10 µL of the suspension in the Neubauer chamber. Count all the cells that are not colored in blue in the center square of the chamber, about 0.1 µL. The cellular concentration is determined by the total number of cells in 0.1 µL multiply by 10,000. Then, multiply the result by the dilution realized with the Trypan Blue. </p>
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                <br>
                <h3>Materials</h3>
                <ul>
                    <li> Prepared tissues </li>
                    <li> Appropriate cell culture medium </li>
                    <li> Microfluidic chips coated with PLL, products of section 1  </li>
                    <li> Pipettes + tips </li>
                    <li> Automated aspiration system </li>
                    <li> Incubator 37◦C, 5% CO2 </li>
                    <li> Ethylenediaminetetraacetic acid solution (EDTA) (Sigma-Aldrich, ref : 03690-100ML) </li>

                </ul>
                <br> 
                <h3>Procedure</h3>
                <br>
                <p> Drain the laminin with aspiration system (Figure 1). </p>

               <figure>
                    <img src="https://static.igem.org/mediawiki/2018/5/5c/T--Pasteur_Paris--Order-HBHB.png" alt="Figure 1: Order without creation of flow">
                    <figcaption>Figure 1: Order without creation of flow</figcaption>
                </figure>

                <p> Dilute cells with appropriate cell culture medium and plate 1.7 µL at 40,000 cells/cm2 (top wells only) (Figure 2). </p>


               <figure>
                    <img src="https://static.igem.org/mediawiki/2018/5/58/T--Pasteur_Paris--Order-HH.png" alt="Figure 2: Cell dilution order">
                    <figcaption>Figure 2: Cell dilution order</figcaption>
                </figure>

                <p>For the microfluidic chip seeding, tilt the tip in the direction of the chamber in order to help the migration of the neurons. Incubate for 5 minutes at room temperature, and add 50 µL of appropriate cell culture medium at 37◦C (Figure 1). The culture medium needs to be prepare at least 1 hour before. Add about 1 mL of EDTA around the chip to avoid contamination and prevent evaporation. Then, incubate the device at 37◦C with 5% CO2. Change half the cell culture medium every 3-4 days, use a P1000 pipette and not the aspiration system. </p>
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                <br>
                <h3>Materials</h3>
                <ul>
                    <li> Microfluidic chips seeded with neurons, products of section 4  </li>
                    <li> Automated aspiration system  </li>
                    <li> Pipettes + tips  </li>
                    <li> Medium B (32% paraformaldehyde solution  (PFA) (Bio-Rad, ref : 15714S) + 4% sucrose)   </li>
                    <li> Medium C (PBS + 0.5% triton X100 (Sigma-Aldrich, ref : T9284-100ML))   </li>
                    <li> Medium D (PBS + 1% bovine serum albumin solution (BSA) (Sigma-Aldrich, ref : A7979-50ML)) </li>
                    <li> Anti-MAP2, clone AP20, Alexa Fluor (Merck, ref : MAB3418A5) </li>
                    <li> Anti-<FONT face="Raleway">β</FONT>-tubulin antibody coupled with Alexa Fluor 448nm  </li>
                    <li> Phosphate buffered saline tablet (PBS) (Sigma-Aldrich, ref : P4417-50TAB) </li>
                </ul>
                <br> 
                <h3>Procedure</h3>
                <br>
                <p> After 6-7 days of incubation, remove the medium with a P1000 pipette (Figure 1) and fixate cells (Figure 2) in 50 µL of fresh medium B (4% PFA) for 30 minutes at room temperature. </p>
               <figure>
                    <img src="https://static.igem.org/mediawiki/2018/5/5c/T--Pasteur_Paris--Order-HBHB.png" alt="Figure 1: Order without creation of flow">
                    <figcaption>Figure 1: Order without creation of flow</figcaption>
                </figure>

               <figure>
                    <img src="https://static.igem.org/mediawiki/2018/e/e9/T--Pasteur_Paris--Order-HHBB.png" alt="Figure 2: Order to fixate the cells">
                    <figcaption>Figure 2: Order to fixate the cells</figcaption>
                </figure>

                <p> Then, drain with the aspiration system (Figure 1), and wash with 50 µL of 1X PBS for 5 minutes (Figure 3) x2 and replace the EDTA around the chip by 1mL of 1X PBS. </p>

               <figure>
                    <img src="https://static.igem.org/mediawiki/2018/5/58/T--Pasteur_Paris--Order-HH.png" alt="Figure 3">
                    <figcaption>Figure 3</figcaption>
                </figure>

                <p>  Drain with the aspiration system (Figure 1), and permeabilize in 50 µL of medium C for 10 minutes at room temperature. Then, drain with the aspiration system (Figure 1), and block with 50 µL of solution D for 30 minutes at room temperature. Prepare in 1X PBS, 500X anti MAP2 antibody coupled with Alexa Fluor 555nm and 500X anti-<FONT face="Raleway">β</FONT>-tubulin antibody coupled with Alexa Fluor 448 nm. Drain with the aspiration system (Figure 1), and drop 20 µL of the suspension on the top wells (Figure 3) and incubate 1 hour at room temperature in the dark. Then, drain with the aspiration system (Figure 1), and wash with 1X PBS and store at 4◦C fluorescent microscopic analysis.



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