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«MED64 Application Note Primary Neuronal Cell Culture Information in this document is subject to change without notice. No part of this document may ...»

-- [ Page 1 ] --

The most sensitive microelectrode array system 

for in vitro extracellular electrophysiology

MED64 Application Note

Primary Neuronal Cell Culture

Information in this document is subject to change without notice.

No part of this document may be reproduced or transmitted

without the expressed written permission of Alpha MED Scientific

Inc.

While every precaution has been taken in the preparation of this

document, the publisher and the authors assume no responsibility for errors, omissions, damages resulting from the use of information contained in this document, or from the use of programs and source code that may accompany it. In no event shall the publisher and/or the author be liable for losses of profit or any other commercial damage caused or alleged to have been caused directly or indirectly by this document.

© 2015 Alpha MED Scientific Inc. All rights reserved Version: 1.10; September 1, 2015 Alpha MED Scientific Inc.

Saito Bio-Incubator 209, 7-7-15, Saito-asagi, Ibaraki, Osaka 567-0085, Japan E-mail: support@med64.com Website: http://www.med64.com Contents

1. Introduction 1 1-1. Acknowledgement

1-2. Disclaimer

2. Pretreatment of the MED Probes and plating neurons 2 2-1. Material to be prepared

2-2. Workflow for preparation

2-3. Pretreatment of the MED Probe

Sterilization

Pre-coating with PEI

* Recipe -PEI solution-

Coating method #1: Coating with Laminin-511

Coating method #2: Coating with poly-D-lysine

2-4. Plating and culturing rat hippocampal neurons

Preparation

Dissection and plating cells onto the MED Probe

* Conditional medium

2-5. Cleaning the used MED Probes

Tryspin method

Breaching method

3. Data acquisition 12 3-1. Recommended experimental environment

1) Use of the CO2 incubator

2) Use of the MED Heated Connector

3-2. Data acquisition

Available Mobius workflow templates

Recommended acquisition settings

MED64 Application Note - Primary Neuronal Cell Culture i Contents

4. Data analysis 14 4-1. Workflow templates available for analyzing neuronal spikes

“Spike_frequency_analysis”, “Spike_frequency_analysis_filter”.................. 14 “Spike_sorting”, “Spike_sorting_filter”

4-2. Setting Thresholds

4-3. Spike frequency analysis

4-4. Spike sorting

4-5. Exporting data

–  –  –

1. Introduction Dissociated neuron cultures offer several distinct advantages for investigating the electrical and chemical communication between neurons. These advantages can be applied to neuronal models of learning and memory, development, aging, disease, and many more. The emergence of the MED64 as the industry leader in micro- electrode array technology has made it a popular platform for studying the electrophysiological properties of neuron cultures.

The MED64's broad acquisition bandwidth combined with superior signal to noise ratio affords impeccable extraction of basic electrophysiological variables. The combination of precise extraction of electrophysiological activity with the ease of culturing cells directly onto the MED Probe (Multi Electrode Dish) makes the MED64 ideal for pharmacological, drug safety screening, and basic scientific applications. The 64 electrodes on the MED Probe have the lowest impedance of any micro-electrode array, making the MED64 ideal for acquiring data from spontaneously spiking neurons or neurons that fire in response to drug application. The MED64 also has high capacitance electrodes, enabling the MED64 to deliver high stimulating current, which is essential for for evoked response studies.

The goal of this application note is to describe how to set up experiments with dissociated neuron cultures, acquire relevant data, and extract the data for presentation or publication. This material has been prepared by scientists with expertise in neuroscience and electrophysiology. A complete protocol for plating, culturing, and carrying out experiments on dissociated neuron cultures has been prepared based on the users' experience.

1-1. Acknowledgement

Alpha MED Scientific would like to thank the MED64 users that have shared their knowledge,:

Ikuro Suzuki, PhD - Tohoku Institute of Technology Aoi Odawara - Tohoku Institute of Technology Keiichi Shirakawa, PhD- Application Scientist, Alpha MED Scientific Michael Trujillo, PhD - Senior Application Scientist, Alpha MED Scientific 1-2. Disclaimer This application note is a summary of information shared by MED64 users and is to be considered marketing material. These methods have been developed, tested, and verified in the course of projects published in peer-reviewed literature. However, Alpha MED Scientific does not guarantee that the information written in this document is correct and is free from all liabilities. Please refer to the scientific literature for further insight on these techniques, as well as the MED64 and Mobius manuals for detailed instructions on use of the MED64 System.

–  –  –

2. Pretreatment of the MED Probes and plating neurons 2-1. Material to be prepared Either of *1 or 2 will be used for pre-coating the MED Probe.





–  –  –

2-2. Workflow for preparation 2-3. Pretreatment of the MED Probe

CAUTION:

Avoid contact with the electrodes in all of following procedures as they are extremely fragile.

Pretreatment of the MED Probe is the most critical step to culture neurons onto the electrodes. The surface of a new MED Probe is hydrophobic. Therefore, hydrophilization of the MED Probe is necessary to enhance the adhesion of neurons to the electrodes. If cells do not adhere to the electrodes, you will not be able to record from them. It should be noted that some coating agents can affect neural activity, viability, degree of neurite outgrowth, extent of migration, and longevity. Improper coating techniques can cause large-scale clumping and/or the death of neurons even if all other cell culture steps are performed properly. The following section contains recommendations for appropriate pretreatment of the MED Probes including 2 different coating methods.

Sterilization

1. Rinse a new MED Probe with sterilized distilled water (SDW) at least three times. Rinse it with 70% ethanol several times (or immerse it in 70% ethanol for 15 minute), and then let it dry naturally on a clean bench. Higher-grade ethanol is recommended to avoid deposits of organic substances onto the MED Probe after drying.

2. Rinse the MED Probe with sterilized distilled water (SDW) at least three times, and then let it dry under ultraviolet irradiation for 15-30 min. Store and handle the MED Probe in a sterilized 90 mm petri dish.

Note:

Above steps are not always required, especially when reusing a MED Probe.

–  –  –

Pre-coating with PEI The surface for the new MED Probe is hydrophobic. Thus, pre-coating with PEI is important for enhancing the hydrophilicity of the MED Probe’s surface and preventing clumping of cells during their growth. While Pre-treatment with PEI is not typically required when re-using MED Probes, some MED Probes may need to be pre-treated again after several uses.

Note:

Pre-coating with PEI needs to be made on the day before plating cells.

1. Treat the surface of a MED Probe with 0.05% Polyethyleneimine (PEI) in 25 mM borate buffer (pH 8.4) for one hour at room temperature. (Make sure the electrodes are covered by the PEI.)

2. Aspirate the PEI and rinse the MED Probe with DSW 4 times.

3. Dry the MED Probe on a clean bench over night.

Figure 1. Rat hippocampal neurons in 5 days after plating with PEI pre-treatment (left) and without PEI pre-treatment (right).

Cells grew evenly on the MED Probe pre-treated with PEI (left).

Recipe -PEI solutionmM borate buffer Dissolve 25 mM Na2B4O7/10H2O (MW: 381.4, Sigma: S9640) in distilled water and adjust the pH to 8.4 with HCl. (500 ml borate buffer solution)

–  –  –

0.05% PEI (polyethyleneimine) solution Since a 50% PEI solution (Sigma: P3143) is so sticky, it is recommended to prepare a 1% stock solution first (1 ml 50% PEI to 49 mls 25mM borate buffer). This 1% stock solutions is then diluted to (0.05% PEI) for final use.

• The PEI solution can be stored in refrigerator up to 1 month.

–  –  –

Coating method #1: coating with Laminin-511

Note:

Coating needs to be made on the day before plating cells.

1. Rinse the MED Probe with PBS and fill the MED Probe with 1 ml of Laminin-511 (2 g/ml). Store the MED Probe in a sterilized petri dish and incubate it for up to 1 hour.

• Alternatively, the Laminin-coated Probe can be left in room temperature for 3 hours or in a refrigerator (4°C) for overnight.

Figure 2. Rinse the MED Probe with PBS (left).

Store the MED Probe filled with Laminin-511 in a sterilized dish and incubate it for up to 1 hr (right).

2. Move the MED Probe into a clean bench. Aspirate the Laminin-511 from the MED Probe, If there are bubbles on the electrodes, carefully remove them using a pipet. (Do this on a clean bench.)

Note:

Bubbles on the electrodes will block cells grown onto electrodes. Make sure to remove all bubbles.

3. Plate neurons as soon as possible before the electrodes are dried (described in the next section).

CAUTION:

Be careful NOT to touch the electrodes.

–  –  –

Coating method #2: Coating with Poly-D-lysine

Note:

Coating needs to be made on the day of plating cells.

1. Rinse the MED Probe with 50 mg/ml poly-D-lysine solution (M.W. is more than 70000) once, and fill the MED Probe with 1 ml of poly-D-lysine. Store the MED Probe in a sterilized petri dish and incubate it for up to 1 hour.

2. Move the MED Probe into a clean bench. Aspirate poly-D-lysine solution and let it dry naturally.

–  –  –

The electrode surface should not repel water and should maintain moisture after the completion of this process.

Figure 2. The MED Probe after completion of the coating.

The electrodes should maintain moisture.

3. Rinse the MED Probe with SDW at least 3 times. Fill the MED Probe with maintenance medium, and incubate the MED Probe at 37°C for 1 hour.

4. Take the MED Probe from the incubator. If there are bubbles on the electrodes, carefully remove them using a pipet. (Do this on a clean bench.)

–  –  –

Bubbles on the electrodes will block cells grown onto electrodes. Make sure to remove all bubbles.

5. Plate neurons as soon as possible (described in the next section).

CAUTION:

Be careful NOT to touch the electrodes.

–  –  –

2-4. Plating and culturing rat hippocampal neurons Preparation

1. Fill four 90 mm petri dish (P#1-4) with 20 ml of treatment medium each. (See Table. 1. for composition of the treatment medium.)

–  –  –

*Adjust pH to 7.2 with NaOH solution Table 1. The composition of the treatment medium (for 2L).

2. Fill six conical tubes (C#1-6) with medium listed in the Table 2-3. Warm up conical tube C#2 with a water bath at 37 C. Chill the other conical tubes (#1, 3-6).

–  –  –

Penicillin-Streotomycin (Life Technologies #15140-148) 5 ml * Dissolve 40.7 mg of L-glutamin with 22 ml of Neurobasal medium, and filter it. Use 20 ml.

Table 3. The composition of modified Neurobasal medium (513 ml).

–  –  –

Dissection and plating cells onto the MED Probe Note: Refer to Ref.1. for further information.

1. Anesthetize a pregnant rat with ether. Dissect both carotid arteries and incise the abdomen.

2. Isolate uterus containing a litter of 18-day-old fetal rats and transfer it into a sterilized petri dish.

3. Isolate fetal rats from the uterus and wash them in petri dish #1 (P#1) containing the treatment medium. Then transfer them into petri dish #2 (P#2).

4. Place the fetal rats on a KimWipe. Isolate brain tissue containing cerebellum and transfer it into petri dish #3 (P#3).

5. Isolate both cerebral hemispheres from the brain tissue in the P#3, and transfer them into petri dish #4 (P#4).

6. Isolate both hippocampi in P#4 chilled with flaked ice under a microscope, and transfer them into conical tube #1 (C#1) chilled with flaked ice.

Figure 4. Isolating hippocampi with flaked ice under microscope (left).

The hippocampal transferred into C#1 (right).

* Anatomical procedure (1-5) with photos is available. Contact support@med64.com.

7. Wait for the hippocampi settled in the bottom of conical vial #1 (C#1), and then aspirate “the supernatant of the medium”. Add the medium contained in conical vial #2 (C#2) to (C#1).

8. Incubate C#1 for 15 min. Take it out from the incubator every 5 minutes, shake it gently, and return it to the incubator. (This process will work to enhance mixing and avoid damaging cells in later processes and pipetting.) Figure 5. Add solution to C#1 (left) and incubate it for 15 min. Take it out to shake gently every 5 minutes (right).

–  –  –

9. Centrifuge C#1 with 1000 rpm at 4°C for 5 min.

10. Aspirate the supernatant and add the medium contained in conical tube #3 (C#3) to C#1. Pipette C#1 gently ten times, then centrifuge it with 1000 rpm at 4°C for 5 min.

11. Aspirate the supernatant and add the medium contained in conical tube #4 to C#1. Pipette C#1 gently ten times, then centrifuge it with 1000 rpm at 4°C for 5 min.



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