Jan 08, 2024
Version 2
Rat organotypic cultures for AAV-mediated vital labeling of the extracellular matrix V.2
- 1Achucarro Basque Center for Neuroscience
Protocol Citation: Federico N. Soria, Mario Fernandez-Ballester 2024. Rat organotypic cultures for AAV-mediated vital labeling of the extracellular matrix. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdnzzlmk/v2Version created by Federico N. Soria
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 08, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 93053
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson
Grant ID: ASAP-020505
Abstract
In this protocol we describe the preparation and maintenance of rat organotypic cultures (cortico-striatal) for AAV-infection and imaging of the extracellular matrix. The AAV, in this case, induces the expression of a exportable hyaluronan-binding protein (the HA-biding domain of Neurocan) fused with GFP, that allows vital labeling (and hence live imaging) of the extracellular matrix. The organotypic cultures can be maintained for several weeks, allowing for repeated imaging and longitudinal studies.
Materials
Organotypic culture medium
50% MEM, HEPES, no glutamineGibco, ThermoFisherCatalog #32360026
25% EBSS, calcium, magnesium, phenol redThermo FisherCatalog #24010043
25% Horse Serum, heat inactivated, New Zealand originThermo FisherCatalog #26050088
1x B-27 SupplementGibco - Thermo FischerCatalog #17504044
1x GlutaMAX™ SupplementThermo Fisher ScientificCatalog #35050061
36 mM Glucose SolutionThermo FisherCatalog #A2494001
50 U/mL Penicillin-Streptomycin (5,000 U/mL)Thermo FisherCatalog #15070063
1.25 ug/mL Fungizone (Amphotericin B)Thermo Fisher ScientificCatalog #15290018
Membrane Inserts
Millipore Cell Culture Inserts (0.4 um pore size, 30 mm diameter)Merck MilliporeSigma (Sigma-Aldrich)Catalog #PICM0RG50
Tissue Chopper
Equipment
McIllwain Tissue Chopper
NAME
Cavey Laboratory Engineering
BRAND
51350
SKU
Protocol materials
Cytosine β-D-arabinofuranoside (AraC)Merck MilliporeSigma (Sigma-Aldrich)Catalog #C1768
Step 11
HBSS, no calcium, no magnesium, no phenol redThermo Fisher ScientificCatalog #14175095
Step 2
Culture preparation
Culture preparation
Prepare a 6-well culture plate with Millipore 0.4 µm culture inserts placed on top of 1 mL of organotypic medium (see Materials) per well. Incubate it at 37 °C and 5% CO2 to warm up.
Note
It is important to minimize air bubbles between the medium and the membrane insert, as this will prevent access of nutrients to the slice.
Prepare a p100 and p60 petri dishes with 25 mL and 5 mL, respectively, ofHBSS, no calcium, no magnesium, no phenol redThermo Fisher ScientificCatalog #14175095 and keep them On ice .
Prepare the McIllwain Tissue Chopper (see Materials) with a fresh blade and clean holder plates.
Sacrifice P5-P7 rat pups by rapid decapitation and extract the brain without damaging the cortex.
Put the brain on a filter paper (to remove excess liquid) and cut through the midline to separate the cerebral hemispheres. Remove the midbrain, separating it from the cortex with a scalpel.
Place both hemispheres in the same holder plate, with the medial face downward. Hold the plate with both hemispheres perpendicular to the blade (coronal sections) and section the brain at 350 um width.
Pour the sliced brain carefully into the p100 petri dish with HBSS On ice . You may use a Pasteur pipette to moisten the brain and slide it more easily.
Separate slices under the magnification microscope with a couple of spatulas (preferentially plastic-ones, which can be custom crafted cutting the back of a Pasteur pipette). Use a blade and clean cuts to remove unwanted regions (such as hippocampus or SVZ). Transfer the slices into the p60 petri dish (you can use an inverted glass pipette with a suction rubber bulb).
Note
It is important to remove the subventricular zone (SVZ) and the meninges with the scalpel to avoid cell proliferation.
Under the laminar flow hood, transfer the selected slices (undamaged slices from the preferred rostro-caudal levels) to preheated culture inserts with medium (Step 1). Each insert can only hold two cortico-striatal slices. Remove excess dissection buffer from the top of the insert with a sterile tip.
Note
Leaving too much HBSS solution on top of the insert will eventually dilute the feeding medium.
From now on, the cultures should always be handled in sterility.
Incubate the culture at 37 °C and 5% CO2.
Expected result
Typically, 4-6 cortico-striatal slices can be obtained from each hemisphere
Maintenance and AAV-infection
Maintenance and AAV-infection
At DIV1 (24h after culture preparation), replace the feeding medium with 1 mL of the same medium but adding antimitoticCytosine β-D-arabinofuranoside (AraC)Merck MilliporeSigma (Sigma-Aldrich)Catalog #C1768 4,4 um per well.
Expected result
AraC will prevent the overproliferation of astrocytes, minimizing the formation of a glial scar on top of the slice.
At DIV3, replace the AraC-containing medium with fresh medium. Then add 1 uL of the virus of choice (in our case, AAV-Ncan-GFP 1012 vg/mL) over the slice.
Safety information
AAVs have to be manipulated in BSL1 facilities.
Note
To improve spatial precision (e.g. when infecting a selected region from the slice), AAV infection can be done with a micromanipulator and a glass pipette.
To maintain the culture, change medium three times a week (1 mL per well).
Expected result
Slices will flatten overtime, and become more transparent. Cortical regions will have more cell density than striatal regions.
AAV expression become visible aprox. at DIV10, with maximum expression from DIV14 onwards (it depends on AAV serotype and promoter).
Imaging
Imaging
Imaging can be performed in two ways:
- IN-PLATE: The insert is not removed from the plate, and the imaging is performed through the plastic. This is usually done in widefield setups, with low magnification. It is not optimal in terms of resolution, but it is very useful for repeated imaging (longitudinal studies) without compromising sterility, since the plate lid is not removed during the imaging process.
- OFF-PLATE: The insert is removed from the plate and placed in a smaller petri-dish or similar holding chamber. Imaging is usually done from above, in upright microscopes (objective on-top), with water-based objectives. Since CO2 is usually not available, a buffer medium with HEPES is recommended. Phenol-red should be avoided as well, since it interferes with fluorescence. This type of configuration is typically used for confocal or 2-photon imaging of the slices. Repeated imaging can be done but special attention should be paid to ensure cleanliness during the procedure (clean objective with 70% EtOH, change medium with fresh antibiotic/antimitotic immediately after imaging, keep an eye for contamination over the following days).