How to Extract CSF-Like Fluid from Choroid Plexus Organoids
The STEMdiffâ„¢ Choroid Plexus Differentiation Kit efficiently and reproducibly generates human pluripotent stem cell-derived choroid plexus organoids featuring cystic structures containing fluid resembling cerebrospinal fluid (CSF). This organoid model permits access to CSF-like fluid without requiring invasive lumbar puncture procedures, and maintains a blood-free sample. Extracting the fluid from these organoids presents a valuable tool for disease-related biomarker discovery and blood-CSF permeability assays. The following protocol describes two options for the extraction of CSF-like fluid from choroid plexus organoids, and was adapted from a previously published protocol developed by Pellegrini et al1.
Table 1. Comparison of CSF-like Fluid Extraction Methods
Protocol Option 1: Syringe Extraction
This method works best with larger choroid plexus organoids (> 1 mm in diameter). Expected extracted volumes are 100 - 1000 µL per organoid.
Materials
- Syringe (1 mL or 3 mL) e.g. (Covidien Catalog #8881501400 or #8881513934)
- Needle (28G or smaller) e.g. (BD Eclipseâ„¢ Catalog #305757)
- 1.7 mL microcentrifuge tubes (e.g. Catalog #38089)
- D-PBS (Without Ca++ and Mg++) (Catalog #37350)
- Pipettors (e.g. Corning® Lambda™ Plus Pipettor, Catalog #38058 and Catalog #38059)
- Pipette tips (e.g. Corning® Filtered Pipette Tips, Catalog #38031 and Catalog #38032)
Procedure
- Transfer a single ChP organoids using a cut P1000 pipette tip (or sterile spatula) to an appropriate vessel to wash (for small to medium ChP organoids these can be transferred to a 12-well plate, for large ChP organoids these can be transferred to a 6-well plate).
- Rinse ChP organoid with enough D-PBS to cover the organoid (0.5 to 3 mL depending on vessel and organoid size).
- Perform wash a minimum of three times to remove excess media, immediately removing wash each time. Note: perform wash steps gently to prevent popping of the ChP organoid cyst.
- Use a 200 μL or 1000 μL pipette to remove as much PBS as possible to prevent dilution of the CSF-like fluid.
- Use a syringe + needle assembly to poke the choroid plexus organoid and slowly extract CSF-like fluid. Aim for the middle of the cysts if possible. Holding the tube at eye-level can make this easier.
- Expel CSF-like fluid into a new microcentrifuge tube and store at -20°C to -80°C for subsequent analysis.
- Note: The total volume of CSF-like fluid extracted from each organoid can range from 10 - 1000 µL, depending on the method.
- The deflated choroid plexus organoid can be used as a control in subsequent experiments.
Protocol Option 2: Centrifugation Extraction
This method works best with smaller choroid plexus organoids (< 1 mm in diameter) mm or when multiple ChP organoids need to be processed per sample. It’s not recommended to use this method with organoid cyst diameter greater than 1 mm due to dimensions of the Amicon Ultra-0.5 mL Centrifugal Filter. Expected extracted volumes are 10 - 500 µL per organoid.
Materials
- Amicon® Ultra-0.5 Centrifugal Filter Unit 100 kDa cut-off (Amicon Catalog #UFC5100)
- 1.7 mL microcentrifuge tubes (e.g. Catalog #38089)
- D-PBS (Without Ca++ and Mg++) (Catalog #37350)
- Pipettors (e.g. Corning® Lambda™ Plus Pipettor, Catalog #38058 and Catalog #38059)
- Pipette tips (e.g. Corning® Filtered Pipette Tips, Catalog #38031 and Catalog #38032)
Procedure
- Transfer ChP organoids using a cut p1000 pipette tip (or sterile spatula) to an appropriate vessel to wash
- Note: Small to medium ChP organoids can be transferred to a 12-well plate, and large ChP organoids can be transferred to a 6-well plate.
- Rinse ChP organoid with enough D-PBS to cover the organoid (0.5 to 3 mL depending on vessel and organoid size).
- Perform wash a minimum of three times to remove excess media, immediately removing wash each time. Note: perform wash steps gently to prevent popping of the ChP organoid cyst.
- Using a cut wide-bore pipette tip, transfer choroid plexus organoid(s) to a Amicon Ultra-0.5 mL Centrifugal Filter. Remove as much excess PBS as possible to prevent dilution of the CSF-like fluid.
Note: 2 to 3 organoids may be loaded into one tube. More than this may clog the filter.
- Centrifuge the tube at 10,000g for 2 minutes.
- CSF-like fluid is collected as the flow-through and can be store at -20°C to -80°C for subsequent analysis. The deflated choroid plexus organoid can be used as a control in subsequent experiments.
Analysis of CSF-Like Fluid
CSF-like fluid may be analyzed by SDS-PAGE and Western blotting using the following antibodies:
- Rabbit anti-IGF-2 (Abcam ab204567) 1:250
- Goat Anti-Rabbit IgG H&L (HRP) (ab205718) (1:2000)
- HRP-Clusterin (Biolegend 848703) 1:250
CSF-like fluid may also be analyzed using other methods such as mass spectrometry, as demonstrated in Pellegrini et al1.
Request Pricing
Thank you for your interest in this product. Please provide us with your contact information and your local representative will contact you with a customized quote. Where appropriate, they can also assist you with a(n):
Estimated delivery time for your area
Product sample or exclusive offer
In-lab demonstration
