Fabrication of Amyloid-Beta-Releasing Polymer Microbeads for Modeling Alzheimer's Disease

Abstract

Source: Almari, B., et al. Fabrication of Amyloid-β-Secreting Alginate Microbeads for Use in Modelling Alzheimer's Disease. J. Vis. Exp. (2019).

This video demonstrates a method for fabricating polymer microbeads that encapsulate neurotoxic amyloid-beta-secreting cells. The cell-polymer suspension is processed through a microbead fabrication device, with polymer gelation achieved via calcium cross-linking, forming spherical microbeads around the cells. The polymer allows cell proliferation while allowing controlled, long-term amyloid-beta release, simulating its accumulation in Alzheimer's disease.

Protocol

1. Preparations

1. Prepare 100 mL of ~4% (w/v) alginate stock solution.
   1. Weigh 4.4 g of alginate sodium salt and add the dry powder to 100 mL of HEPES-buffered saline (HBS, 20 mM hydroxyethylpiperazine ethane sulfonic acid [HEPES] (0.477 g) with 150 mM NaCl (0.877 g) in deionized water allowing hydration.
   2. Use a magnetic stirrer at ~500 revolutions per minute (rpm) and heat up to 50 °C to allow complete alginate hydration.
      NOTE: The alginate may need one or two hours to be completely hydrated. To save time, alginate solution can be made up a day in advance of the encapsulation protocol and stored at 4 °C until the next day. When alginate has been refrigerated, heat gently to 37 °C using a water bath or a hot plate and agitate with a magnetic stirrer immediately before use.
   3. Sterile filter the alginate solution using a 0.22 µm pore polyethersulfone (PES) filter before use. Filtering is recommended at 37 °C to allow easy flow of alginate. The viscosity will increase if the temperature is allowed to drop.
2. Prepare 1,000 mL of 0.5 M CaCl₂.
   1. Weigh 55.49 g of anhydrous CaCl₂ and dissolve in 1,000 mL of HBS (20 mM HEPES and 150 mM NaCl in 1,000 mL of deionized water).
   2. Sterile filter using a 0.22 µm pore PES filter.
3. Prepare 100 mL of dissolution mix.
   1. Prepare a 100 mM HEPES (23.83 g) and 500 mM trisodium citrate dihydrate (147.05 g) solution in phosphate-buffered saline.
   2. Adjust to pH 7.4 using NaOH or HCl.

2. Setting up the encapsulation system

1. Clean the laminar flow hood.
   1. Sterilize the laminar flow hood containing the encapsulator using UV exposure followed by spraying with 70% v/v ethanol solution.
   2. Flush the encapsulator system with 10 mL of 70% v/v ethanol solution followed by 10 mL of sterile deionized water.
   3. Attach the 300 µm nozzle and repeat step 2.1.2.
   4. Spray the bottle containing the filtered alginate solution, the bottle containing the filtered calcium chloride solution and any tools, equipment and empty culture plates that will be used with 70% v/v ethanol solution and place them into the laminar flow hood. Prior to use, switch on a UV light again for 30 min to thoroughly sterilize.
   5. Prepare a waste beaker filled with a biological grade disinfectant solution and place the beaker into the hood.
   6. Fill a beaker with 100 mL of sterile CaCl₂ (aqueous) solution and add in a magnetic stirrer. Set the stirring speed to 100 rpm. Place the beaker on a magnetic platform at a height of 18 cm from the tip of the nozzle. This beaker will be used to collect alginate beads and allow their gelation.
2. Prepare cells for encapsulation.
   1. Remove cells from the incubator and detach from the near-confluent flask using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution and incubate at 37 °C for 5-10 min.
   2. Isolate a sample for estimation of cell density and then centrifuge the remaining cells at 1,000 rpm for 5 min to obtain a cell pellet.
   3. Resuspend the pellet in HBS (20 mM HEPES and 150 mM NaCl) to double the final desired cell concentration (e.g., for a final concentration of 1.5 x 10⁶ cells/mL, resuspend the cells in HBS to achieve a concentration of 3 x 10⁶ cells/mL).
   4. In a 50 mL centrifuge tube, mix the cell suspension in a 1:1 ratio with ~4% (w/v) alginate solution to obtain a final suspension containing the desired cell concentration (e.g., 1.5 x 10⁶ cells/mL) in a ~2% (w/v) alginate solution.
3. Set the encapsulation parameters.
   1. Set the speed of the encapsulator machine to the maximum extrusion speed (8.9 mL/min), the voltage to 1.0 kV and the frequency to 5,500 Hz.
      NOTE: These parameters were previously optimized to obtain microbeads of 550 µm in diameter.

3. Fabrication

1. Fabricate the microbeads.
   1. In a 20 mL syringe, load 5 mL of the cell-alginate suspension and attach a syringe to the encapsulator.
   2. Start the encapsulator by activating the flow which will push the cell-alginate suspension through the feeder. A stream of droplets will be extruded through the nozzle.
   3. Collect the first 1 mL in the waste beaker to avoid the initial non-uniform stream.
   4. Continue to run the remaining 4 mL allowing the droplets to fall into the CaCl₂ gelation bath. Each milliliter can be run separately (but successively) and collected in four different gelation baths if required.
      NOTE: Upon contact with the gelation bath, the alginate in the droplets will instantly cross-link with the calcium ions in the gelation bath and form spherical microbeads.
   5. After one minute, remove the gelation beaker from the magnetic platform and allow the microbeads to sit for a further 4 min without agitation (time necessary to allow complete gelation across the microbeads at room temperature).
2. Retrieve microbeads.
   1. Remove any large alginate debris or artefacts using a pair of sterile tweezers, and then use a sterile plastic pipette to transfer to a 74 µm mesh filter to retrieve the microbeads from the gelation bath.
   2. Transfer the microbeads into a centrifuge tube using the appropriate culture medium and allow them to equilibrate for 5 min in the cell culture medium.
   3. Transfer to a flask, plate or Petri dish for incubation and further experiments.
      NOTE: Gelation bath should not be reused.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 um PES filter	Merck, UK	SLGP033RS
15mm Netwell insert 74 um mesh filter	Constar, usa	#3477
Alginic acid sodium salt from brown algae	Sigma-Aldrich,uk	A0682
Calcium chloride	Sigma-Aldrich,uk	C1016
CellTiter96 AQueous One Solution cell proliferation assay	Promega, USA	G3580
Encapsulator	Inotech	IE-50	serial no. 05.002.01-2005
HEPES	Sigma-Aldrich,uk	H4024
Phosphate buffered saline	Sigma-Aldrich,uk	D1408
Sodium chloride	Sigma-Aldrich,uk	433209
Trispdium citrate dihydrate	Sigma-Aldrich,uk	W302600-K
Trypsin-EDTA solution	Sigma-Aldrich,uk	T4049