This protocol explains fabrication of microbeads, which will be used to help control the rate of release and the amount of amyloid which is a protein of interest. The microbeads will immobilize cells in a suitable biomaterial and it will shelter them from their surrounding environment, as well as allowing them to exchange byproducts and nutrients with their surrounding environment. Encapsulating cells using this technique ensures tight control of microbead size as well as cell number, and we do this by adjusting various fabrication parameters.
So encapsulating the cells described in this protocol will give us more of a chronic release of amyloid to use in both in vitro and in vivo systems, and the idea would be this would give us a better understanding of the mechanisms of disease and also allow us to test new treatments. This method can be used to formulate controlled system and study the release of any biomolecules for many cell types, whether alone or in combination. To begin, remove a near-confluent flask from the incubator.
Treat the cells with 0.25%trypsin-EDTA solution and incubate at 37 C for five to 10 minutes to detach the cells. After adding DMEM/F12 medium, collect the cells into a 50 milliliter tube. Centrifuge the cells at 1000 RPM for five minutes.
Remove the supernatant and re-suspend the pellet in HEPES buffered saline to double the final desired cell concentration. In a 50 milliliter centrifuge tube, mix this cell suspension in a one to one ratio with 4%weight and volume alginate solution to obtain a final suspension containing the desired the cell concentration in a 2%weight and volume alginate solution. To set the encapsulation parameters, set the speed of the encapsulator machine to the maximum extrusion speed, and then set the voltage and the frequency.
To fabricate the microbeads, in a 20 milliliter syringe, load five milliliters of the cell-alginate suspension and attach a syringe to the encapsulator. To start the encapsulator, activate the flow which will push the cell-alginate suspension through the feeder, and a stream of droplets will be extruded through the nozzle. Collect the first one milliliter in the waste beaker to avoid the initial non-uniform stream.
Then continue to run the remaining four milliliters allowing the droplets to fall into the calcium chloride gelation bath. Upon contact with the gelation bath, the alginate in the droplets instantly cross-links with the calcium ions in the gelation bath, forming spherical microbeads. After one minute, remove the gelation beaker from the magnetic platform and allow the microbeads to rest for a further four minutes without agitation to complete their gelation at room temperature.
To retrieve the microbeads, first use a pair of sterile tweezers to remove any large alginate debris or artifacts. After cutting the end of a plastic pipette, use it to transfer the microbeads from the gelation bath to a 74 micrometer mesh filter held over a waste beaker. To ensure success, we always cut the end of a plastic pipette to avoid damaging the microbeads and we do this every time we transfer beads from one vessel to another after encapsulation.
Invert the mesh filter over a centrifuge tube. Pipette the appropriate culture medium over it to wash the beads down the tube and allow them to equilibrate in that medium for five minutes. Then transfer them to a flask previously filled with the appropriate medium for incubation and further experiments.
To assess the cell viability after encapsulation and culture, add dissolution mix to gently disrupt the microbeads and release the encapsulated cells. Incubate the cells in a cell culture incubator supplemented with 5%carbon dioxide at 37 C for 10 minutes with gentle agitation. Estimate cell viability of these cells by staining them with trypan blue and using a hemocytometer chamber.
To assess microbead stability, measure the average diameter for a sample from each microbead population over a time course using a microscope and imaging software. After preparation, uniform and spherical alginate microbeads were successfully generated using this protocol. After one day in standard cell culture conditions, encapsulated 7PA2 cells were evenly distributed in microbeads.
When 7PA2 cell proliferation was tested using a MTS assay, there was no significant difference between the behavior of 7PA2 cells grown with or without alginate over a seven day period. Conditioned media analyzed from 2D and 3D cultures of 7PA2 cells revealed a constant increase in amyloid-beta 1-42 levels in both cultures. The rate of release of amyloid-beta 1-42 from microbeads, or 3D culture, is similar in profile to that released from the 2D culture.
7PA2 cells encapsulated in alginate microbeads can be effectively used for the sustained release of amyloid-beta. Microbeads for engraftment in the rat brain must be small enough to be embedded without creating a large lesion. Implanting a millimeter-scaled bead within the brain for in vivo purposes would not work, whereas a microbead fabricated using this protocol has a suitable size for insertion within the hippocampus of the rat.
To make sure we've got an even distribution of cells in final product, it's important to thoroughly mix the cells in the cell alginate suspension, and this guarantees uniform release of amyloid from each microbead.
