Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

The overall goal of this procedure is to embed stem cells into alginate, hydrogel capsules safely and effectively, and to culture them within the capsules to control For aggregation and microenvironment, this is accomplished by first mixing a filtered stem cell suspension and a sodium alginate solution. The mixture is then transferred to a syringe and a coaxial nozzle is installed on the syringe before setting them on a syringe pump. Next, the droplets are prepared by expelling the cell suspension with co flowing nitrogen gas, followed by collection and calcium chloride solution.

The capsules are then treated with poly L lysine to prevent cellular leakage and EDTA to fluidized the inner capsules before culturing the encapsulated cells in a rotary shaking condition. Finally, the cells are collected from the capsules by EDTA and physical destruction of the membrane. Ultimately, alginate encapsulation of pluripotent stem cells prevents the cells from aggregating excessively and limits the sheer stress experienced by the cells in suspension culture.

This method can help to answer the key questions in the biological and biomedical field, such as large expansion of cells transplantation and researching on stem cell niche. Begin cell encapsulation by collecting the cells as described in the text protocol following centrifugation at 160 times G for three minutes. Wash the induced pluripotent stem or IPS cell pellet by Resus, suspending in heaps buffered saline and centrifugation thrice after resus, suspending the cells and heaps buffered saline.

Filter the cell suspension through a 40 micron cell strainer in order to remove large aggregates, which could otherwise clog the nozzle. Next, mix two milliliters of filtered cell suspension and three milliliters of 5%sodium alginate solution. To obtain a five milliliter cell suspension in 3%sodium alginate cell density is DIR more than 1 million cells per milliliter.

After collecting the cell suspension into a five milliliter syringe, install a coaxial nozzle on the syringe and set them on a syringe pump emit nitrogen gas flow through the outer needle of the coaxial nozzle and expel the cell suspension through the inner nozzle. Collect droplets into 250 milliliters of 0.5%calcium chloride solution, and wait 10 to 20 minutes for dation while stirring at 60 to 90 RPM. After washing with heaps buffered saline, collected capsules are utilized as naked capsules to treat the capsules incubate calcium alginate capsules in 0.05%weight per volume.

Poly L lysine solution for five minutes at 37 degrees Celsius and 5%carbon dioxide with shaking. After collecting the capsules into a tube and washing with heaps buffered saline, incubate them in DMEM containing 10%FBS. In order to neutralize the electrical charge on their surface, utilize the resulting capsules as coated capsules to generate hollow capsules.

Incubate the capsules in heaps buffered saline containing 10 millimolar EDTA for five minutes. The EDTA treated capsules are utilized as hollow capsules. Incubate the encapsulated cells at 75 RPM with shaking at 37 degrees Celsius and 5%carbon dioxide for 10 days following incubation, collect and incubate the capsules in 10 millimolar EDTA at 37 degrees Celsius and 5%carbon dioxide for 10 minutes.

Collect the cells from the alginate capsules without poly l lycine treatment by centrifugation at 1000 times G for three minutes. If alginate capsules are treated with poly L lysine, break the alginate poly L lysine membrane by pipetting up and down around 10 times with a needle attached to a syringe. Needle diameter should be lower than capsule size, and 25 gauge needles are used in this experiment.

After centrifugation at 1000 to 5, 000 times G for five minutes, collect the cell pellet strictly from broken alginate poly L lycine membranes If mRNA samples are collected from cells, note that remaining alginate poly L lycine membranes may prevent mRNA purification as visualized by a high speed camera. The expelled alginate solution forms a spherical shape immediately after expulsion. If the suspension is expelled with a nitrogen flow rate lower than one liter per minute, the size of the droplets is uniform.

However, if the nitrogen flow is higher than one liter per minute, the droplet breaks down and the size of the droplets becomes heterogeneous. Given this, it is difficult to prepare droplets smaller than 500 microns using this method. In the gelling process, sodium alginate droplets form spherical shaped calcium alginate capsules in calcium chloride solution.

If the concentration of sodium alginate is high enough, however, low concentration of sodium alginate causes nons, spherical and nonoo shape of capsules, which have a problem in the coding process. Even if the sodium alginate concentration is not enough, spherical capsules can be formed by increasing the calcium concentration. However, around 50 millimolar of calcium chloride is appropriate for encapsulation because too high, a concentration of calcium chloride decreases cellular growth and viability.

Encapsulated cells can grow in the capsules, but cellular leakage can occur in capsules without the alginate poly L lysine coating. In the case of mouse IPS cells, the cells form disc shaped aggregates in each calcium alginate capsule, whereas the cells clump together and form single spherical aggregates in each hollow capsule. After watching this video, you should have a good understanding of how to encapsulate and culture cells into size control.

Hydrogen captures without any special equipment, no organic absorbent.