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

Summary

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Abstract

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N₂ gas. The controllable capsule diameter must be larger than 500 µm because too high a flow rate of N₂ gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl₂ causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

Introduction

Induced pluripotent stem cells (iPS cells) are currently the source of intense research due to their role in regenerative medicine. However, huge amounts of cells are required for tissue regeneration. For instance approximately one billion pancreatic cells required for a type 1 diabetic patient¹. However, conventional dish culture is only able to obtain 1 × 10⁵ cells/cm², thus requiring 1 m² of culture area to obtain enough stem cell-derived pancreatic cells to treat a type 1 diabetic patient. The development of a system for the mass-culture of pluripotent stem cells, such as microcarrier² and suspension culture is therefore required for regenerative medicine. Suspension culture represents a promising method of mass culture but controlling the aggregation of cells is challenging in direct suspension cultures of human iPS cells³. Indeed, suspended cells are exposed to shear stress, which causes cell damage³ or differentiation⁴.

Research into hydrogel-based encapsulation has been conducted to solve problems associated with suspension culture. In hydrogel capsules, cells are protected from the flow of the medium. Previous reports have documented the use of various types of hydrogel, including agarose⁵, PEG⁶, and alginate (Alg), for cellular encapsulation. Alg-Ca hydrogel is one of the most useful hydrogels for cell encapsulation because Alg−Ca hydrogel is formed immediately after dropping alginate solution into a CaCl₂ solution and is also readily digested by enzymes or chelating reagents.

Here, we have established a stable alginate encapsulation process for iPS cells using a co-axial nozzle. By using N₂ gas flow for forming droplets, it is possible to encapsulate cells into uniform capsules without the need for other reagents such as oil. In this method, the flow rate of N₂ and concentration of both CaCl₂ and alginate are the major operating conditions affecting the size, shape, and uniformity of capsules. This report demonstrates the optimization of these operating conditions through the use of a hi-speed camera and a microscope.

Protocol

1. Preparing Materials

1. Prepare 10 mM HEPES buffer. Adjust the pH to 7.0 at RT and add NaCl to 0.9%.
2. Prepare 5% alginate solution and 10 mM EDTA solution by mixing HEPES-buffered saline prepared in 1.1. Adjust the pH to 7.0 at RT.
3. Autoclave the reagents (1.1, 1.2) for 20 min at 121 °C.
4. Prepare gelatin-coated 60 mm dishes layered with 1.2 − 2.0 × 10⁶ mouse embryonic fibroblast (MEF) cells, which are treated as feeder cells by incubation within DMEM containing 10% ES-qualified FBS and 10 µg/ml of mitomycin C for 90 - 120 min.
5. Maintain mouse induced pluripotent stem (iPS) cells on the dish with the feeder cells in the presence of 5 ml DMEM high glucose containing 20% of ES-qualified FBS, 50 µM of 2-mercaptoethanol, non-essential amino acid, and antibiotics (100 Units/ml of Penicillin G Sodium, 100 µg/ml of streptomycin sulfate, and 250 ng/ml of amphotericin B).
6. Seed 4 × 10⁵ iPS cells on a gelatin-coated 60 mm dish and incubate them at 37 °C and 5% CO₂. Change the culture medium every day during incubation. After 3 – 4 days of culture, trypsinize cells for 1 min with 500 µl of 0.05% Trypsin containing 0.02% EDTA at 37 °C and 5% CO₂. After that, detach cells by tapping the culture dish ten times.
7. Add 2.5 ml of DMEM with 10% of ES-qualified FBS and antibiotics (following dissociation mouse iPS cells into single cells by pipetting with 1,000 µl micropipette three times) centrifuge cell suspension at 160 × g for 3 min and remove supernatant and count the cells. Following cell counting, reseed the collected cells on a gelatin-coated dish and incubate for 30 min to isolate the iPS cells from the MEF cells. After counting of the cells (usually 1 – 3 × 10⁶ cells/dish can be collected), reseed 4 × 10⁵ iPS cells on the dish.

2. Cell Encapsulation into Alginate Hydrogel Capsules

1. Collect the cells by the same procedure described in 1.5 and 1.6. Following centrifugation at 160 × g for 3 min, wash the iPS cell pellet with HEPES-buffered saline thrice. After re-suspending the cells in HEPES-buffered saline, filter the cell suspension through a 40 µm cell strainer in order to remove large aggregates, which could otherwise clog the nozzle.
2. Mix 2 ml of cell suspension within HEPES-buffered saline and 3 ml of 5% Alg-Na solution. Finally 5 ml of cell suspension within 3% Alg-Na solution is obtained. Cell density is desirably more than 10⁶ cells/ml.
3. After collecting the cell suspension into a 5 ml syringe, install a co-axial nozzle (Figure 1A, B) on the syringe and set them on syringe pump. Emit N₂ gas flow (lower than 1L/min) through the outer needle of the co-axial nozzle and expel the cell suspension through the inner nozzle.
4. Collect droplets into 250 ml of 0.5% CaCl₂ solution and wait 10 – 20 min for gelation while stirring at 60-90 rpm.

3. Treatment of Alginate Capsules (optional)

1. Incubate Alg-Ca capsules in 0.05 % (w/v) poly-l-lysine (PLL) solution for 5 min at 37 °C and 5% CO₂.
2. After washing the capsules with HEPES-buffered saline, incubate them in DMEM containing 10% FBS in order to neutralize the electrical charge on their surface. Utilize them as coated capsules (Figure 1C).
3. Incubate the capsules in HEPES-buffered saline containing 10 mM EDTA for 5 min. The EDTA-treated capsules are utilized as hollow capsules (Figure 1C).

4. Culture and Collect Cells in Alginate Capsules

1. Incubate the encapsulated cells at 75 rpm with shaking at 37 °C and 5% CO₂ for 10 days.
2. Collect and incubate the capsules in 10 mM EDTA at 37 °C and 5% CO₂ for 10 min. Collect cells from the alginate capsules without PLL treatment by centrifugation at 1,000 × g for 3 min.
3. If alginate capsules are treated with PLL, break the Alg-PLL membrane by pipetting up and down around ten times with a needle attached to a syringe and centrifuge it at 1,000 - 5,000 × g for 5 min. Needle diameter should be lower than capsule size and 25 G (260 µm) needles are used in this experiment (600 µm)
4. After centrifugation, collect cell pellet strictly from broken Alg-PLL membranes if you want to collect mRNA samples from cells. Remaining Alg-PLL membranes possibly prevent mRNA purification.

Results

At protocol 2.5, the expelled alginate solution forms a spherical shape immediately after expulsion (Figure 2A – H). If the suspension is expelled with a N₂ flow rate lower than 1L/min, the size of the droplets is uniform (Figure 2I). However, if the N₂ flow is higher than 1L/min, the droplet breaks down (Figure 2G, white arrowed) and the size of the droplets becomes heterogeneous (Figure 2J). Given this, it is difficult to pr...

Discussion

Encapsulation culture can be compared with direct suspension cultures. Suspension culture is a simpler method to obtain large quantities of pluripotent stem cells than encapsulation methods. However, controlling the aggregation of cells in suspension culture is still challenging. In encapsulation method, cellular aggregation is limited in capsules and can therefore be well controlled. A previous publication showed that encapsulated cells formed aggregates of uniform size, whereas large cell clumps appeared in a free-susp...

Materials

Name	Company	Catalog Number	Comments
Mouse embryo fibroblast	Cell Biolabs	SNL 76/7
Mouse induced pluripotent stem cell	RIKEN Bio resorce centre	iPS-MEF-Ng-20D-17
DMEM high-glucose	GIBCO	11995
ES qualified  FBS	GIBCO	16141079
Antibacterial Antibiotics	GIBCO	15240
Nonessential Amino Acid	GIBCO	11140
2-mercaptoethanol	GIBCO	21985-023
ESGRO Leukemia Inhibitory Factor	Merck Millipore	ESG1107
Trypsin/EDTA	GIBCO	25300
26 G/16 G needle	Hoshiseido
10 ml Syringe	TERUMO	SS-10ESZ
Sodium  Chloride	Wako	191-01665
HEPES	SIGMA	H4034
Sodium Alginate	Wako	194-09955
Calcium Chloride	Wako	039-00475
Poly-L-lysine (MW = 15,000 - 30,000)	SIGMA	P7890
EDTA	DOJINDO	345-01865
Sylinge pump	AS ONE
Microscope	Olympus	IX71
Microscope	Leica	DM IRB
Hispeed camera	nac image technology	Memrecam HX-3