Recent development in cell encapsulations and their therapeutic applications

1. Introduction

With the increasing life expectancy over the past century due to better nutrition and medical advances, another problem arises. The increased life expectancy brought about new problems, one of them being chronic and degenerative disease such as degenerative bone disease, diabetes, Alzheimer's disease, gene related diseases and cancer to name a few. This new enemy does not have an absolute counter and many people fall victim to it every day. In 1933, Vincenzo Bisceglie discovered the first step of a possible solution through his experiments. In his experiment, he proposed that since his transplant of tumour cells into the host body were still surviving after a long period of time, the polymer structure that is used to encase the tumour cells showed an ability to prevent immunoresponse in the host body to foreign cells [1]. In 1964, Thomas Chang proposed the concept of ‘artificial cells’ for his idea of encapsulating cells to prevent the immunoresponse of the body [2]. This lead to a surge in encapsulation technology in the 70s and 80s where it is used to test islet cells to control diabetes in small animals [3]. Cell encapsulation is based on the concept of immobilizing the cells in a matrix which is surrounded by a semi permeable and biocompatible membrane. The membrane serves to protect the cell from immunoresponse and a pathway for nutrients in the body to enter the matrix. The matrix, on the other hand, serves as a scaffold to promote cell proliferation, provides mechanical strength and control the release of drugs from the matrix to the body (Fig. 1) [4].

2. Semi-permeable membrane in cell encapsulation

The membrane has one main function which is to prevent immunoresponse from the host body to the encapsulated cells. However, there are also other factors that should be considered when choosing the membrane, such as morphology, biocompatibility, degradability and mechanical strength. In this section, different factors of the membrane materials that have significant influence in the cell encapsulation are discussed.

2.1. Semi permeable membrane factors

The main function of the membrane is to prevent immunoresponse from the body reaching the cells in the encapsulation. The morphology of the membrane such as wall thickness, pore size distribution, surface structure and porosity will affect the transport mechanism of nutrient and drugs [5]. As the membrane must be selective in its permeability whereby only nutrients are able to diffuse past the membrane and block out all immunoresponse, the surface morphologywould be very important in determining the permeability. The permeability of the membrane determines what kind of therapeutic drugs are able to diffuse into the host body as different drugs have different sizes and the sizes of the drugs have to be taken into account for when choosing the permeability of the membrane. In addition, the membrane has to be biocompatible as it is in contact with the host body and the encapsulated cells. As the membrane is in contact with the host tissues, it should not cause any immunoresponse by the body to the encapsulated cells such as inflammatory response or tissue encapsulation. It should also be non-cytotoxic as there should not be any tissue or cell death caused by the encapsulated cells.

In another aspect, some membranes are degradable over time and this will lead to immunoresponse breaching the membrane to reach the matrix and then the cells. If this is not planned, it could lead to the failure of the encapsulated cells. However, in some cases, the encapsulated cells are meant to degrade over time so as to remove the requirement for another surgery to remove the encapsulated cells. It should be noted that when the membrane starts to degrade, it will lose mechanical strength and increased permeability of the membrane, hence, plans should be made if the membrane were to degrade. On top of the degradation behaviour, the mechanical strength is also considered as different sites of the body experience different amount of stress from daily activities, hence, a suitable membrane with the required mechanical strength is needed for the success of the implant. If the membrane was to break due to lack of mechanical strength, it will lead to the failure of the encapsulated cells as immunoresponse will be able to affect the cells.

2.2. Representative types of semi permeable membranes

The semi permeable membrane is made of many different materials and these materials are chosen to suit the function of the encapsulated cell for drug releases [6]. In general, there are three main types of materials used as semi permeable membranes, including hydrogels, thermoplastic polymers and non-polymeric materials. Hydrogels are made of hydrophilic polymers that are able to absorb water without dissolving [7], [8], [9], [10], [11], [12], [13]. They are used due to their high viscoelasticity and water content which is similar to biological tissues in a living organism [14], [15], [16]. The close resemblance makes hydrogels have a less inflammatory response in vivo compared to the rest. The permeability properties of hydrogels can be controlled by changing the amount of crosslinks within the hydrogels where a higher crosslinking will lead to higher perm-selectivity and lower diffusion rates [8], [17], [18], [19], [20], [21], [22], [23]. In addition to hydrogels, thermoplastic polymers are polymers that are able to change its physical state by altering its temperature. They are used as the membrane due to its superior physical and chemical properties compared to hydrogels. This makes them very durable in vivo and thus, be able to withstand the stress from the body and resist interactions with blood and immunoresponse. A popular thermoplastic used for the semi permeable membrane would be acrylonitrile vinyl chloride copolymer (PAN-PVC). However, even though they have good physical and chemical properties, they have a low permeability for water soluble molecules hence, it has a limited use in cell encapsulation membrane.

On the other hand, non-polymeric materials are also considered for the semi permeable membrane due to the ability to control its properties such as porosity which will allow the control of permeability and perm-selectivity and purity compared to polymeric materials. Silicon and titanium oxide are two of the most commonly used materials due to their high stability and high mechanical strength [24]. However, using non-polymeric materials may have implications on the viability of the cells as they usually have harsh treatment conditions for them to be used. An example would be that titanium oxide has to be sintered after using the drip-coating method for cell encapsulation and this temperature needed could easily destroy the encapsulated cells if it is higher than the survival temperature of the cells [25].

3. Matrix in cell encapsulation

The factors to consider when creating the matrix for cell encapsulation includes architecture, biocompatibility due to non-cytotoxic to the cells it cultivates and good membrane and host compatibility, cell types and cell proliferation. All these factors will be discussed in this section.

3.1. Matrix factors

The architecture of the matrix is important as it determines the porosity of the matrix for cell growth and for nutrient and waste transport. A highly porous matrix would promote cell growth as there is a lot more areas within the matrix for the cells to grow. It also determines the rate of nutrient and waste transport where a highly porous structure would increase the rate of transport. The architecture also determines the mechanical strength of the matrix as if the porosity increases, mechanical strength decreases, vice versa [26]. Therefore, if the encapsulated cells are meant to be placed in areas where high mechanical strength is needed, the porosity of the matrix has to be sacrificed. However, it should also be taken note that too high of a porosity may leave to aggregation of cells and this might cause the death of cells which are at the centre of the aggregation as they do not receive enough nutrients, hence, the porosity should be carefully determined. Lastly, the matrix's porosity can also serve as a second layer of immunoprotection after the semi permeable membrane, for example, hydrogel alginate [27], [28].

In addition to the architecture, good biocompatibility is important for the matrix as the matrix is in contact with the cells and potentially the host tissues as well if the semi permeable membrane were to break or fail. As the matrix is in contact with the cells, it has to be biocompatible and non-cytotoxic to the cells or it will cause cell death. Although the matrix is not in direct contact with the host tissues, there are instances where the semi permeable membrane may fail its function, either degrading or breaking leading to contact between the host tissue and the matrix. There can also be instances where the matrix would degrade over time, leading to some of its molecules diffusing into the host body, hence with these reasons, good biocompatibility and non-cytotoxicity is important [24], [29], [30]. Moreover, the type of cells that is encapsulated should also be taken into account. Some cells that are grown using a suspension method might not need a substrate to grow while anchorage dependant cells would require a substrate. Fibroblast secretes its own matrix to grow on and for such cells which are able to create their own environment to grow, might not need an extensively designed matrix, only a conducive environment for them to slowly create its own local environment. There are also cells which depend on neighbouring cells to proliferate and in these cases, the matrix may be required to include such cells [31]. Overall, the cell type would determine the type of matrix that is needed for cell encapsulation. As the cell proliferation determines the cell population over time, it will be a factor in controlling therapeutic rate and nutrient absorption rate. A high cell proliferation would lead to more nutrients needed to be absorbed by the encapsulation and this may result in the lack of nutrients to the cells within the encapsulation if the rate of transfer of nutrients from the host body through the semi permeable membrane and matrix to the encapsulation is slower than the rate of consumption of the nutrients by the cells, leading to cell death [30].

3.2. Materials of matrix

The Matrix uses mainly two different types of materials, synthetic and naturally derived [24], [29], [32]. Most of these polymers are able to form hydrogels. Hydrogels are polymers that swell in water without dissolving due to their three-dimensional networks and are usually used for the matrix due to its properties [33]. Hydrogels are highly porous and they have low protein adsorption due to the low interfacial energy with surrounding body fluids [34]. As natural hydrogels structures are very similar to the extracellular matrix of humans and they can be processed using mild conditions which are suitable for cell survival [35], hence, they are widely used a the matrix for encapsulated cells. The immobilization of cells by a hydrogel is by first suspending the cells in a solution based hydrogel precursor. It is then cured uses different methods based on which hydrogel is used. The different methods used to form hydrogels are illustrated in Fig. 2 [32]. It should be noted that each of the processes especially the curing process are suitable for the cells that are in suspension as if the process could cause damage to the cell, the encapsulated cells may not be able to carry out its functions.

Particularly, thermogels form cross links when there is a change in temperature [12], [13], [36]. In general, there are two types of thermogels, one that forms a gel when heated above a certain temperature called transition temperature like elastin and some collagens and the other is when it is cooled to a certain temperature like gelatin and agarose. Thermogels that forms a gel when heated have a lower critical solution temperature (LCST) whereby the thermogel is miscible with water below this temperature. The same goes for thermogels that gel upon cooling, they have an upper critical solution temperature (UCST) [19], [37], [38]. There are three types of interactions between the polymer and water and that by thermo curing it, the polymer-water interactions become the least favourable and hence, a gel is formed. For thermogel with an LCST, the change in entropy of water causes the gelling while in UCST, the change in enthalpy causes the reaction [39]. For thermogels that are suitable for cell encapsulation, they should have transition temperatures close to body temperature. In addition, ionic hydrogels are called polyelectrolytes and they are hydrogels that form cross-links when exposed to charged species [40]. Polyelectrolytes have a net charge on their polymer backbone and when combined with oppositely charged ions, they will form insoluble complexes [41], [42], [43], [44], [45], [46], [47]. Polyelectrolytes are soluble due to their charged backbone, however, when oppositely charged ions are introduced, there will be a shielding effect on the polymer backbone to the water molecules, rendering it insoluble [48]. Some polymers that are charged include alginate and carrageenan. Thermogels and polyelectrolytes form reversible cross-links, where under suitable condition, the crosslinking can be removed. However, hydrogels, which are cross-, linked through irradiation of ultra violet light (UV) form irreversible cross-links. These types of hydrogels contain photo-initiators that when irradiated by UV, form free radicals that will cause a chemical bond with other functional groups on the polymer.

Hydrogel is an excellent platform to achieve not only cell encapsulation and delivery but also therapeutic agent delivery. For example, Paclitaxel (PTX) and doxorubicin (DOX), tow common used chemotherapeutic drug were increased their localisation and sustained release by using a multiblock poly(ether ester urethane)s thermo-gels comprising poly(ε-caprolactone), poly(ethylene glycol), and poly(propylene glycol) in a nude mice model [49]. Such drug delivery system could be a very promoting approach to treat chemoresistant cancers [42], [50]. The therapeutic application of bioresponsive hydrogel are well summarized in another reviewer which include their potential applications in restorative therapies, biosensors, and drug-conveyance applications [51].

There are some cases where the matrix material can function as the semi permeable membrane and in such cases, there would not be a need to coat the encapsulated cells with another material. However, for the better interaction between the encapsulated cells and the host body, sometimes a change in the morphology would be much better to prevent immunoresponse from the body. For example, alginate is usually taken from seaweed and Pseudomonas and Aztobacter, two kinds of bacteria [52], [53], [54]. It is a negatively charged hydrogel with polysaccharide chains. Alginate is used as a matrix due to its easy cross-linking by positively charged divalent ions, resulting in a rapid sol-gel transformation with a high survival rate of encapsulated cells [55], [56], [57]. The permeability of alginate depends on the concentration of the added ions where higher concentrations lead to a denser structure with less permeability. This method is used to immobilize cells such as bovine adrenal chromaffin cells for the treatment of chronic pain. In addition, Agarose is another polysaccharide taken from red algae, which is much better than Alginate in terms of purity control and stability in vivo [58]. Agarose has a UCST and thus will form a gel when cooled [59], [60]. The gelling process is caused by the formation of a double helix which causes hydrogen bonds to be formed when agarose is cooled and the gelling temperature is dependent on the average molecular weight, the concentration of the polymers in the solution and by the structure that it forms [61], [62]. Due to agarose ability to change its gelling temperature by changing some parameters, it is widely used in the industry and has a long history of use in cell culture. The pore size of agarose is determined by the concentration of the polymer and the gelling temperature where a higher concentration leads to a smaller pore size [63] and a lower gelling temperature would lead to smaller pores [64]. Agarose is a non-biodegradable polymer only through agarase, hence, there is no degradable factor when using agarose as the matrix. In another aspect, chitosan is taken from chitin through partial deacetylation of the exoskeletons of shrimp and crabs [65], [66], [67]. It is a polysaccharide with N-acetyl-β-d-glucosamine chains in a semi-crystalline structure. In acidic conditions, the primary amine group on the chitosan becomes protonated which allows chitosan to be soluble and used in the layer-by-layer technique to from polyelectrolyte complexes with negatively charged polymers such as alginate and even with DNA [68], [69], [70]. The collagen matrix is another interesting material that contain not only collagen but also elastin and fibronectin [71], [72], [73]. Collagen is the major protein composition in this matrix, hence it is referred to as a collagen matrix. Collagen is the most common protein found in humans and it is synthesized naturally by fibroblast and osteoblast [74], [75], [76], [77]. During cell encapsulation, the cells are first suspended in a neutral solution of collagen before moving it into an incubator [73]. Collagen also contains a tripeptide (Arg-Gly-Asp) that is important for the interaction between a large number of cells and the matrix. Due to its nature, collagen is used to simulate the natural environment of an extra cellular matrix and it can take different forms such as soluble hydrogels, cross-linked with glutaraldehyde and collagen sponges. Collagen is degradable by collagenases and the rate of degradation can be regulated using enzymatic treatment or cross-linking [78].

4. Cell encapsulation techniques

There are many methods to encapsulate cells, this paper would discuss six different methods for cell encapsulation, mainly through extrusion, lithography, emulsion, microfluidics, bioprinting and using superhydrophobic surfaces. In this section, the recently reported techniques and approaches in cell encapsulation are summarized.

4.1. Extrusion

The most common way to encapsulate cells is through the extrusion method, also known as gravitational dripping [79]. The cells are first suspended in a hydrogel precursor and they are extruded through a small needle. The cells with the precursor will slowly grow in size as constant pressure is applied to extrude the solution until the droplet of cells and hydrogel precursor grows to a critical size and will fall due to gravity into an appropriate hardening bath that will harden the hydrogel precursor. The diameter of the encapsulation is due to the surface tension of the droplet, viscosity of the hydrogel precursor, flow rate and the diameter of the needle [80]. Surface tension affects the diameter as when the surface tension is low, the droplet will not be able to form properly and instead will be released like a jet [81]. Therefore, the decreasing surface tension of the droplet will lead to a decreasing encapsulation diameter. The higher the viscosity of the hydrogel, the larger the diameter of the encapsulation as when viscosity increases, the hydrogel will flow at a slower rate and will be able to form a much bigger droplet than at lower viscosity. This bigger droplet will result in a much bigger encapsulation. The flow rate of the hydrogel through the needle tip affects the diameter of the encapsulation where a higher flow rate will lead to a smaller encapsulation [82]. This is because at a higher flow rate, the droplet does not have enough time to coagulate to its maximum size before dropping into the hardening bath. The bigger the diameter of the needle, the bigger the size of the encapsulation as a bigger needle size would mean a larger area for the droplet to coagulate and thus, it will result in a larger droplet and larger encapsulation [83]. Lastly, the surface tension of the hardening bath can lead to a distortion in shape of the encapsulation. If the surface tension for the hardening bath is too high, when the droplet falls into the bath, it will distort the droplet into a tear shaped or result in a breaking of the droplet into irregular smaller sizes.

Extrusion method usually leads to a spherical encapsulation if there is an air gap between the needle tip and the hardening bath. However, if this air gap was to be removed, the encapsulation will form fibres instead of spheres and this is called wet spinning [84]. If smaller encapsulations are needed and the parameters mentioned above cannot be changed to allow smaller encapsulations, there are a few techniques that are called ‘breaking’ the jet techniques, which would allow smaller encapsulations. The techniques include coaxial air flow, vibrational encapsulator, the JetCutter and using a bio-electrospray approach etc.

Coaxial air flow method is by applying compressed gas to force the droplet to fall into the hardening bath rather than waiting for gravity to pull the droplet down [82], [85]. This method has been used to encapsulate IB3-1 cells inside alginate using a hardening bath of barium chloride and the method did not alter the viability of the encapsulated cells (Fig. 3) [86].

In addition, vibrational encapsulator uses a vibrating nozzle to break the droplets into smaller sized droplets [87]. By controlling the vibration of the nozzle in resonance with the Rayleigh instability, smaller droplets can be produced and the droplets have been reported to be more uniformed in size [88]. However, there is a limit to the maximum frequency of vibration whereby increasing the frequency does not decrease the size but increases it due to coagulation of the droplets. The flow rate of the hydrogel precursor also affects the droplet sizes where an increase flow rate decreases its size. The distance between the nozzle and the hardening bath will affect the geometry of the cell encapsulation whereby increasing the distance to a certain limit will elongate the cell encapsulation [89]. Similarly, the JetCutter uses the concept of rotating cutting wires to cut the liquid jet. It is usually used for highly viscous liquids as the coaxial air flow and vibrational encapsulator has not much effect on a highly viscous liquid. The encapsulated cell size depends on the diameter of the nozzle, the rotation frequency of the JetCutter and the number and diameter of the JetCutter wires [90]. The smaller the diameter of the nozzle, the smaller the encapsulated cell size. A higher frequency of rotation of the JetCutter, a larger number of wires and a larger diameter of the JetCutter wires will lead to a smaller encapsulated cell size. The JetCutter have been used by Schwinger et al. to encapsulate murine fibroblast inside alginate/poly-l-lysine layer by layer complexes and it has been reported that it did not result in the change of any properties [91].

Another interesting technique used for cell encapsulation is bio-electrospraying, which applies electrical potential as a way for liquid atomization [92], [93]. By applying an electrical potential across the tip of the nozzle, the droplets at the tip of the nozzle would react to the charged nozzle due to the potential difference applied and charges of the opposite signs would accumulate at the surface of the hydrogel precursor droplet in response to the electrical potential [94]. As similar charges would start to accumulate on the surface of the hydrogel precursor, a repulsive force between the similar charges on the hydrogel precursor would concentrate and would deform the shape of the droplet into a Taylor cone. Once a charge limit is reached when the repulsive force is stronger than the surface tension of the droplet at the nozzle, the droplet would be repelled off the nozzle into the hardening bath. This technique is used to disperse droplets into very small particles of diameters ranging from micrometres to nanometres. The encapsulated cell diameter is controlled by the flow rate of the hydrogel precursor and the electrical potential on the nozzle [95]. Recently, He et al. developed a novel coaxial electrospray technology for one-step generation of hundreds of thousands of microcapsules with a hydrogel shell of alginate and an aqueous liquid core of living cells (Fig. 4) [96]. The average murine embryonic stem (ES) cells encapsulated in the core is 50 μm with viability more than 92.3%. The encapsulated cells within the microcapsule can further proliferate to form a single ES cell aggregate with an approximate size of 128.9 μm after 7 days culture. The gene and protein expression analyses showed that ES cells cultured in the as-developed core–shell microcapsules have significantly higher pluripotency when compared with the cells cultured on the 2D substrate or in 3D alginate hydrogel microbeads. This result was further confirmed by their significantly higher differentiation capability into beating cardiomyocytes, as well as the higher expression level of cardiomyocyte specific gene markers. Due to its essential characteristics in wide availability, easiness to set up and operate, reusability, and high production rate, the novel coaxial electrospray technology show high potential in the mass production of ES cells and could facilitate the translation of the emerging pluripotent stem cell-based regenerative medicine into the clinic applications [96].

4.2. Lithography

There are two different type of lithography for cell encapsulation, soft lithography which involves the use of elastomeric materials, mainly organic and polymeric materials and photolithography, which uses photo-crosslinks to create moulds and stamps so as to replicate a shape for cell encapsulation (Fig. 5) [32]. Soft lithography is a technique that uses organic and polymer structuresto replicate moulding and self-assembly techniques which include micromoulding and capillary moulding [97], [98]. It usually uses a polymer which can chemically crosslink to form the mould such as poly(dimethysiloxane) (PDMS). The polymer is first poured or spin coated onto the master and then crosslinked to form the mould. For polymers such as PDMS which uses a hardening/crosslinking agent, the polymer is mixed with the hardening agent and then poured into the master. As the hardening process takes some time, the polymer will be able to conform to the shape of the mould before it hardens and form the mould [99]. If the polymer does not use a hardening agent for curing, the polymer solution is then first poured into the master and then crosslinked with the required methods. For PDMS, it may have biological implications if the crosslinked was not done correctly as uncrosslinked PDMS can have potential interactions with cells or other mediums such as the semipermeable membrane or the matrix. To ensure that there are no uncrosslinked PDMS, a Soxhlet extractor uses ethanol could be used [100]. PDMS still remains a good choice even with its unknown interaction with cells which can be removed as PDMS can also undergo increased cellular attachment through functionalizing by surface patterning with fibronectin. After the mould has been made, the replica is filled with the hydrogel precursor and cells by pressing the mould into the hydrogel suspension and using the required techniques to harden the hydrogel precursor with the cells. This method has been used to encapsulate NIH-3T3 using hyaluronic acid as the hydrogel precursor which uses UV to harden. It should be taken note of that using UV to cure the hydrogel with encapsulated cells might cause harm to the cells, hence, the UV wavelength used and the free radicals that are formed during the reaction must be carefully engineered such that it does not cause harm to the cells. It has been found that after 6 h of UV exposure, 85% of the cells were still viable [101].

On the other hand, some polymers require UV light to crosslink and such polymers can be used in photolithography [26]. These polymers when coming into contact with UV light, will undergo a free radical reaction to form chemical bonds which will act as the hydrogel crosslinks. Using this concept, a mask can be placed on the hydrogel precursor with the cells and then subject the hydrogel to UV light through the mask. Areas that are covered by the mask will not be exposed to the UV light and will stay uncrosslinked while areas that are exposed will be crosslinked. The crosslinked areas will form the encapsulated cell structure, while the uncrosslinked areas washed away. This technique can form very thin encapsulates and the process can be repeated to form layered structures if needed or different layers of polymers [102]. Another advantage of the technique is that you can form the encapsulated cells on a substrate instead of the being in the hardening bath like the extrusion methods. It should be taken note of that like the UV curing in the lithography, using UV to cure the hydrogel with encapsulated cells might cause harm to the cells, hence, the UV wavelength used and the free radicals that are formed during the reaction must be carefully engineered such that it does not cause harm to the cells. The usual mechanism for photolithography for encapsulated cells is using photo initiators. However, there are other photolithography reactions which use a donor/acceptor pair instead and it should be noted that these techniques are not suitable for cell encapsulation as if there are insufficient crosslinking, the monomers which are not crosslinked can be cytotoxic to the cells [103]. Photolithography is usually used to make thin layers of encapsulated cells, but there is a two-photon lithography that allows the creation of 3D structures instantly and not through the layer on layer methods. The two-photon lithography method uses polymers that are able to absorb two photons to reach the transitional energetic state. While usual photoinitiators have a linear relationship of increasing absorption rate with increasing light intensity, polymers that use two-photon lithography has a relationship of increasing absorption rate with increasing light intensity square. This will increase the crosslinking process in areas where there is increased light intensity and by using the laser to determine which areas should harden faster, a 3D crosslinking can be made (Fig. 5) [32].

4.3. Microfluidics

Microfluidics is used to create controlled spherical, cylindrical and hollow fibre cell encapsulates by altering the flow rate of the hydrogel precursor [104], [105]. It is a technique used to control fluids in microenvironments where the hydrogel is usually flowing in a laminar orientation [106]. The cell encapsulations are created using the emulsion method of the hydrogel precursor encapsulates in a non-miscible phase [107]. The hydrogel precursor with the cells is first injected through a microchannel and the encapsulated cells would start to form when the hydrogel precursor meets the non-miscible phase at the T-junction or flow-focusing interaction [108]. For the T-junction micro channel, when the hydrogel precursor reaches the channel, there would be a physical interaction between the non-miscible phases as since they are non-miscible, they would not be able to dissolve in each other and thus, they would apply physical interactions on each other [108]. The pressure and the flow rate of the non-miscible phase would apply a force onto the hydrogel precursor that causes the hydrogel precursor to be sheared off and follow the channel and create the cell encapsulations [109]. The size of the cell encapsulation would be determined by the size of the T-junction and the flow rate of the hydrogel precursor and the non-miscible phase. For the flow focusing interaction, the non-miscible phase flows from the side channel and intersect the main channel of the hydrogel precursor [110]. The flow focusing channels can also create encapsulate with different hydrogels such as Janus particles. These particles can be used in co-culture cells applications [111]. After the hydrogel precursor and the non-miscible phase have mixed, the hydrogel encapsulations are then collected and hardened accordingly. If the hardening agent is non-miscible with the hydrogel precursor, it can be used as the non-miscible phase and this would save a step in creating a cell encapsulation. The advantages in using microfluidics are high throughput capacity, uniform average cell encapsulated sizes, highly regulated cell encapsulate size and distance between the cell encapsulations from flow rate adjustments. However, there is a problem with cell viability where the non-miscible phase may affect the cells by not allowing it to have any nutrients, causing them to die if it is not washed properly after the procedure. In a recent work, Jiang et al. developed a microfluidic-based cell encapsulation technique which could allow polyethylene glycol norbornene (PEGNB) to be employed as the cell encapsulation [112]. In the designed approach, a flow-focusing microfluidic device was used to encapsulate cells within PEGNB droplets, which were then collected and exposed to UV light in bulk solution for photopolymerization (Fig. 6) [112]. This can lead to a fast polymerization kinetics of PEGNB and achieve high post-encapsulation cell viability over the course of 30 days. In addition, this approach was found to be superior to vortex-based cell encapsulation in terms of controlling hydrogel microsphere size and uniformity, showing high potential for practical use.