**1. Introduction**

Cell encapsulation is the process of entrapping cells into a matrix. In general, the matrix is spherical in shape and in the form of a polymeric hydrogel. Cell encapsulation technology has shown great promise for immunoisolation and controlled release of therapeutic products towards gene therapy. Figure 1 demonstrates the mechanism of encapsulated transgenic cells for gene therapy.

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#### **1.1. Encapsulation materials**

Both natural and synthetic polymers have been utilized for cell encapsulation. Natural polymers that have been used include alginate, agarose, collagen, and hyaluronic acid, while synthetic polymers, including poly(vinyl alcohol), poly(lactic-co-glycolic acid), polyacrylates, HEMA-MMA-MAA, polyphosphazines, and polyepoxides, have been studied.[1] Natural polymers are more commonly used because of their biocompatibility and are easily accepted by the public. However, their product quality and characteristics can vary greatly between companies and batches compared to synthetic polymers. Alginate, agarose, and polylactideco-glycolide (PLGA), the most commonly used encapsulation materials, are introduced here.

#### *1.1.1. Alginate*

Alginates, polysaccharides, are linear block polymers consisting of α-l-guluronic acid (G) and β-d-manuronic acid (M) blocks (Figure 2). Divalent cations, such as Ca2+, Ba2+, and Sr2+, can link alginate molecules together (i.e. through ionic cross-linking) forming alginate hydrogel capsules while encapsulating cells inside. The G and M contents of the alginate molecules can affect the gel properties including mechanical strength, biocompatibility, and permeability.[2– 6] Recently, it has also been shown that oligochitosan could be used as a cross-linker for polysaccharide-based gel formations.[7]

**Figure 1.** A conceptual schematic demonstrating cell encapsulation for gene therapy.

**Figure 2.** Chemical structure of alginate (A) and alginate-based hydrogel formation mechanism (B).

#### *1.1.2. Agarose*

**1.1. Encapsulation materials**

192 Gene Therapy - Principles and Challenges

polysaccharide-based gel formations.[7]

**Figure 1.** A conceptual schematic demonstrating cell encapsulation for gene therapy.

*1.1.1. Alginate*

Both natural and synthetic polymers have been utilized for cell encapsulation. Natural polymers that have been used include alginate, agarose, collagen, and hyaluronic acid, while synthetic polymers, including poly(vinyl alcohol), poly(lactic-co-glycolic acid), polyacrylates, HEMA-MMA-MAA, polyphosphazines, and polyepoxides, have been studied.[1] Natural polymers are more commonly used because of their biocompatibility and are easily accepted by the public. However, their product quality and characteristics can vary greatly between companies and batches compared to synthetic polymers. Alginate, agarose, and polylactideco-glycolide (PLGA), the most commonly used encapsulation materials, are introduced here.

Alginates, polysaccharides, are linear block polymers consisting of α-l-guluronic acid (G) and β-d-manuronic acid (M) blocks (Figure 2). Divalent cations, such as Ca2+, Ba2+, and Sr2+, can link alginate molecules together (i.e. through ionic cross-linking) forming alginate hydrogel capsules while encapsulating cells inside. The G and M contents of the alginate molecules can affect the gel properties including mechanical strength, biocompatibility, and permeability.[2– 6] Recently, it has also been shown that oligochitosan could be used as a cross-linker for

> Agarose, a thermal-responsive polymer, consists of β-d-galactopyranose and 3,6-anhydro-αl-galactopyranose units which can undergo a sol–gel transition upon cooling (i.e. through thermal cross-linking) (Figure 3). Some agarose products have a transition temperature close to body temperature, making it a good candidate for cell encapsulation.[8]

#### *1.1.3. Polylactide-co-Glycolide (PLGA)*

PLGA polymers belong to aliphatic polyesters and are biodegradable (Figure 4). To prepare the capsules, PLGA is dissolved in methylene chloride, and then a second component is added to precipitate the polymer molecules (interfacial precipitation).[1,9]

#### **1.2. Encapsulation technologies**

Different technologies have been used for preparing macro/microcapsules, which include airjet encapsulation, electrostatic spray, laminar jet breakup, and microfluidic channel/nozzle. Among them, electrostatic spray and microfluidic channel/nozzle are two of the most fre‐ quently used encapsulation approaches.[10]

#### *1.2.1. Electrostatic spray method*

The electrostatic spray method has a significant appeal due to its ease of operation, scale-up capabilities, negligible damage to cells, and allowance for sterile operation conditions.[10] The mechanism of cell encapsulation by using the electrostatic pray method is shown in Figure

**Figure 3.** Chemical structure of agarose (A) and agarose-based hydrogel formation mechanism (B).

**Figure 4.** Chemical structure of PLGA.

5A. In general, a cell polymer mixture is extruded through a nozzle by using a pump or compressed air. The droplets are broken down into smaller ones under electrostatic force and/ or other introduced forces (e.g. vibration). Once the droplets reach the gelling bath containing the cross-linkers, the cell-loaded hydrogel capsules form immediately through various forces, such as ionotropic reaction between divalent ions and alginate molecules. Moreover, the system could be modified to prepare the core-shell structure hydrogel capsules, as depicted in Figure 5B.[11]

#### *1.2.2. Microfluidics channel/nozzle method*

Microfluidics devices can be used to generate micrometer-scale droplets with a narrow size distribution and controlled morphology.[12–14] This method shows great promise for cell encapsulation, especially for single cell encapsulation.[15] In general, capsules are formed by allowing a core fluid to be surrounded by a flowing sheath stream.[16] Recently, these devices have also been successfully applied for the generation of cell-loaded core-shell capsules (Figure 6).[14] Besides the relatively low encapsulation efficiency, a significant drawback of the current microfluidic technologies is that the oil used for shearing may leave a residual adhesive oil layer on the capsule which affects subsequent coating processes.[10,17] a residual adhesive oil layer on the capsule which affects subsequent coating processes.[10,17]

 Figure 5. A sketch of the electrostatic spray device used for generating polymeric hydrogel capsules (A).[10] *Reproduced by permission of The American Society of Mechanical Engineering* **Figure 5.** A sketch of the electrostatic spray device used for generating polymeric hydrogel capsules (A).[10] *Repro‐ duced by permission of The American Society of Mechanical Engineering (ASME)*; A modified electrostatic spray setup for fabricating the core-shell structure hydrogel capsules (B).[11] *Reproduced by permission of The Royal Society of Chemistry.*

*(ASME)*; A modified electrostatic spray setup for fabricating the core‐shell structure hydrogel capsules (B).[11] *Reproduced by permission of The Royal Society of Chemistry.*

5A. In general, a cell polymer mixture is extruded through a nozzle by using a pump or compressed air. The droplets are broken down into smaller ones under electrostatic force and/ or other introduced forces (e.g. vibration). Once the droplets reach the gelling bath containing the cross-linkers, the cell-loaded hydrogel capsules form immediately through various forces, such as ionotropic reaction between divalent ions and alginate molecules. Moreover, the system could be modified to prepare the core-shell structure hydrogel capsules, as depicted

**Figure 3.** Chemical structure of agarose (A) and agarose-based hydrogel formation mechanism (B).

Microfluidics devices can be used to generate micrometer-scale droplets with a narrow size distribution and controlled morphology.[12–14] This method shows great promise for cell

in Figure 5B.[11]

*1.2.2. Microfluidics channel/nozzle method*

**Figure 4.** Chemical structure of PLGA.

194 Gene Therapy - Principles and Challenges

Figure 6. A sketch of the **Figure 6.** A sketch of the microfluidics device for generating core-shell hydrogel capsules. The core channel height (H1) is the lowest. H: height and W: width.[14] *Reproduced by permission of The Royal Society of Chemistry.*

microfluidics device for generating core‐shell hydrogel capsules. The core channel height (H1) is the lowest. H: height and W: width.[14] *Reproduced by permission of The Royal Society of*

*Chemistry.*

5

### **2. Recent progress on transgenic cell encapsulation for gene therapy**

Encapsulation of genetically modified cells has been conducted for the treatment of central nervous system diseases, cardiovascular disorders, mucopolysaccharidosis type VII (MPSVII) disease, wounds, bone fractures, and cancer.[18–30] Considering most genetically engineered cells are from allogeneic or xenogeneic sources, immunoisolation is a critical factor when using these cells.[5]

#### **2.1. Bone-related diseases**

Bone morphogenic protein-2 (BMP-2) is a member of the transforming growth factor-β (TGF-β) superfamily and has been widely reported to have osteoinductive activity. Ding *et al.* [31] studied the behaviour of BMP-2 gene-transfected bone marrow-derived mesenchy‐ mal stem cells in alginate-poly-l-lysine-alginate (APA) microcapsules. The results showed that encapsulated transfected cells could secrete BMP-2 proteins for at least 30 days and the APA microcapsules could be used for immunoisolation. Olabisi *et al.* [28] investigated microencapsulation of AdBMP-2-transduced MRC-5 cells (human diploid fetal lung fibroblasts) in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. After injecting the encapsulated cells intramuscularly, the volume of the bone formed was about twice that of the control group (unencapsulated cells). Recently, rapid heterotrophic ossification by using cryopreserved PEGDA encapsulated BMP-2 expressing mesenchymal stem cells (MSCs) was also observed (as shown in Figure 7).[32] Additionally, human calcitonin delivered by microencapsulated recombinant myoblasts showed potential for allergenic gene therapy for postmenopausal osteoporosis. [33] Furthermore, transplantation of fibrin glue-compound‐ ing hepatocyte growth factor-transgenic MSCs is a promising novel method for avascular necrosis of the femoral head (ANFH) therapy.[34]

#### **2.2. Cancer**

Both mouse myoblasts (C2C12 cells) and human embryonic kidney 293 (HEK293) cells were engineered to continuously secrete angiostatin, and were encapsulated into alginate-based microcapsules for cancer treatment. The *in vivo* experimental results demonstrated the potential for angiostatin-mediated cancer therapy by using an encapsulated transgenic cellbased approach.[35,36] Considering immunotherapies have been proven to be alternative strategies for malignancy treatment[37], combined immunotherapy (an interleukin 2 fusion protein, sFvIL-2) and antiangiogenic therapy (angiostatin) were tested. It was shown that transplantation of angiostatin expression and sFvIL-2-expressing C2C12 cells encapsulated in APA microcapsules improved the survival rate of experimental animals.[38] Recently, microencapsulation of therapeutic antibodies producing cells in APA microcapsules was tested for cancer treatment. [39] Additionally, with the advancement of stem cell research, there is an increased potential for cancer therapy by using encapsulated stem cells.[40]

**Figure 7.** Microencapsulated BMP2-transduced MSCs in a mouse model for heterotopic ossification. X-ray and Mi‐ croCT images of the resulting heterotopic ossification for freshly prepared BMP2 microencapsulated MSCs (a and b) and for cryopreserved BMP2 microencapsulated MSCs (d and e).[32]

#### **2.3. Neural diseases**

**2. Recent progress on transgenic cell encapsulation for gene therapy**

these cells.[5]

**2.2. Cancer**

**2.1. Bone-related diseases**

196 Gene Therapy - Principles and Challenges

necrosis of the femoral head (ANFH) therapy.[34]

Encapsulation of genetically modified cells has been conducted for the treatment of central nervous system diseases, cardiovascular disorders, mucopolysaccharidosis type VII (MPSVII) disease, wounds, bone fractures, and cancer.[18–30] Considering most genetically engineered cells are from allogeneic or xenogeneic sources, immunoisolation is a critical factor when using

Bone morphogenic protein-2 (BMP-2) is a member of the transforming growth factor-β (TGF-β) superfamily and has been widely reported to have osteoinductive activity. Ding *et al.* [31] studied the behaviour of BMP-2 gene-transfected bone marrow-derived mesenchy‐ mal stem cells in alginate-poly-l-lysine-alginate (APA) microcapsules. The results showed that encapsulated transfected cells could secrete BMP-2 proteins for at least 30 days and the APA microcapsules could be used for immunoisolation. Olabisi *et al.* [28] investigated microencapsulation of AdBMP-2-transduced MRC-5 cells (human diploid fetal lung fibroblasts) in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. After injecting the encapsulated cells intramuscularly, the volume of the bone formed was about twice that of the control group (unencapsulated cells). Recently, rapid heterotrophic ossification by using cryopreserved PEGDA encapsulated BMP-2 expressing mesenchymal stem cells (MSCs) was also observed (as shown in Figure 7).[32] Additionally, human calcitonin delivered by microencapsulated recombinant myoblasts showed potential for allergenic gene therapy for postmenopausal osteoporosis. [33] Furthermore, transplantation of fibrin glue-compound‐ ing hepatocyte growth factor-transgenic MSCs is a promising novel method for avascular

Both mouse myoblasts (C2C12 cells) and human embryonic kidney 293 (HEK293) cells were engineered to continuously secrete angiostatin, and were encapsulated into alginate-based microcapsules for cancer treatment. The *in vivo* experimental results demonstrated the potential for angiostatin-mediated cancer therapy by using an encapsulated transgenic cellbased approach.[35,36] Considering immunotherapies have been proven to be alternative strategies for malignancy treatment[37], combined immunotherapy (an interleukin 2 fusion protein, sFvIL-2) and antiangiogenic therapy (angiostatin) were tested. It was shown that transplantation of angiostatin expression and sFvIL-2-expressing C2C12 cells encapsulated in APA microcapsules improved the survival rate of experimental animals.[38] Recently, microencapsulation of therapeutic antibodies producing cells in APA microcapsules was tested for cancer treatment. [39] Additionally, with the advancement of stem cell research, there is an increased potential for cancer therapy by using encapsulated stem cells.[40]

Parkinson's disease (PD) belongs to a group of conditions called motor system disorders, resulting from the loss of dopamine-producing brain cells.[41] This disease could be amenable to gene product replacement strategies including implantation of encapsulated transgenic cells.[42] There are several publications regarding encapsulated cell biodelivery of glial cell line-derived neurotrophic factor (GDNF) for PD treatment; GDNF has been proven to have neuroprotective and neurotrophic properties on dopaminergic neurons.[26,43,44] Further‐ more, encapsulated transgenic cells could be utilized in brain tumour treatment.[45,46]

Small capsules (<200 µm) have been developed for the delivery of gene products, secreted by encapsulated transgenic cells, to the brain, bypassing the blood–brain barrier (BBB). To date, several alginate-based microcapsule systems, Ca-alginate, APA, and alginate-chitosanalginate (ACA), have been reported.[10,47,48] Encapsulation of transgenic cells has also been used for other disease treatments, such as mucopolysaccharidosis VII and myocardial infarction. Table 1 summarizes the recent gene therapy studies based on encapsulated transgenic cells, with the exception of bone-related and neural diseases and cancer treatment.


**Table 1.** Recent gene therapy studies by using encapsulated transgenic cells
