**7. Cell immobilisation**

The process of localising intact cells onto a surface without compromising their essential biological function is known as cell immobilisation. This technique allows the cell system to be reused multiple times and eliminates the negative feedback inhibition that metabolic products have on cells [116]. Cell cultures enable scientists to understand the mechanism behind the disease, the action of drugs, tissue morphology, cell biology, protein synthesis, and tissue engineering [117].

In 1906, for the first time, Harrison cultured cells as part of his investigation into the development of nerve fibres [118]. Since then, cells are mainly cultivated in two dimensions (2D). In 2D cultures, cells develop as a monolayer adhering to a plastic or glass surface in a culture flask or flat petri dish [116]. Although 2D adherent cultures are simple and cost-effective, they have many drawbacks, including the inability to mimic the native structures of tissues in both health and disease. In the 1970s, Hamburg and Salmon conducted one of the earliest threedimensional (3D) cultures [119]. The 3D systems sustain cell development, organisation, and differentiation like what is found in the human body. A variety of materials enable the 3D cell culture. Among these materials, alginate hydrogels are practical as a framework for immobilising cells in 3D cell culture [120]. In 1980, alginate was first used as an artificial semipermeable membrane enclosing viable islets [121]. Since then, alginate microbeads have been employed with many cell types *in vivo* and *in vitro* [120]. Alginate offers a fantastic toolset for design and optimisation, even though one system is likely to fit only some research or cell types [120]. The hydrogels used for *in vitro* 3D cell culturing have specific physicochemical characteristics, such as hardness, water holding capacity (WHC), swelling-erosion ratio, and swelling rate, to mirror the natural extracellular matrices (ECMs) found in living things [122].

#### **7.1 Encapsulation in beads**

Lim and Sun were the first to develop the encapsulation method for immobilising cells [49]. The researchers encapsulated pancreatic islet cells in calcium alginate matrices (**Figure 13**). Alginates have relatively little natural cell attachment and cellular contact, which is a crucial property [123]. This can benefit cell encapsulation applications but may be a drawback for other applications in tissue engineering. Alginate can be altered by including peptides for cell adhesion [124] or other bioactive components [120]. Also, the strength of the surface coating and the capsule porosity can be regulated by wrapping the alginate gel matrix with polycations such as poly-Lornithine, poly-L-lysine, or chitosan [120, 125].

Encapsulating cells in an alginate gel is a safe, and adaptable approach for immobilising cells [125]. Alginate and cells are combined once the osmolality is regulated, and the mixture is then ejected (extruded or dripped) into a calcium chloride bath [120]. The instantaneous ionic crosslinking reaction traps live cells within an alginate hydrogel bead. The development of artificial organs via cell encapsulation is being researched to treat many different ailments [126]. The artificial pancreas used to treat diabetes is perhaps the best-known example (encapsulated pancreatic islets) [127]. By injecting encapsulated canine islet allografts intraperitoneally, Soon-Shiong and colleagues formed mechanically stable microcapsules with alginate, high in guluronic acid, and reported extended remission of diabetes in the diabetic dog model [128]. In other reports, the brains of dogs receiving treatment for spontaneous brain tumours were transplanted with alginate-encapsulated cells that produce the antiangiogenic protein endostatin [129, 130]. Alginate has been used to immobilise a wide variety of other cell types, including chondrocytes [131, 16], mesenchymal stem cells [124, 132], and adipose-derived stem cells [133, 134], as summarised in **Table 2**.

According to the cell encapsulation approach, cells are enclosed within an artificial enclosure and separated from the host immune system by a semipermeable barrier that protects the transplanted cells from the host immunological response [137]. However, the membrane allows for the flow of small molecules such as glucose, oxygen, therapeutic molecules, and waste materials while isolating cells from the immune reaction [138]. The encapsulation technique eliminates the need for harmful immunosuppressant drugs after transplantation [128] and overcomes the shortage of available donors by enabling allogeneic and xenogeneic transplants [126, 137]. Most techniques for encapsulation cells in alginate consist of two phases. The first step is the development of an internal phase, during which the alginate or composite is divided into tiny droplets. The droplets are solidified in the second step, either by gelling or creating a membrane at the surface of the droplets [120].

#### **7.2 Cell entrapment by self-gelling**

Systems for self-gelling (or delayed gelation) is the one in which the gelling of the gels happens inside the body (*in situ*) as shown in **Figure 14**. This method enables

*Alginate-Based Applications in Biotechnology with a Special Mention to Biosensors DOI: http://dx.doi.org/10.5772/intechopen.110737*


#### **Table 2.**

*Different cell immobilisation techniques and their applications.*

**Figure 14.** *Cell entrapment by self-gelling.*

implantation with less invasive surgical procedures, thus making delivery easier since they precisely occupy tissue spaces and defects [120]. Herlofsen *et al*. in their study of human mesenchymal stem cells (hMSCs) used the self-gelling system [135]. In their study, calcium ions from the calcium alginate particles diffused into the sodium alginate solution, forming an alginate hydrogel that entraps the cells. Self-gelling alginate hydrogel enables the homogenous distribution of the cells within a hydrogel with specific dimensions and shapes. Herlofsen *et al*.'s study showed how the hMSC differentiation led to the upregulation of many genes related to hyaline chondrogenesis, which might be exploited to repair possible lesions of hyaline cartilage. Available data also indicate that the self-gelling approach might get around some of the drawbacks of the 3D scaffolds that are now available, including retrieval of cells and the staining and imaging of cells in situ [139]. Andersen and colleagues [139] used dried calcium alginate foams as a scaffold, which supplies the gelling ions for the alginate solution that occupies the foam's pores and subsequently forms a gel. Cells were evenly distributed throughout the scaffold and entrapped by *in situ* gelations initiated by calcium ions that diffuse from the foam while the alginate solution is rehydrating it.

The formation of tiny microbeads to reduce the mass transfer resistance problem associated with big-diameter beads has been a critical concern in cell immobilisation [140]. The conventional method used for a long time includes swiftly passing the cell/ gel solution through a nozzle with compressed air to produce alginate beads [141]. Different techniques for forming droplets have been described, such as extrusion through a needle, Coaxial air (or liquid flow), electrostatic potential, vibrating capillary jet breakage, a pressurised-vessel generated droplets from a vibrating nozzle, and rotating capillary jet breakage [142].

Attempts to use electric fields to form cell immobilisation beads have been successful [136, 140]. For example, electrostatic droplet generation can produce significantly smaller beads than an air jet extruder [140]. Additionally, bead size can be

*Alginate-Based Applications in Biotechnology with a Special Mention to Biosensors DOI: http://dx.doi.org/10.5772/intechopen.110737*

easily controlled by adjusting the applied potential. This application's fundamental idea is the electrostatic force that disturbs a liquid surface to generate a charged stream of tiny droplets [136]. Lord Rayleigh was the first to thoroughly investigate the impact of electrostatic forces on atomised liquid droplets as he looked at the stability of a jet of liquid both with and without an applied electric field [143]. When a liquid is exposed to an electric field, a charge is generated on its surface, and due to mutual charge repulsion, a force that pushes outward is produced [140]. The electrostatic surface pressure can drive a drop of liquid into a conical shape under the right circumstances, such as when a liquid is forced through a needle [136], **Figure 15**. The discharge of charged droplets from the liquid's tip causes excess charge to be discharged [136, 140]. The electrode geometry, applied voltage, collecting solution distance, and needle diameter all affect the emission process [116]. After being exposed to strong electrostatic potentials, there was no discernible reduction in the survival of immobilised cell cultures [136].
