In the early 1960s, in vitro CF was performed to describe the effect of viruses such as hemagglutinating virus of Japan (HVJ) on murine cell cultures [26–28]. Currently, there are three major CF methods: chemical, electrical, and viral. Advantages and disadvantages of each method were compiled and are presented in Table 72.1. New methods of CF applying different agents such as cephalin, bispecific nanoparticles, fusogenic cell lines, or v-fusion are investigated [29–32]. Combining different fusion methods and introducing additional modifications of conventional techniques have been studied to improve the fusion efficacy, either by increasing the cell-to-cell contact capability or permeabilization area. The selection of proper fusion method and protocol optimization depends on the cell type, cell number, culture conditions (monolayer vs. cell suspension), their sensitivity to unfavorable conditions, and equipment availability. The efficacy of polykaryons creation can be controlled by factors such as time and/or fusing agent concentration. Higher numbers of polykaryons can be also generated by repeating the fusion procedure [33]. Additionally, the application of supporting agents such as dimethyl sulfoxide (DMSO), in case of polyethylene glycol (PEG) mediated fusion, was confirmed to be more efficient in creating higher number of fused cells than using PEG alone [34, 35]. PEG, due to its hydrophobic properties, decreases the distance between cells by removing water (thermodynamically unfavourable environment) and causing their aggregation. Dehydration leads to asymmetry in the lipid membrane bilayers leading to formation of a single bilayer septum at a point of close apposition of two cell membranes and facilitates formation of the pore following septum decay (Fig. 72.2).

Ex vivo CF can be a crucial tool in creating new cell types or improving research models used in vitro in the basic mechanistic studies (e.g. cancer research) or for more efficient drug/treatment development. Recently ex vivo CF was applied to create human insulin-releasing 1.1B4 cells, which are able to form pseudoislets [36]. Most of the studies describing interactions and insulin secretion were performed using B cells routinely grown in the monolayers, which can impact their secreting properties. The use of fused cells in this model may improve the knowledge of regulatory mechanism underlying insulin secretion and survival of human pancreatic B cells. Additionally, it can help develop better types of insulin secreting cell therapies against diabetes [36]. CF is a well-established procedure in the process of producing monoclonal antibodies, which are widely used in research application such as protein or cell type detection, blocking cell activity, studying cross-reactivity, and antigen purification. Monoclonal antibodies are produced by immortalized hybridoma cells, which are created by ex vivo CF between antibody secreting B cells and myeloma cells. The first report describing the creation of hybridoma was published in 1973 by Schwaber and Cohen [37] and was soon followed by a procedure of fusion between myeloma cells and B lymphocytes isolated from spleen of immunized animal, in 1975 by Kohler and Milstein [12]. Generated cells were characterized by both lymphocyte properties to produce specific antibodies and the immortal character of the myeloma cells. CF played a critical role in a variety of studies exploring processes such as cancer pathogenesis, explaining trans-differentiation of committed somatic cells through cell reprogramming, or even assessing the effects of nuclear transfer of nucleus of one cell transferred to the cytoplasm of enucleated oocyte [38]. Examples of application of CF in research are presented in Table 72.2.

The infusion of bone marrow-derived cells, presence of chimerism in peripheral blood, and following migration of donor-derived cell to lymphoid organs was associated with prolonged survival of the transplants or even tolerance induction [53–57]. Bonde et al. [58] reported that during co-culturing spontaneous fusion of bone marrow cells derived from two different mice strains occurred. In vivo study confirmed this result following allogenic and syngenic transplantation. Further, it has been shown that fused cells, on their surface, expressed both donor and recipient MHC antigens.

Siemionow’s group also performed experiments on CF of bone marrow derived cells [59]. Results of this preliminary study were in line with results of Bonde et al. [58] and showed that there is a possibility of creating in vivo donor-recipient fused cells that can facilitate face allograft survival (Article in press). Short immunomodulatory protocol of anti-αβ-TCR monoclonal antibody and cyclosporine A was used to facilitate engraftment of spontaneously created donor-recipient chimeric cells (DRCC).

The successful establishment of protocol creating in vivo DRCC either via the mechanism of trogocytosis or spontaneous CF is opening many possibilities of using bone marrow derived cells as a tool for development of novel therapeutic products. Although, further research on DRCC is necessary in order to fully understand the underlying mechanisms of their creation and action in vivo, the creation of tolerance-inducting cells could be a breakthrough modality in solid organ and CTA transplantation.

There are several disadvantages for creating DRCC in vivo in the clinical scenario as described in the previous chapter (Chap.​ 71). The most challenging issue in clinical execution of the primary chimera creation protocol is the critical time frame following bone marrow infusion required for development of chimeric pro-tolerogenic environment in the transplant recipient. Pre-treatment of the transplant recipient in order to create in vivo DRCC will be possible only for the living organ donor. To overcome this hindrance and move forward to a more clinically applicable model, Siemionow’s group adopted a new approach to create DRCC via ex vivo CF. DRCC therapy was created by fusion of donor and recipient bone marrow derived cells by polyethylene glycol (PEG) technique. Briefly, bone marrow cells were harvested from the tibia and femur bones of two fully MHC mismatched ACI (RT1^a) and Lewis (RT1^l

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