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Live-cell imaging in the field of cell therapy

Cell therapy is the therapeutic use of live cells isolated from the patient (autologous) or donor (allogenic) sources to treat disease and repair damaged tissue1–4. The technique has broad applications that range from treating cancer, diabetes, cardiovascular and neurogenerative conditions to tissue regeneration and replacement. Considering the scope of its application, a variety of live cells have been purposed for cell therapies, including allogeneic mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), hematopoietic stem cells, cancer cells, and autologous genetically modified immune cells4. The use of cell therapies is rapidly growing, and the uptake of this technology is evident with over 1200 cell therapy clinical trials in progress globally in 20215–8.

Extensive research goes into making viable cell therapy products; this is because, being living cells, they are sensitive to even the slightest variation, thus potentially rendering the therapy ineffective4. Technologies that can optimize upstream research and manufacturing are therefore crucial for the production of quality cell therapies9. Thus, there is a particular need for non-invasive live-cell imaging and tracking to allow researchers to identify and study potential cell therapies. Furthermore, non-invasive live-cell imaging can minimize the risk of contamination introduced through manipulating and sampling cultures4,10. CytoSMART offers an excellent range of live imaging systems that can be used to research and manufacture cell therapies. The applications and examples of these live-cell imaging systems will be discussed below.

Cell profiling through high-content imaging

The progress made in imaging and computational technologies has enabled high-content approaches in high-throughput settings. One such application of this high-content imaging has been in regenerative medicine. Protocols used in this field to generate iPSC-derived cells, such as endothelial cells, often require extensive optimization to produce the desired tissue-specific cells. High-content imaging can be applied here to determine the state of cell activation and specialization through analysis of cell morphology, cell-cell junctions, and NOTCH1 activation status. These parameters are readily viewed at both the levels of the cell population or as single cells11,12. In the example of iPSC-derived endothelial cells, profiled cells can then be benchmarked against primary endothelial cells to develop novel differentiation protocols11.

High-content imaging has been further adapted from analyzing fixed cells at one time-point to live-cell imaging. Real-time imaging is beneficial as often specific cell processes and phenotypes are time-dependent. Live-cell imaging allows the user to capture these chronological events in real-time and has the added advantage of enabling the user to identify changes in the cell cycle and differentiation in heterogeneous cell populations. The CytoSMART offers a range of live-cell imaging systems capable of cell culturing monitoring and high-content imaging. The CytoSMART Omni is a robust and straightforward automated live-cell imaging system that can be readily applied to cell therapy research11.

Studying tissue regeneration using live-cell imaging

MSCs have been identified as a promising regeneration treatment for hearing loss. When tissue damage occurs, MSCs are naturally released into the circulatory system. They then migrate to the injury site and promote regeneration. Understanding this migration process is crucial for developing MSC treatments. Unfortunately, in vivo analysis of MSC migration is a highly sophisticated undertaking that is labor-intensive and time-consuming. As a result, alternative in vitro methods have been devised that involve tracking the two-dimensional path of MSCs in a wound healing method13.

One such method, focusing on regenerating hearing loss, used a modified wound healing assay. In this study, researchers dissected the Organ of Corti located within the cochlear of the inner ear. The tissue was then placed onto a confocal dish and surrounded by a glass cloning cylinder. Fluorescently tagged MSCs were then plated on the outside of the glass cloning cylinder. The glass cylinder was then removed once the cells had attached, and the boundary between the Organ of Corti and the MSCs was imaged in real-time. This technique successfully aided researchers in tracking the migration of MSCs into the damaged tissue, thereby acting as a helpful test in the development of MSC treatments, particularly in the study of hearing loss13.

The CytoSMART imaging systems are designed explicitly for capturing wound healing assays and single-cell migration. In addition, the CytoSMART Lux3 FL is capable of fluorescence live-cell imaging with one brightfield and two fluorescent channels, making this device well-suited for capturing fluorescently labeled cells used in wound healing assays.

Ensuring monoclonality in standard well plate formats

Cell therapies often require the isolation and cloning of single cells. Single cells in cell culture can be isolated using limiting dilutions; in this method, cells are pipetted into microplates at a distribution of less than one cell per well. In practice, this method is often repeated to increase monoclonality confidence; however, this adds additional time and cost to the protocol. Furthermore, according to regulatory authorities, demonstrating a single cell colony is insufficient to prove monoclonality, and the researcher must establish that a single cell was plated into the well14.

Various techniques exist to show the isolation of single cells; this includes fluorescently labeled cell sorting that, while effective, can impact cell viability. Alternative methods, such as manual cell picking, can confirm single cells were isolated but cannot confirm single-cell status after plating into the well. Therefore, the limiting dilution remains the best method to ensure clonality. When using this method, the most effective way to establish that a single cell was plated is to image the entire surface of the well. However, imaging the surface of the well can be complicated by the ‘edge effect.’ This phenomenon is observed at the boundary of the wall and base of flat-bottomed wells. Images collected at this interface can appear blurred and darkened, making identifying cells challenging and time-consuming14.

A recent publication described a helpful technique that can be used to circumvent the ‘edge effect.’ The method involves depositing dilutions of limited volume into the center of the plate well and overlaying these droplets with an immiscible liquid with a refractive index close to that of the cell medium. Using a microscope such as the CytoSMART Omni, the drop can be easily imaged to see whether it contains a single cell. This method of preparing limiting dilutions for cell therapies can be readily adapted to high throughput14.

Quantification of biological therapeutics

Adipose tissue is a valuable source of MSCs as the tissue has been shown to have a far greater concentration of MSCs per volume when compared to bone marrow. MSCs are isolated from adipose tissue as part of the stromal vascular fraction— a heterogeneous mixture of cells. Obtaining this fraction containing the adipose-derived stem cells can be performed either enzymatically or mechanically. Once isolated, these free cells are counted manually using the Trypan Blue exclusion assay and the Burker chamber or with an automated system. After that, the stromal vascular fraction containing MSCs is ready for further characterization15.

Automated cell counting has several advantages over manual counting. Hemocytometers are considered the gold standard for cell counting; however, the process has a high element of subjectivity due to handling errors, lack of precision, and inter-user variability. Manual counting is also time-consuming and not suited to high throughput particularly when patient samples are being handled. Automated cell counters are designed for high reproducibility and precision to reduce inconsistency and variability. These features make automated cell counters particularly well-suited to the cell therapy laboratories that routinely isolate stem cells16. The CytoSMART has a range of automated cell counters that can be applied to the field of cell therapy. The CytoSMART cell counter has already found use in counting cells from isolated stromal vascular fractions and determining the cell proliferation capacity of these cells15.

Three-dimensional organoid and spheroid applications in cell therapy research

Organoids are three-dimensional cell culture structures that resemble organ-like tissues. In comparison, spheroids are cell aggregates that have been cultured on non-adherent substrates. In both cases, stem cells are the primary source for generating these structures17. The advantage that organoids and spheroids have over traditional two-dimensional cell culture is that these structures more closely represent the in vivo environment. This advantage allows researchers to predict biochemical and physiological responses of stem cells used in cell therapies with greater confidence18.

A recent comparison between two regenerative treatments for periapical tooth lesions looked at stem cells cultured as spheroids or as a monolayer. The formation of spheroids was closely monitored with the aid of a CytoSMART live-cell imaging system. The study found that spheroids had greater viability and uniform cellularity in the lesion cavity than monolayer cultured cells. In addition, these spheroids also had higher levels of markers that indicate osteoblastic differentiation. These results suggest that patients may benefit from the use of spheroids to treat periapical lesions18.

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