In the field of disease biology, cell therapy, and developmental biology, the experimental approaches do not result in reproducible efficiency upon using cell lines as limitations of genetic difference and characterization morphing occur in such cases. Therefore, scientists have shifted towards using primary cells and stem cells for in vitro cell culture studies in order to preserve the genetic integrity, biochemical signaling, and cell phenotype morphology. While primary cells are tissue-extracted cells that have not been immortalized and retain the native functionality of body cells, stem cells are special!
Stem cells have the potential to differentiate into various cell lineages and can also self-proliferate like normal primary cells. There are two broad types of stem cells: embryonic stem cells (pluripotent stem cells) and adult stem cells (multipotent stem cells). Embryonic stem cells help the body to develop from the blastocyst stage as they can differentiate into ectoderm, endoderm, and mesoderm cell lineages. Adult stem cells, on the other hand, replenish adult body tissues and help in repair and rejuvenation.
Stem Cell Culture
Stem cells have been used in research for various studies ranging from genomics to regenerative medicine, but culturing stem cells has been a fair challenge for many researchers. Let us discuss the two common stem cell culture systems and how to characterize stem cells for efficient culturing.
Types of Stem Cell Culture System
Feeder-dependent Stem Cell Culture System
Feeder-dependent stem cell culture is used for Stem Cells which are grown in co-culture with feeder cells like fibroblasts. The feeder cells support the attachment and proliferative potentials of the stem cells and support their growth by providing a great extra-cellular culture medium environment. In most co-culture systems, mouse embryonic fibroblasts are used after being growth-inactivated by mitomycin C to arrest the cell cycle. In some other cases, human foreskin fibroblasts are also used as a xeno-free alternative for clinical applications. These fibroblast feeder cells are usually cultured on plates pre-coated with a 0.1% gelatin.
Feeder-free Stem Cell Culture System
In such systems, feeder layers are replaced by an optimum extracellular matrix. These systems maintain a balance between promoting stemness of the culture and inhibiting spontaneous differentiation by fine-tuning of the media with essential growth factors and amino acids.
For the feeder-dependent stem cell culture system, fibroblasts play an important role in attachment and stemness regulation but limitations to this system include rigorous culture models and developing a mixed culture of stem cells and fibroblasts. Whereas in the case of feeder-free stem culture system, there are advantages of easier handling, optimum for large-scale use, no issues of mixed culture yield for experiments.
Characterization of Stem Cells
In stem cell culture, the issue of mixed cultures and cross-contamination is prominent, and therefore, with advanced technology, various characterization methods have been practiced by researchers to detect stemness and abnormalities affecting cell morphology and functionality.
- Observation of cellular morphology to detect stem cells from differentiated cells.
- Alkaline phosphatase staining to discriminate feeder cells from stem cells and maintain cell viability.
- Immunostaining and flow cytometry to provide marker expression results. Stem cells have positive markers like SSEA4, TRA-1-60, TRA-1-81, and negative markers like CD44 and SSEA1. These markers can be detected to recognize stem cells.
- Evaluation of stem cell differentiation potential by molecular or cellular analysis. In the case of molecular analysis, qPCR and immunostaining come to the rescue as they help researchers in detecting specific cell lineage markers in order to understand the differentiation lineage and stage. In the case of cellular analysis, several markers specific for mesoderm (SMA), endoderm (AFP), and endoderm (TUBB3/TUJ1) can be detected by using advanced immunochemistry methods.
Applications of Stem Cells
Due to the potential of Stem Cells to self-renew and trans-differentiate into other cell lineages, researchers have considered these cells as candidates in several biomedical and clinical research domains like Repair and rejuvenation of body tissues, drug development and testing, drug toxicity, gene functioning, and genomics, cell therapy, gene therapy, immunotherapy, cancer research, neurodevelopmental biology research, pharmacology, organ imprinting and 3D tissue culture, patient-derived xenograft, and organoid modeling, etc.
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