Stem Cell Analysis – Quality control assays for stem cell therapies


Stem Cells are extraordinary. They can differentiate into any cell in the organism and are capable of self-renewal which is why stem cell therapy holds such high expectations for treating numerous diseases. But…working with stem cells in research and development laboratories is not easy, the cells are fragile and prone to decreased viability after each experimental step.


This article introduces the basic types of stem cells, how they can be used to treat diseases and how Cellix’s Inish Analyser is used as a quality check for cell counting and viability assays – key metrics when working with stem cells.


Stem Cell Basics

There are different types of stem cells, as follows:

  • Totipotent stem cells - they can divide and differentiate into cells of the whole organism.

  • Pluripotent stem cells (PSCs) - they form cells of all germ layers except extraembryonic structures.

  • Multipotent stem cells - they can differentiate into cells of specific cell lineages, such as hematopoietic stem cells, found in the peripheral blood and the bone marrow.

  • Oligopotent stem cells – they can differentiate into several cell types.

  • Unipotent stem cells – they are only able to form one cell type.

After fertilization, a blastocyst is formed. Its inner wall contains short-lived embryonic stem cells. The blastocyst contains two cell types, the inner cell mass (ICM) and the trophectoderm (TE), [1]. The TE forms a specialized support structure while the ICM remains undifferentiated, fully pluripotent, and proliferative. Human embryonic stem cells (hESCs) derive from the ICM. After hESCs differentiate into one of the germ layers, they become multipotent cells.


Pluripotent cells occur all over the organism, and they can proliferate and differentiate into specialized cells under the right conditions. In the body, adult stem cells enable the healing, growth, and replacement of lost cells. It is also possible to reprogram adult stem cells back to their pluripotent state, resulting in induced pluripotent stem cells (iPSCs).


Stem Cells Applications

Stem cell-based therapy can improve the health and quality of life of people with genetic disorders, cancer, and neurodegenerative diseases. The applications in medicine are vast and some examples include:

  • Hematopoietic Stem Cells Transplantation

Hematopoietic stem cell transplantation is one of the most popular stem cell therapies. It helps treat diseases in which the hematopoietic system is not working correctly, like leukemia and anemias. Target cells for this treatment usually derive from the bone marrow, peripheral blood, or umbilical cord blood. The procedure can be autologous (when it uses the patient's cells) or allogenic (when the stem cell comes from a donor).

  • Cell-based therapies

Researchers can induce stem cells to become a specific cell type to repair damaged tissue. People with macular degeneration, osteoarthritis, neurodegenerative disorders, and diabetes can benefit from this type of therapy.

This technique, for example, enables the creation of healthy heart muscle cells to be transplanted into patients with heart diseases. It can also improve diabetes treatment by inducing stem cells to differentiate into insulin-producing cells. It could also slow down the progression of incurable neurodegenerative disorders like Parkinson's or Alzheimer's and improve cognitive function in these patients.

  • Target for pharmacological testing

An exciting application of stem cells is in drug development. Pluripotent stem cells allow researchers to test drugs on living tissues safely. If any undesirable effects appear, it is possible to change the formula until it reaches the desired efficacy. Thus, the drug can enter the market without the need for animal testing.


Why is Quality Control so Important for Stem Cells?

For stem cells to be used in therapies, they must undergo rigorous quality control, [2]. This process guarantees the maximum effectiveness and safety of stem cells for use in humans. These include:


1. Tests for assessing the starting material:

  • Karyotype – long-term hESCs cultures can accumulate mutations. Thus, researchers must test them for genomic integrity.

  • Phenotypic pluripotency assays – this technique enables recognizing undifferentiated cells. Overall, stem cells show a high nucleus to cytoplasm ratio and a prominent nucleolus. The cells are flat with defined borders while differentiating colonies contain loosely located cells with rough walls.

2. Tests for single-cell transcriptomics (examining the gene expression level of individual cells),

  • Molecular analysis – it includes genotyping the source tissue, cells, and master cell banks to ensure the comparability of stem cell lines from different individuals. Single nucleotide polymorphism arrays (SNPs) are also helpful in detecting population polymorphisms.

  • Epigenetics analysis - the methylation process silences pluripotency genes when stem cells differentiate while other genes lose methylation markers.

3. Tests for tumorigenicity (i.e. capable of producing tumours):

  • Teratoma formation- this assay is important for demonstrating the pluripotency of human iPSCs by measuring their ability to form tumors.

  • Residual vector testing – to test for the presence of vectors integrated into the host genome, which can be dangerous.

4. Sterility tests:

  • Microbiology – it involves testing for the presence of viruses, bacteria, and fungi, ensuring cells are not contaminated with harmful microorganisms.

  • Identity analysis – for cell line identification, avoiding unintentional switching of lines and contamination.

5. Cell counts and viability analysis: cell counts and cell viability are vital metrics of every cell-based analysis. As stem cell therapies involve many quality control tests, it is necessary to repeat cell counts and viability analysis at several points during the development of these stem cell therapies. These metrics ensuring quality control during cell development and manufacturing processes.


How can The Inish Analyser Help You?

Traditional cell count and viability assays can be cumbersome as they require several steps due to the use of dyes. And worse, stains can affect your cell's functionality. So, commonly used assays are not preferable for stem cell therapy.


But it is now possible to perform fast analyses label-free, i.e. dye-free. Cellix’s Inish Analyser is an automated method for cell counting and viability analysis based on microfluidic impedance spectroscopy. This means it’s fast, enabling you to do quick viability checks several times throughout your workflow without hassle. Key advantages include:

  • Time Saving

The Inish Analyser gives you an automatic cell count in minutes. Unlike manual cell counting with hemocytometers, the Inish Analyser requires no cell staining, reducing the steps in your workflow.

  • Accuracy and reproducibility

Automated cell analysers like the Inish Analyser help to avoid user-to-user variability for more reliable results.

  • Cost-effective

The Inish Analyser is a cost-effective option for your lab since it doesn't require cell staining and it also doesn't require expensive slides/chambers.


How does it work?

The process is pretty straightforward; you only need to pipette 40 mL of your cell sample into a tube, add 360 mL of the Inish Analyser buffer, and select “Run Assay” on the Inish Analyser's touchscreen. The results are available to you in a few seconds.

Cell counting & viability of stem cells (Human Bone Marrow-derived Mesenchymal Stem Cells)

Depending on your results, it will generally show 3 visible populations:

  • Live cells, represented as green dots.

  • Dead cells, represented as red dots.

  • Debris population


Results are saved on the Inish Analyser and also easily exportable as standard FCS or CSV format.

If you'd like to learn more about the Inish Analyser, contact Cellix to book an online demo or request a quote.


References

1. Zakrzewski, W., Dobrzyński, M., Szymonowicz, M., & Rybak, Z. (2019). Stem cells: past, present, and future. Stem cell research & therapy, 10(1), 1-22.

2. Michele Trott, 28 April 2021. Key Techniques in Cell Therapy Quality. Technology Networks Biopharma. https://www.technologynetworks.com/biopharma/lists/key-techniques-in-cell-therapy-quality-control-331890



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