How to Determine Cell Viability and Vitality

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Many physicians and patients get pitched on amniotic and cord “stem cell” products based on a document that purports to show that these cells are “viable”. However, there’s much more to the story than a simple viability percentage. You need to know a lot more to determine if cells are vital and healthy.

What Is Cell Viability and Cell Vitality?

Viability simply means the percentage of cells that are living. Viability is not the same thing as cell vitality, so it doesn’t reveal the full story of a cell’s health. Let’s use you as an analogy for cells. Since you are reading this right now, you would be counted as alive, or viable, in a live/dead test. However, so would the guy at your local hospital in the ICU on his deathbed. In addition, that same live/dead test would count the 90-year-old across the street with severe Alzheimer’s and who can barely walk as viable. You get the point, viability doesn’t equal health.

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How to Determine Cell Viability and Vitality

There are four assays that need to be performed to determine viability and vitality:

  • Simple viability
  • Apoptosis
  • Cell proliferation
  • Cell function

While the simple viability assay is the test you most often see, it’s important to understand that all four tests must be done to properly determine not just cell viability or live cells, but also vitality or healthy cells.

Simple Viability Assay

The most common type of simple viability assay is called a “dye exclusion” test. Cells that are living have an intact membrane. Cells that are dead have a damaged membrane that is not intact. When dye (e.g., trypan blue, eosin, or propidium) is added to a bunch of cells, the living cells will exclude, or not take up, the dye due to their intact and functional membrane. The dead cells will take up the dye because their membrane is not intact.

Apoptosis Assay

Cells also have a process called apoptosis, which means they’re dying but not dead yet. In apoptotic cells, the membrane is still intact, so they typically don’t take up the dye in dye exclusion tests and are counted as viable cells in a simple cell viability assay.

Consider a high school wrestler in the ER with a fractured arm and an unconscious guy upstairs in ICU who’s nearing his final breath. For one of them, life goes on; for the other, death is imminent. In both cases, however, they are currently alive. However, an apoptosis test can differentiate that the guy in the ICU shouldn’t be counted as healthy.

The apoptosis assay, uses an “I’m dying” marker. How? Using flow cytometry is a technology that measures whether a fluorescent marker attaches to the surface of a cell at a specific receptor. In this case, the molecule used is usually Annexin that binds to a phosphatidylserine molecule (the ‘I’m dying marker). The Annexin has a fluorescent dye on it that will light up when it passes through the LASER light in the flow cytometer.

So credible cell viability and health studies always include measuring the level of dying or apoptotic cells as well as the number of viable cells via flow cytometry. If we only use the simple viability assay, we won’t get a clear picture of which viable cells are truly healthy and which are dying. Somehow, amniotic and cord blood tissue manufacturers always manage to skip this test.

Cell Proliferation Assay

The cell proliferation assay is another essential test for viability and vitality. It involves growing the cells in culture to see if they form colonies and propagate. Live, viable, healthy cells will thrive during this test, while dead and apoptotic cells will not grow or reproduce. This is a very simple and inexpensive test to perform that doesn’t require specialized equipment, so there is absolutely no reason to skip this test. However, just like the apoptosis assay, it’s almost never performed by amniotic and cord tissue vendors.

Cell Function Assay

Finally, for stem cells, a functional assay is needed. And this is usually via a trilineage differentiation test for mesenchymal stem cells (MSCs) to make sure that they can still function and differentiate (i.e., become osteoblasts, adipocytes, and chondrocytes). This makes sure that cells that can grow haven’t been damaged and can still carry out one of their important key tasks.

The upshot? As you can see, there’s a lot more to know about cell health than just simple viability. Yet somehow, we never seem to see a complete test of cell viability and vitality from amniotic and cord blood tissue vendors. Why? That is, of course, the right question be asking. The IOF asked this question and found that it couldn’t reproduce the viability numbers being reported. In addition, in the four amniotic and one cord blood samples it has tested, everything died and wasn’t able to propagate. Hence, don’t accept simple viability as proof that the cells you’re being sold will live to do some good for your patients.

This blog post provides general information to help the reader better understand regenerative medicine, musculoskeletal health, and related subjects. All content provided in this blog, website, or any linked materials, including text, graphics, images, patient profiles, outcomes, and information, are not intended and should not be considered or used as a substitute for medical advice, diagnosis, or treatment. Please always consult with a professional and certified healthcare provider to discuss if a treatment is right for you.

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6 thoughts on “How to Determine Cell Viability and Vitality

  1. Gloria Tangco

    Thks so much for educational information. We started using the cord blood and amniotic fluid for our aesthetic and Pain Therapy combined with Prp . Kindly update us concerning the regenerative medicine approach therapy. God bless

    1. Regenexx Team Post author

      Both we and the Interventional Orthopedics Foundation (a nonprofit) have tested every aminiotic and cord blood product we’ve been able to access, and have found no live viable cells in any of them. Please see: and

  2. Michael Moloney

    Good information.

    Thank you

  3. Robert Levin

    Does this apply to the use of bone marrow stem cells that other practitioners do?
    Another question: Is it possible to get good stem cells from bone marrow from other parts of the body other than the hip, such as the Femur or the Tibia?

    1. Regenexx Team Post author

      Robert Levin,
      Yes. The amount of viable and healthy stem cells that are harvested affect outcome as stem cell treatment is dose dependent. Some doctors, for conveinience perform BMA on the tibia or femur, however it does affect what they will be able to harvest and it’s also more painful. Please see:

    2. Chris Centeno Post author

      Most practitioners who use bone marrow don’t measure viability, but we do. Bone marrow stem cell yields from sites other than the back of the pelvis (PSIS) are less.

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