One of the most confusing areas for doctors and patients alike is the idea of what data indicates viable and functional stem cells in frozen umbilical cord and amniotic products. Most have no idea what to make of numbers thrown around by vendors, and many are easily confused. So if you’re a physician practicing in this space and don’t understand this topic, you are an easy mark for the scammers. Let me explain.
One of the problems with commercially available birth tissue products is that the initial viability of the cells is often in question. Meaning, there is a big difference between a birth product that is harvested from the OB ward and then brought to the lab to be immediately processed versus tissues that are saved in the hospital and then eventually shipped, processed, and frozen. As a rule, the longer the tissues are outside the body, the more cell death occurs.
In addition, when you freeze cells and then thaw them, this is a tremendous shock to the cells. Hence, properly frozen cells use certain strategies, like DMSO and controlled-rate freezing, to reduce ice crystal formation that can destroy cells. When these cells are thawed, they are often placed into an ideal cell-culture environment for days to make sure they recover. However, in the commercially available umbilical cord or amniotic products, the cells are shock-thawed in the hand and then injected.
These less-than-ideal conditions for sourcing and recovering cells from cryopreservation can result in lower initial viability numbers. For example, properly sourced, cryopreserved, and thawed cells have viability numbers that exceed 90%. However, when these numbers drop into the 80s, this generally indicates problems with cell health. When the numbers drop far lower, into what we often see with birth tissue products, such as viability numbers in the 40s to 70s, there are serious issues with cell health.
Viability in the context of cells simply means what percentage of the cells are alive. There are four levels of viability tests that go from the least meaningful to the most important. Regrettably, the companies claiming to sell vials of live and functional stem cells often quote only the least meaningful.
These levels are:
These tests can give wildly different results on the same sample. As an example, the first level of dye exclusion testing produces results that often don’t match up with the most meaningful test, a cell culture assay, giving a false positive viability test. To explain why, let’s review each test.
The simplest test that can be performed to determine a snapshot of viability is a dye exclusion test. Here are the pros and cons:
The test is based on the principle that living cells have intact cell membranes that exclude certain dyes (such as trypan blue, eosin, or propidium), whereas dead cells tend to have broken membranes that will allow the dye into the cell. So dead cells stain and live cells do not.
The biggest problem that we’ve seen with dye exclusion stains is that it’s possible for dead tissue fragments to exclude the dye because they aren’t permeable to the dye. We’ve seen this with testing amniotic products that have fragments of the extracellar matrix from macerated and finely chopped membranes.
The next test up from a simple dye exclusion stain is one that relies on the metabolic activity of the cell. Here are the pros and cons:
These tests usually work by taking advantage of natural cell enzymes to metabolize a chemical from colorless to colorful or fluorescent. Hence, unlike the dye exclusion tests, since they rely on metabolic activity to be positive, they are less likely to produce an immediate false positive. A commonly used test here is Sytox Red/Green.
The next step up is an apoptosis stain. Apoptosis is the process by which cells die. Here are the pros and cons:
When cells go into preprogrammed cell death (apoptosis), they put up a marker on their surface (phosphatidylserine-PS) that identifies them to the clean-up machinery of the body. This apoptosis marker can be bound by a fluorescent antibody (usually annexin). Hence, this test can tell if the cell is alive but dying.
Finally, the most reliable way to test if you have viable and functional cells is a cell culture assay. These are the pros and cons:
Here cells are placed in culture to determine how many will ultimately survive. Hence, this is the ultimate test of cell viability. However, the cells still need to be identified. For example, if the manufacturer claims mesenchymal stem cells, since they are adherent, one can simply rely on finding cells that attach to plastic. See my video below to see what this assay (also known as a CFU-f test) looks like. A second step would be identifying these cells via flow cytometry or differentiation assays.
The upshot? As a physician using these products and claiming that you’re injecting live and functional stem cells, it’s irresponsible to rely only on simple live/dead test results. That’s why you get paid the big bucks, to know the finer points of the science behind the wild claims of vendors selling products. Hence, please educate yourself. Right now, unless you have apoptosis testing and culture assays, relying on simple viability tests is a recipe for serious problems!
*DISCLAIMER: Like all medical procedures, Regenexx® Procedures have a success and failure rate. Patient reviews and testimonials on this site should not be interpreted as a statement on the effectiveness of our treatments for anyone else.
Providers listed on the Regenexx website are for informational purposes only and are not a recommendation from Regenexx for a specific provider or a guarantee of the outcome of any treatment you receive.
About the Author
Christopher J. Centeno, M.D. is an international expert and specialist in regenerative medicine and the clinical use of mesenchymal stem cells in orthopedics. He is board certified in physical medicine as well as rehabilitation and in pain management through The American Board of Physical Medicine and Rehabilitation.…