A stem cell certificate of analysis: how cells are tested before they reach you

How are stem cells tested before they reach a patient?

1. Sterility: nothing else in the vial

   The first job of the testing panel is to prove that the only living things in the vial are MSCs. Anything else (bacteria, fungi, mycoplasma, viral contaminants) would be injected directly into a patient, often into the bloodstream. The consequences range from local infection to systemic sepsis, with a real mortality rate. cGMP exists primarily to prevent this.

   Day1234567891011121314

   BacT/ALERT Rapid · early read

   Day 7 · No growth

   USP <71> Gates release · 14d

   Day 14 · No growth

   Release gate

   Day 7 · early readDay 14 · batch released

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   Bacteria + fungi

   Detected by 14-day USP <71> culture and 7-day rapid systems.

   Spec · No growth

   Mycoplasma

   The most common cell-culture contaminant. Invisible to USP <71>. PCR-detected.

   Spec · Negative

   Endotoxin

   Bacterial-cell-wall fragment. Detected by LAL assay even when no living bacteria remain.

   Spec · < 0.5 EU/mL

   Viral panel

   Donor pre-screen: HIV, HBV, HCV, HTLV, syphilis. PCR + serology.

   Spec · Negative

2. Identity: that they are MSCs

   A cell count tells you how many cells. An identity assay tells you what kind of cells. The International Society for Cellular Therapy (ISCT) published minimal criteria for what counts as a mesenchymal stem cell in 2006; these are the global standard. Every cGMP MSC product is released against them.

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   Positive markers Must express

   CD73 Ecto-5′-nucleotidase

   98.4%

   CD90 Thy-1 glycoprotein

   99.1%

   CD105 Endoglin

   97.6%

   Release spec · ≥ 95% positive

   Negative markers Must lack

   CD45 Pan-leukocyte

   0.3%

   CD34 Hematopoietic SCs

   0.1%

   CD14 · HLA-DR Monocyte / activated

   0.4%

   Release spec · ≤ 2% positive

   The ISCT criteria are not just "has these markers." They are "has these AND lacks these." A cell with the right positive markers but the wrong negative markers is contaminated. A vial with 15% CD45 is contaminated with white blood cells; with 10% CD34, with hematopoietic stem cells. Neither is a pure MSC product. This is why the "100M cells" headline is meaningless without a flow panel.

3. Viability: that the cells are still alive

   Identity proves the cells are MSCs. Viability proves they're alive and metabolically active when they reach the patient. Therapeutic effect depends on living cells: dead cells don't signal, don't release a useful secretome, and in fact can produce a pro-inflammatory response, the opposite of what's intended. Viability is measured after thaw, right before treatment, on the actual lot being released.

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   The physician team's laboratory holds every batch to a post-thaw viability standard of 95% or above before any treatment proceeds, well above the regulatory minimum for cell viability. Batches that do not meet this threshold are not used. They are discarded, not "released with a note." Viability is a release criterion, not a marketing claim. The number on the Certificate of Analysis is the number from the bag the patient receives, not a number from before freezing.

   Ask: "Is viability tested on the day of treatment, post-thaw, on the same lot I'm receiving, and what is the release floor?"

   The answer should be yes, post-thaw, and a specific number the lab commits to. A clinic quoting only a manufacturing-time viability number is quoting from before freezing, which is not the number that matters.

4. Potency: that they still function

   Identity proves the cells are MSCs. Potency proves they still do the things MSCs are supposed to do. Cells can be alive, the right phenotype, and yet senescent: they no longer divide, no longer secrete the cytokines that drive immunomodulation, no longer differentiate. Potency assays catch this.

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   Tri-lineage differentiation Osteo · Chondro · Adipo

   All 3 Pass

   IDO induction Immunomodulation

   2.4× threshold Pass

   T-cell suppression Co-culture assay

   76% suppression Pass

   Cytokine secretion VEGF · HGF · IL-10 · TGF-β

   Within range Pass

   Sterility and identity are binary. Potency is graded: a cell can be 30% as potent as a fresh-from-tissue cell and still pass an identity panel. The newest FDA draft guidance on potency for cell therapies (2023) asks sponsors to define and validate a potency assay relevant to the proposed mechanism of action. Operations selling MSCs with no potency data are operating below the regulatory standard required for clinical trials.

   "Our cells are viable and pass identity" is not a complete answer. Both can be true while the cells are functionally dead, alive but no longer secreting the cytokines that make MSC therapy work.

   Ask: "What's the potency spec for this batch, and what was the measured result?"

5. Residuals: what's in the vial that isn't cells

   Manufacturing MSCs requires reagents: growth media, antibiotics, dissociation enzymes, cryoprotectants, serum. By release, these should be reduced to trace amounts or removed entirely. Residual testing quantifies whatever remains. These checks, with karyotype and tumorigenicity, are qualification work on the process and the cell bank. They sit beside the six release tests, not among them.

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   What gets quantified, and limited.

   Residual BSA	Bovine serum albumin from culture media · ELISA quantitation	< defined limit
   Residual antibiotics	Gentamicin or other antibiotics from culture · HPLC or immunoassay	< defined limit
   Residual DMSO	Cryoprotectant · removed by a post-thaw wash, then quantified before clinical use	< 10% v/v
   Residual trypsin	Dissociation enzymes · ELISA · should be removed by wash steps	< defined limit
   Karyotype	When a cell bank is made · G-banded chromosome analysis · confirms no abnormalities during expansion	46, XX / XY · bank only
   Tumorigenicity	Master Cell Bank · soft agar + in-vivo immunodeficient mouse assay	Negative · MCB only

   MSCs grown through many passages can accumulate chromosomal changes. Most don't, but some can. A karyotype, run when a cell bank is made, confirms the banked cells have a normal chromosome complement. Same-day, non-expanded preps don't need it because the cells haven't been through enough divisions to develop abnormalities, but they also haven't been characterized at all.