What Is Passage Number
In cell culture, "passage" refers to the process of detaching, diluting, and reseeding cells when a culture flask becomes confluent. Each time cells are passaged, they undergo cell division to re-populate the flask. "Passage number" (P) is the count of how many times this process has occurred since the cells were originally isolated from tissue.
A cell at passage 2 (P2) has been subcultured twice from the original isolation. A cell at passage 10 (P10) has been through ten rounds of expansion. In terms of cell biology, these are not equivalent states. Every cell division carries biological cost.
The relationship between passage number and MSC function is well-established in the published literature. The trajectory is consistent: early passage cells are more potent, more secretory, and more representative of native MSC biology. Later passage cells accumulate replicative stress, lose multipotency, and shift toward a senescent phenotype that has fundamentally different paracrine activity.
Replicative Senescence in MSCs
What Happens as Cells Divide
Each cell division introduces potential for error. DNA damage responses accumulate. Telomeres — the protective caps on chromosome ends — shorten with each replication cycle. Epigenetic drift occurs: methylation patterns that regulate gene expression shift away from their original state. The cumulative weight of these changes is replicative senescence: a state in which cells remain metabolically active but have fundamentally altered their gene expression program.
For MSCs specifically, the senescence trajectory is characterized by:
- Loss of multipotent differentiation capacity (reduced adipogenic and osteogenic potential)
- Increased cell size and morphological irregularity
- Elevated beta-galactosidase activity (a classic senescence biomarker)
- Upregulation of p21 and p16 cell cycle inhibitors
- Activation of the NF-kB inflammatory signaling pathway
- Shift in secretome composition toward pro-inflammatory cytokines
The Senescence-Associated Secretory Phenotype (SASP)
The most clinically relevant consequence of MSC senescence for exosome manufacturing is the emergence of the senescence-associated secretory phenotype (SASP). SASP is the term for the shift in paracrine output that accompanies cellular senescence — a shift away from growth-promoting, immunomodulatory, and tissue-supportive factors toward a pattern dominated by pro-inflammatory cytokines and matrix-degrading enzymes.
SASP in senescent MSCs includes upregulation of IL-6, IL-8, MCP-1, and matrix metalloproteinases (MMPs). These are not the molecules associated with the therapeutic rationale for MSC-derived exosome therapy. They represent a shift in cell identity — the cell is no longer behaving as a healthy MSC. It is behaving as a senescent cell, and its exosomes reflect that state.
"By passage 8 and beyond, the exosomes produced by MSCs carry a cargo that increasingly reflects senescent cell biology — not peak MSC secretory activity. These are different products, regardless of what is on the label."
What the Research Shows Across Passages
| Passage | Secretory Status | SASP | Growth Factors | Inflammatory Markers | Clinical Relevance |
|---|---|---|---|---|---|
| P1–P2 | Peak (post-isolation) | Absent | Highest (VEGF, HGF, IGF-1) | Low | Optimal |
| P3–P4 | High | Absent | High | Low | Optimal |
| P5–P6 | Moderate | Emerging | Declining | Increasing | Borderline |
| P7–P8 | Declining | Present | Reduced | Elevated | Not recommended |
| P9+ | Low (senescent) | Established | Low | High (IL-6, IL-8, MMPs) | Unsuitable |
Specific Cargo Changes Documented by Research
The passage-dependent decline in MSC quality is not a qualitative impression — it has been characterized at the molecular level in peer-reviewed literature. Key findings include:
Growth factors: VEGF (vascular endothelial growth factor), HGF (hepatocyte growth factor), and IGF-1 (insulin-like growth factor 1) are all significantly higher in exosomes from early-passage MSCs compared to late-passage preparations. These are among the most studied growth factors in the context of tissue repair and cellular signaling.
miRNA cargo: The miRNA payload of MSC-derived exosomes changes dramatically with passage. Early-passage cells produce exosomes enriched in miRNAs associated with proliferation, angiogenesis, and anti-apoptotic signaling. Late-passage cells show upregulation of senescence-associated miRNAs that can actually promote senescent phenotype transfer to recipient cells — a phenomenon sometimes called "senescence bystander effect via exosomes."
Surface markers: While core tetraspanin markers (CD9, CD63, CD81) remain relatively stable, functional surface molecules that facilitate exosome uptake and signaling — including CD44, CD73, and integrin expression profiles — shift with passage in ways that affect interaction with recipient cells.
The Scale Problem
The challenge for commercial exosome manufacturing is that cell numbers — and therefore passage number — are directly linked to production volume. Starting from a primary isolation that yields a limited number of cells, reaching the quantities required for commercial production requires extensive expansion. Every additional passage of expansion is another step toward senescence.
This creates a fundamental tension in exosome manufacturing between scale and quality. A manufacturer can:
- Use high-passage cells to reach large production volumes cheaply — accepting SASP emergence and declining secretory quality as a trade-off
- Maintain strict low-passage limits (P2–P4), accept smaller batch sizes, and manage the cost of more frequent cell sourcing and banking operations
- Develop working cell bank (WCB) and master cell bank (MCB) infrastructure that allows production from cryopreserved low-passage stocks — adding process complexity but maintaining passage control at commercial scale
The first approach is the path of least resistance. The second and third approaches are what quality-focused manufacturing requires. The passage number used in production is a proxy for which path a manufacturer has chosen.
Passage number is a useful shorthand but is an imprecise measure of cellular age. The biologically meaningful metric is population doubling level (PDL) — the total number of times the cell population has doubled since initial isolation. A cell at P3 may have undergone 6–12 total population doublings depending on seeding density and growth conditions.
Rigorous quality-focused manufacturers track PDL alongside passage number because PDL is more directly connected to the accumulation of replicative stress. A specification that states "production cells must be at P2–P4 with PDL not exceeding 12" is more meaningful than a simple passage number cutoff.
When evaluating products, ask whether the manufacturer tracks PDL — not just passage number.
What P2–P4 Means in Practice
Specifying passage 2–4 for production cells is not an arbitrary quality parameter. It represents the window during which MSCs, as extensively characterized in the published literature, exhibit:
- Maximal multipotent differentiation capacity
- Highest expression of immunomodulatory surface markers (CD73, CD90, CD105)
- Peak secretory output of growth factors and anti-inflammatory mediators
- Minimal p21/p16 senescence marker expression
- Lowest NF-kB activation and SASP cytokine secretion
- Telomere lengths consistent with robust replicative capacity
Exosomes produced from cells within this passage window carry a cargo that reflects this phenotype. The molecular memory of cell age is encoded in the vesicle.
Key References
- Choudhery MS, Khan M, Mahmood R, Mehmood A, Khan SN, Riazuddin S. Bone marrow derived mesenchymal stem cells from aged mice have reduced capacity to support cardiac repair. J Cell Mol Med. 2012 Jul;16(7):1551-60.
- Behrmann A, Khan A, Xu S, et al. Passage-dependent decrease in human MSC paracrine activity is associated with alteration of mitochondrial status and exosome secretion. Stem Cell Rev Rep. 2021;17(5):1862-1876.
- Turinetto V, Vitale E, Giachino C. Senescence in human mesenchymal stem cells: functional changes and implications in stem cell-based therapy. Int J Mol Sci. 2016 Jul;17(7):1164.
- Galipeau J, Sensebe L. Mesenchymal stromal cells: clinical challenges and therapeutic opportunities. Cell Stem Cell. 2018 Jun 1;22(6):824-833.
- Coppe JP, Desprez PY, Krtolica A, Campisi J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu Rev Pathol. 2010;5:99-118.
Next: Molecular Cargo
Now that the manufacturing variables are clear, look at what the exosome actually carries.