This article in 30 seconds
- Cell enlargement is a sign of senescence. The shift in which repeatedly divided cells flatten and grow larger is reported as a feature of cellular senescence, which makes cell size a usable gauge of the condition of the source cells.
- The measured gap is 1.5-fold in diameter, about 3.4-fold in volume. The conventional medium gave 22.3 μm in diameter after 30 days of culture; our proprietary medium gave 14.9 μm after 20 days.
- The aim is manufacturing quality control, not a claim of therapeutic efficacy. These are records kept so that the condition of the cells at harvest stays consistent from lot (a batch manufactured in one run under the same conditions) to lot.
1. What enlargement indicates — the ticket book of cell division
The short answer first: cell enlargement is reported as a sign of aging that appears in cells which have divided many times. It is not a change of appearance alone — it is an indicator of the condition of the cells.
The number of times a cell can divide is known to have a ceiling, much like a ticket book holding a fixed number of tickets. In cells that have worked their way through those tickets — that is, cells that have divided again and again — morphological changes appear: flattening (spreading out thin and wide) and enlargement. These are among the features that accompany replicative senescence.
In manufacturing a secretome (the totality of components a cell secretes during culture), that morphology carries practical weight. Cook to the same recipe with ingredients in a different state and the dish comes out differently; in the same way, if the condition of the source cells varies from lot to lot, the composition of the secreted components collected from them becomes harder to keep consistent.
The key point
Cell size is an objective measure that returns the same number whoever takes it. We measure and record it as one of the inputs to deciding when to harvest.
2. The measured data — diameters of 22.3 μm and 14.9 μm
The result first. Cells cultured for 30 days in a conventional medium measured 22.3 μm in diameter; cells cultured for 20 days in our proprietary medium measured 14.9 μm. Below are the actual cells, photographed on a hemocytometer (a counting chamber for counting cells under a microscope) and shown at the same magnification.
Cell diameter 22.3 ± 0.75 μm
Cell diameter 14.9 ± 0.21 μm
On the left, cell sizes vary, with broken outlines and clumps visible. On the right, round cells are evenly and uniformly distributed. The "±" attached to the values is the standard error of the mean (SEM).
A 1.5-fold gap in diameter becomes roughly a 3.4-fold gap in volume — a much larger difference than the raw numbers suggest.
The key point
Make a balloon 1.5 times wider and the air inside goes up by the cube, to about 3.4 times. Cells behave the same way, so "1.5 times the diameter" means about 3.4 times the contents. A diameter figure has to be read in volume terms.
3. Why the gap arises — days to reach the target count
The short answer: what creates the gap is the number of days needed to reach the required cell count. There is no special treatment that stops enlargement itself.
Reaching the required cell count in a shorter culture period means the cells can be harvested before enlargement progresses. Arrive at the same destination ten days earlier and the cells you collect are that much younger.
The key point
This is not a technology that stops aging; it is one that shortens the wait. Nothing is done to the cells themselves — the fact that fewer days are needed before harvest is what produces the difference in diameter shown in Figure 1.
Growth over time
The difference in speed shows up along the way as well. These are phase-contrast microscope images at day 6 and day 10 of culture; in each pair the first image is the conventional medium and the second our proprietary medium.
Day 6 of culture
Day 10 of culture
Comparing the same day side by side makes the difference in how quickly the field fills clear. By day 10, cells in our proprietary medium cover almost the entire surface.
4. Corroboration beyond morphology — surface markers
The short answer: a measurement independent of morphology shows a difference in the same direction. Impressions taken from photographs invite subjectivity, so we also measure the surface markers used to identify mesenchymal stem cells (stem cells that can differentiate into bone, fat, and other tissues).
| Item | Competitor medium | Proprietary medium | Criterion |
|---|---|---|---|
| Viability | 97.2% | 99.1% | Higher is better |
| CD44 | 100% | 100% | Positive |
| CD73 | 99.5% | 100% | Positive |
| CD90 | 99.2% | 100% | Positive |
| CD105 | 85.3% | 99.2% | Positive |
| HLA-ABC | 98.6% | 98.6% | Positive |
| CD31 | 0.6% | 0.5% | Negative |
| CD45 | 0.3% | 0.2% | Negative |
| HLA-DR | 0.3% | 0.4% | Negative |
Positive/negative classifications follow the minimal criteria for mesenchymal stem cells set out by the International Society for Cell and Gene Therapy (ISCT).
The one to watch is CD105: 85.3% against 99.2%, a gap of 13.9 percentage points. CD105 is a marker known to decline with repeated passaging (splitting expanded cells and re-seeding them) — a yardstick separate from cell size, yet it points in the same direction.
The other positive markers are high in both media, with only marginal differences. The negative markers are sufficiently low in both; HLA-DR alone is slightly higher in our proprietary medium, but both values remain below 1%.
5. How to read this data — three caveats
There are three things a reader should know about this data. In the interest of fairness, we state them up front.
- This is not a controlled experiment at a single time point. The conventional medium reached the equivalent of 100 million cells in 30 days; our medium reached the equivalent of 500 million cells in 20 days — both the culture duration and the final cell count differ. This data does not answer what would happen if the durations were matched
- The competitor product used for comparison is not disclosed. This is not a comparison a third party could reproduce under the same conditions
- The "±" attached to the cell diameters is the standard error of the mean (SEM)
With that said, what this article demonstrates is control of the source cells' condition in the manufacturing process. The aim is to record cell size at harvest and keep it consistent across lots. It is not data showing that one product is therapeutically better than another.
Contract culture services available
The medium and culture technology shown here are available not only in our own production but also as contract culture services. We can accommodate requests such as expanding cells without animal-derived components, or keeping the condition of cells at harvest consistent.
- Services
- Expansion culture of mesenchymal stem cells, consultation on culture conditions, and measurement of cell count, cell diameter, and surface markers
- Culture environment
- Class 100 biosafety cabinets inside the Class 10,000 cleanroom at our Ginza manufacturing facility
- Medium
- Our in-house medium — xeno-free, serum-free, and protein-free
- Getting started
- Tell us the cell type, quantity, and schedule, and we will confirm feasibility and provide a quote
Depending on the conditions, we may not be able to accept a request, so please start with a consultation. Select "Contract culture consultation" on the contact form and our staff will get back to you.
For the difference between conditioned medium and secretome, see Conditioned Medium vs. Stem Cell Secretome.
Frequently Asked Questions
What is the problem with cells getting larger?
Flattening and enlargement are reported as morphological changes seen in cells that have undergone repeated divisions. If the condition of the source cells varies from lot to lot, the composition of the secreted components collected from them becomes harder to keep consistent. We record cell size as one of the criteria for deciding when to harvest.
How much difference does a 1.5-fold difference in diameter make?
Assuming a spherical cell, volume scales with the cube of the diameter. Diameters of 22.3 μm and 14.9 μm differ by a factor of about 1.50, which corresponds to a roughly 3.4-fold difference in volume.
Do you offer contract culture services?
Yes. We offer expansion culture (growing cells to the required number) under conditions free of animal-derived components, along with measurement of cell count, cell diameter, and surface markers. Depending on the cell type, quantity, and schedule, we may not be able to accept a request, so please contact us via the "Contract culture consultation" option on the contact form.
Do you disclose compositional data?
We measure cytokine levels and the miRNA content of exosomes for every production lot, and provide a Certificate of Analysis on request. For details, see Safety & Quality.
This article describes how the product is manufactured and how its quality is controlled. Our stem cell secretome is supplied for research use only and is not intended to demonstrate efficacy against any specific disease.
Sources and primary literature
- Minimal criteria for mesenchymal stromal cells: Dominici M, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-317. (Basis for classifying the surface markers as positive or negative)
- Morphological changes accompanying cellular senescence: Wagner W, et al. Replicative senescence of mesenchymal stem cells: a continuous and organized process. PLoS ONE. 2008;3(5):e2213. (Reports the enlargement and flattening of cells over serial passaging)
- Measured values: The cell diameters, growth over time, surface markers, viability figures, and micrographs in this article come from our own measurement data.
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