Selection of Cell Passages for Clinical Application of Stem Cells

Looking back at the history of human development over the past few hundred years, the 19th century was the century of the industrial revolution represented by steam engines, the 20th century was the age of information technology represented by computers and communications, and the 21st century will be the age of life sciences represented by biotechnology. century with technology.

Stem cell technology is one of the core contents of biotechnology. Regenerative medicine or tissue regeneration treatment based on stem cells is another revolution in medical technology after drug treatment and surgical treatment. Stem cell treatment has more important significance in the recovery stage of the disease. With the continuous exploration of scientific researchers, there is new hope in the treatment of refractory diseases such as diabetes, heart disease, and Alzheimer's disease.

When using stem cells to treat diseases, attention should be paid to various issues such as treatment timing, administration route, cell type selection, and quantity used. Only by finding the best feasible solution among multiple factors can we help this technology to be implemented faster .

When applying stem cell therapy clinically, it is often easy to ask which passage of stem cell therapy is more appropriate. These are questions that should be thought about and yet to be solved. We all know that people are divided into men, women, old and young, and stem cells are also divided into young and old. Therefore, the passage of cells is an important factor affecting the activity, clinical effectiveness and safety of stem cells .

Theoretically, the higher the cell passage, the stronger the stemness of the stem cells. However, during the process of stem cell culture in vitro, stem cells undergo an adjustment and adaptation process from the tissue microenvironment to the in vitro culture environment. Some cells that do not adapt will be eliminated. Therefore, in the first two or three passages, the stem cell genome has unstable factors, which may Not suitable for clinical application.

Studies have shown that through karyotype analysis, stem cells have a higher proportion of abnormal karyotypes in the first three passages of cells. For mesenchymal stem cells ( MSCs ), the optimal passage number for clinical application should be 4 to 6 passages.

In current clinical research, the most common sources of MSC are bone marrow, fat, umbilical cord, placenta, etc. This article summarizes the three common sources of umbilical cord, fat, and placenta. For mesenchymal stem cells from different sources, the differences after in vitro subculture are different. Changes in some biological characteristics of passages (such as cell morphology, cell cycle detection, surface marker detection, karyotype analysis, related gene expression and quantitative analysis of cytokines) are insufficient due to limited reference materials. Please understand.

umbilical cord

Among various types of mesenchymal stem cells from different sources, human umbilical cord mesenchymal stem cells ( hUC -MSCs), as the "younger" adult mesenchymal stem cells, have prenatal and postnatal capabilities. The comprehensive advantages of MSC have attracted more and more widespread attention.

After long-term in vitro subculture of human umbilical cord mesenchymal stem cells, their basic stem cell biological properties, such as cell surface immune markers and induction of multilineage differentiation potential, did not change significantly. After long-term culture up to 20 passages, no pathogenic or tumorigenic gene mutations were found. There was no significant difference in cell proliferation activity and telomerase activity between high passage (P20) hUC-MSCs and early low passage (P5) cells. As the number of culture passages increases, the cells begin to age, especially in the 20th passage cells .

After in vitro subculture, hUC-MSCs showed exactly the same cell morphology and growth arrangement at the 5th and 10th passage, showing long spindle-like adherent growth and mild spiral flow-like arrangement. The 20th passage cells showed very similar long spindle-like adherent growth, but the cell body was slightly enlarged and the local arrangement was disordered after fusion.

Studies have shown that although the karyotype of hUC-MSCs cultured at high passages remains stable, their telomerase activity will decrease by the 7th passage. We detected the expression of the human telomerase reverse transcriptase activity subunit gene and showed that there was no significant difference between the telomerase activities of hUC-MSCs at passages 5, 10 and 20; and cells of different passages showed similar growth. The proliferation curves showed that there was no difference in their cell proliferation activity.

Detection of proliferation activity of human umbilical cord mesenchymal stem cells of different passage

hUC-MSCs of different passages (5th, 10th, and 20th passage) were seeded in 6-well culture plates at the same density, and hUC-MSCs of different passages were collected from 3 wells every day to count and draw the growth curve. The illustration represents three replicate experiments.

In addition, due to the strong professionalism and limited space, similar procedures (conventional karyotype analysis, chromosomal karyotype analysis, surface marker detection, gene expression and cytokine quantitative analysis) will not be described again. In short, although hUC-MSCs at the 20th passage still maintain stem cell biological properties, gene stability and growth characteristics similar to those of lower-passage cells, as the passage and culture time increases, the cells begin to show a certain degree of aging. In view of the fact that excessive subculture will lead to aging signs of hUC-MSCs, it is recommended that the in vitro culture of hUC-MSCs used for stem cell clinical treatment should not exceed the 10th passage .

placenta

Mesenchymal stem cells are rich in sources, and placental mesenchymal stem cells also have the advantage of being easy to obtain. The full-term placenta is a waste product after fetal delivery. It is easy to obtain and large in volume. The cost of scientific research is low and does not involve social and ethical issues. Moreover, placental mesenchymal stem cells have functions and characteristics similar to bone marrow mesenchymal stem cells, and have a better proliferation ability than bone marrow mesenchymal stem cells, and can greatly expand the number of mesenchymal stem cells.

Some studies have tested the proliferation ability of P5, P10 and P15 passage cells respectively. The results show that the proliferation ability of P5 passage mesenchymal stem cells is significantly higher than that of P10 and P15 passage. The experimental results show that when placental mesenchymal stem cells are cultured in vitro, , as the number of cell passages increases, cells will gradually age. Since the proliferation ability of placental stem mesenchymal cells will gradually decrease after the P5 passage, it is best to choose mesenchymal stem cells below the P5 passage for in vitro culture for clinical application.

Growth curve, proliferation ability and passage results of placental mesenchymal stem cells

Note: Figure A shows that the doubling time of P5 passage cells is significantly shorter than that of P10 and P15 passage cells; Figure B shows that the proliferation abilities of placental mesenchymal stem cells of different passages are different, and the proliferation ability of P5 passage is significantly higher than that of P10 cells. and P15 passage, compared with P5 passage, aP < 0.01, bP < 0.001, compared with P10 passage, c < 0.05; C is placental mesenchymal stem cell specimens from 5 cases passed to 25th passage, with no adherent cells or weak adhesion. , but the proliferation rate is fast.

In this study, placental mesenchymal stem cells can be passaged for a long time to the 25th passage. Placental mesenchymal stem cells of different passages have different proliferation abilities. The proliferation ability of the P5 passage is significantly higher than that of the P10 and P15 passages, revealing that clinical application should be based on Preferable before P5 passage.

Fat

Adipose derived mesenchymal stem cells ( ADSCs) are a type of stem cells that exist in adipose tissue and have multi-directional differentiation potential. Cell sources are more abundant and materials are more convenient to obtain. Moreover, its immunogenicity is very low and it is easier to amplify in vitro. These advantages make it have better application prospects in tissue engineering, wound repair and gene therapy.

Studies have cultured ADSCs in vitro to passages 1, 3, 5, 7, 10, 14, and 15. It was found that after passage of ADSCs to the 7th passage, the cell proliferation rate began to slow down, and the cell proliferation rate slowed down significantly after the 10th passage. The cell morphology is long spindle-shaped and remains stable.

There are no obvious changes in the karyotype of chromosomes such as translocations and deletions, the expression levels of related genes are slightly different, and the cytokines secreted in the culture medium remain stable.

Studies have shown that during the passage of adipose mesenchymal stem cells (cultured in serum-free medium) to 5 passages, the biological and genetic characteristics are basically stable without significant changes. It has been initially revealed that adipose mesenchymal stem cells can be safely used up to the 5th passage in vitro .

Conclusion

It is not difficult to see from the above studies that mesenchymal stem cells can theoretically be cultured for a long time, but it is not recommended to select batches of stem cells with higher passages in clinical applications. MSC cells that have not been expanded in vitro are safe and harmless unless they have some genetic defects. The biggest risk in in vitro expansion is genetic mutation. A research team found that when bone marrow MSCs were cultured in vitro for 18 passages, gene mutations and chromosomal abnormalities appeared. A large number of other studies have confirmed that chromosomal abnormalities will only occur when MSC are cultured in vitro for too high a number of passages (more than 10 passages) .

As mentioned in previous articles, another issue that affects the safety of stem cell reinfusion is additives , such as using serum-free medium instead of animal serum culture; avoiding the use of antibiotics through strict aseptic operation. However, for irreplaceable components of cell culture such as trypsin, it is also crucial to strictly inspect the quality during reinfusion to ensure that no components remain.

Postscript

As the effective component of cell preparations, mesenchymal stem cells have their own safety issues that cannot be ignored; the quality control system of cell preparations during the production and preparation process also affects the safety of their reinfusion.