This article was written by YANG, Fujian Medical University
Review expert of this article: Professor Li Jing from the Affiliated Hospital of Jiangsu University
introduction
In recent years, stem cells have become a hot field in the field of cell therapy, involving more and more diseases. Cryopreservation is the most common method of stem cell storage and transportation, and has become the current gold standard. Long-term storage of cells through freezing technology provides resources for subsequent scientific research, disease treatment, etc. So, after a long period of freezing, does the viability and function of stem cells change? According to the published literature, mesenchymal stem cells with good differentiation ability can still be obtained after more than 20 years of cryopreservation. There are also multiple domestic literatures confirming that the biological characteristics of umbilical cord/placental mesenchymal stem cells remain stable after cryoresuscitation. These scientific publications provide strong evidence for the future application of cryostored mesenchymal stem cells.
In recent years, the development of cryogenic storage technology has been widely used in stem cell preservation, which has promoted the development of stem cell storage and laid the foundation for the clinical development and application of stem cell therapy. Cryopreservation is increasingly being used in the clinical development of cell therapies, and new clinical trial applications for cryopreservation-based cell therapies have been approved.
With the gradual popularity of cryopreservation stem cells, there is also a lot of interest and curiosity about the function of stem cells after cryopreservation. For example, after 20 or 30 years of cryopreservation, will the activity and function of stem cells remain the same as before? Today we will give you the answer through the latest literature.
The literature confirms:
Mesenchymal stem cells that are cryopreserved for more than 20 years still have a strong differentiation potential
In one study [1], mesenchymal stem cells were obtained by thawing and expanding human cell samples that had been cryopreserved for 23-25 years, and comparing the recovery efficiency, growth characteristics, and differentiation potential of freshly collected human cell samples as a control.
The results showed that the ability of mesenchymal stem cells to differentiate into osteoblasts, adipocytes and neuronal cells in vitro was similar. It has been confirmed that mesenchymal stem cells with good differentiation ability can be obtained even after long-term cryopreservation (23-25 years).
In this study, the researchers evaluated the viability and differentiation potential of long-term cryopreserved mesenchymal stem cells (MSCs). It was found that:
(1) Although some cells are damaged after long-term cryopreservation and thawing, an appropriate amount of adherent healthy cells can still be obtained after culture.
(2) Mesenchymal stem cells can be obtained from primary cultures that have been cryopreserved for a long time.
For example, in the study, small prismatic adherent cells were seen in both the cryopreservation group and the control group after 3 days, and the number increased after 10 days of culture. At day 14, both groups of adherent cells showed spiral growth. After 14 days, the percentages of fibroblast-like cells in the frozen and control groups were 88.2±4.3% and 89.1±3.8%, respectively (P>0.05). When calculated based on the number of viable cells, both groups had similar proliferative capacity. By measuring the expression of common molecular markers of MSCs, it was shown that the above fibroblast-like cells were indeed MSCs.
Image from Ref. [1]
(3) Long-term cryopreservation of mesenchymal stem cells still has strong differentiation potential: The results showed that there was no significant difference in the ability of P3 cultures obtained from cryopreservation and control groups to differentiate into osteoblasts, adipocytes and neuronal cells, indicating that long-term cryopreservation and thawing did not damage the differentiation potential of mesenchymal stem cells.
The review provides high-level evidence:
Cryopreservation does not affect stem cell activity and function
In order to further verify the results of the above experiments, cryopreservation does not affect the activity and function of stem cells. Another research team conducted a systematic review [2], published in the international journal Journal of Translational Medicine, which included 41 in vitro studies and concluded that cryopreservation does not affect the morphology, surface marker expression, differentiation or proliferation potential of mesenchymal stem cells. A higher level of evidence is provided for the above results.
The study used PubMed, Science direct, and Google Scholar to systematically search the literature, and finally included 41 studies that detailed information about the composition of the freezing solution, freezing protocol, storage time, frozen cell concentration, freezing passage number, and thawing method for each article. and extract information on cell surface marker expression, differentiation potential, proliferation and growth, attachment and migration potential, genomic stability, and paracrine function. In addition, the viability and morphological information after thawing were organized. (The following figure is a flow chart)
Image from Ref. [2]
The final analysis results [2] showed that cryopreservation did not affect the morphology, surface marker expression, differentiation or proliferation potential of mesenchymal stem cells. However, there are mixed results regarding the effect on colony-forming capacity, and the effects on viability, attachment and migration, genome stability, and paracrine function have not been determined, mainly due to the large differences in cryopreservation processes in different studies and the lack of standardized detection methods. Therefore, further evaluation and the establishment of a unified cryopreservation system are needed in the future.
brief summary
There is no doubt that stem cells have become a research hotspot in the treatment of diseases, and cryostorage technology plays an important role in it. The development of cell therapies using cryostored mesenchymal stem cells has been adopted by more and more domestic and foreign research teams. Therefore, the viability and function of stem cells after cryopreservation have always been the focus of many scholars. Previous studies have shown the safety and efficacy of cryopreservation, which does not affect the viability and function of stem cells after thawing.
At present, the cryostorage of mesenchymal stem cells from umbilical cord and placental tissues is relatively popular in China, and the biological function of such mesenchymal stem cells remains stable after long-term cryopreservation, which has been confirmed by the literature [3]. The cryostorage technology of mesenchymal stem cells is of great significance for the development of cell therapy, which can improve the feasibility and effectiveness of stem cell therapy and provide innovative treatment options for more patients. However, stem cell cryopreservation requires high-quality standards and technical support systems for the whole process to provide safety for clinical applications!
Bibliography:
[1] Shen JL, Huang YZ, Xu SX, Zheng PH, Yin WJ, Cen J, Gong LZ. Effectiveness of human mesenchymal stem cells derived from bone marrow cryopreserved for 23-25 years. Cryobiology. 2012 Jun; 64(3):167-75. doi: 10.1016/j.cryobiol.2012.01.004. Epub 2012 Jan 18. PMID: 22280954.
[2] Bahsoun S, Coopman K, Akam EC. The impact of cryopreservation on bone marrow-derived mesenchymal stem cells: a systematic review. J Transl Med. 2019 Nov 29; 17(1):397. doi: 10.1186/s12967-019-02136-7. PMID: 31783866; PMCID: PMC6883667.
[3] Wang Youchen, Chai Liang, Shi Enxiang, et al. Study on biological traits of human umbilical cord mesenchymal stem cells after cryopreservation and resuscitation[J].PLA Medical Journal, 2011(A04):4.DOI:10.3969/j.issn.2095-140X.2011.04.001.