Freezing Amnion-Derived Stem Cells in Xeno-Free Media Feasible

Amnion-derived stem cells show a dizzying set of regenerative properties, at least in the laboratory (see Miki T, Stem Cell Res Ther 2011, 2:25; Miki T, Grubbs B, J Obstet Gynaecol Res 2014, 40:360-8). Bringing these cells into the clinic will definitely take a lot more work, but a new paper examines a culture system that does not use any animal products. Such culture systems (known as “xeno-free) are vitally important if stem cells are going to be used in human trials.

When stem cells are grown in culture, typically serum from animal blood is used to provide the cells with the growth factors and things they need to kick the cells into the growth phase. However, occasionally, some animal-based products have animal viruses that can infect human cells with unpredictable consequences (see Karlsson JOM, Toner M, Biomaterials 1996, 17:243-56). Also, cells grown in animal-based culture media can have animal proteins around them that are very hard to get rid of. Implanting such cells into a human patient would cause their immune system to react against the animal proteins. Such immune responses might result in something as innocuous as itching at the site of injection or as dangerous as anaphylactic shock. Therefore, growing stem cells under xeno-free conditions is important for many stem cell applications.  Expanding and preserving stem cells under so-called Good Manufacturing Pracctices (GMP) is essential for their use in human patients.

However, now we have just opened another can of worms. If all the characterizations of stem cells have been under culture conditions that utilize animal-based culture media, will the cells have the same capabilities if grown under xeno-free conditions?

Herein is the reason why this paper by Toshio Miki from the Keck School of Medicine at the University of Southern California and his colleagues is so important. In this paper, which was published in the journal Stem Cell Research and Therapy (Stem Cell Research & Therapy 2016, 7:8 doi:10.1186/s13287-015-0258-z), Miki and others examined the characteristics of amniotic stem cells grown in xeno-free media (see Mitry RR, Lehec SC, Hughes RD, Methods Mol Biol 2010, 640:107-13; Polchow B, et al. J Transl Med 2012, 10:98; Stacy GN, Masters JR, Nat Protoc 2008, 3:1981-89; Miki T, et al. Curr Protoc Stem Cell Biol 2010, Chapter 1:Unit 1E.3) and compared them to stem cells grown in standard culture media. This is a very important exercise, since amniotic stem cells are easily accessible as source of material for regenerative treatments that can be banked and immunotype matched for clinical applications.

Miki and others isolated human amniotic epithelial cells from newborn babies with the consent of their parents and then stored the cells at −160 °C in one of five commercially available culture media. Miki and his group used cells frozen in standard media containing fetal bovine serum as controls to which all the other cells were compared. Then they thawed the cells, and tested their viability, mitochondrial integrity, and senescence status (tendency to fall asleep in culture and stop growing). They also examined gene expression profiles in these cells by using quantitative real-time PCR. Flow cytometry was used to identify the stem cell surface markers.

The results were encouraging and interesting. There were no significant differences in viability and growth in cells grown and preserved in xeno-free media versus standard cryopreservation medium. Additionally, comparisons of the cells grown in the different cryopreservation media did not reveal significant differences in the senescence status, mitochondria, or overall morphology of the cells. The upshot is that the cells preserved in standard or xeno-free media looked and grew the same after being thawed. There were some differences in the expression of stem cell marker genes (e.g., OCT4, SOX2, and NANOG) and a particular cell surface marker (TRA1-60) following cryopreservation in different xeno-free media. However, it turns out these differences were slight and, overall, not statistically significant. Again, the upshot is that there were small differences in gene expression, but these differences did not amount to a hill of beans.

Miki and his colleagues have nicely shown that cryopreserving amnion-derived stem cells in xeno-free media is feasible and does not affect the characteristics of the cells. This paper also suggests that such xeno-free media can be used to establish a bio-bank of human amnion-derived stem cells for future clinical application.

Well that’s one hurdle vaulted. Now Miki and others need to figure out which of the xeno-free media is the best and optimize that medium to for improved preservation of stem cell-like characteristics in these cells.


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Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).