Mesenchymal stem cells (MSCs) are multipotent stromal cells that have the ability to self-renew and can differentiate into other mesenchymal tissue lineages such as adipocytes, osteocytes, and chondrocytes. Additionally, they are known to secrete trophic factors involved in immunoregulation. These MSC features make them valuable tools for T cell therapy and tissue engineering applications, and have led to increased demand for their large-scale expansion. This article comments on some of the different human tissue sources used for isolating MSCs and shares tips for successful scale-up.
Human mesenchymal stem cells (hMSCs) can be isolated from a variety of sources. These include adult tissues such as bone marrow, peripheral blood, and adipose tissue, as well as neonatal birth-associated tissues like umbilical cord, placenta, and amniotic fluid. Notably, different sources offer different advantages. For example, bone marrow represents the first identified source of MSCs and has established a reputation as the gold standard, while an advantage of using umbilical cord is that it circumvents the need for complex collection methods. Critically, it should be noted that, depending on the source, hMSCs differ significantly in terms of their growth rate and doubling capacity.
Following isolation, hMSCs are typically scaled up. However, because their expansion potential varies according to medium composition, it is important to identify a substrate that promotes optimal growth. A key consideration here is whether to use serum; although traditional hMSC culturing methods use serum-containing media, the undefined nature of serum is a concern in clinical applications. To address this issue, chemically defined, serum-free medium formulations are now available, which, when used in conjunction with specialized surfaces designed to facilitate cell binding, have been shown to provide attachment and growth comparable to that of serum-containing cultures.
Image: Bone marrow derived human mesenchymal stem cells at 40x total magnification
When optimizing growth conditions for hMSCs, it is advised that researchers use population doubling rather than passage number to track cell growth. The main difference between these two parameters is that population doubling can vary depending on the length of time between passages. For example, if cells are seeded at a low density and allowed to culture for 7 days, they may undergo 5–6 doublings, but if they are seeded at a higher density and cultured for 5 days they may instead achieve 4–5 doublings. Since hMSCs can go through only a limited number of doublings in their lifespan, the density at which they are maintained determines the length of time they can expand. Because the number of population doublings cells will go through before they stop dividing is independent from passage number, population doubling is a more reliable measure.
Once hMSCs have been isolated and expanded in culture, researchers must perform meticulous characterization before proceeding with further studies. This includes testing for both cell identity and functionality, with cell identity being largely based on flow cytometric analysis of cell surface markers. To identify hMSCs, CD105, CD166, and CD29 are typically used as positive markers, with CD14 and CD34 acting as negative controls. Additionally, microscopic examination of defining cellular features increases confidence in results. Determining hMSC functionality involves differentiating the cells into a specific lineage using appropriate culture conditions; following differentiation, flow cytometry is again used for cellular identification.
Because isolating, culturing, and differentiating hMSCs can be both complex and time-consuming, it is advised that researchers consider using tools designed with scale-up in mind. Included among the available options, chemically defined, serum-free medium formulations support transition from a research environment to the clinic, while culture vessels featuring specialized surfaces can save time and money by eliminating the need to perform laborious (and often inconsistent) coating procedures in-house. Other tools aimed at streamlining scale-up comprise advanced, multi-layer culture vessels that enable production of high cell numbers without requiring large amounts of shelf-space in CO2 incubators. For example, using just a single Corning® HYPERStack® 36-layer cell culture vessel, it has proven possible to obtain as many as 870 million viable human bone marrow-derived MSCs, and over 1 billion human umbilical cord-derived MSCs, with high viability and characteristic marker expression.
Corning offers a broad selection of tools to facilitate hMSC scale-up, including Corning® HYPERStack® 36-layer Cell Culture Vessels. To learn more, visit corning.com