Stem cells are one of the more challenging cell types to culture. Maintaining successful iPSC cultures requires a skilled eye and careful considerations to the culture conditions. Minor adjustments can have large impacts, positive or negative, on culture outcome. Knowledge about how each factor impacts proliferation and differentiation is key to improving the quality of your cultures. Understanding the growth factor over time in culture, through controlled-release technology, achieves better culture outcomes.
Since Dr. Shinya Yamanaka was granted the Nobel Prize for his discovery of iPSCs less than 15 years ago, there have been multiple culture protocols that have emerged. Specific feeding schedules, passaging methods, and materials can be mixed-and-matched to create the culture protocols that work best for you. Ultimately, these conditions should be optimized by defining and controlling growth factor levels.
Two common types of culture methods for iPSCs are feeder and feeder free methods. Feeder methods were the first to be adopted but have since become less popular since the addition of cell types such as mouse fibroblasts introduce variability. Because of this, feeder-free iPSC cultures are now most commonly employed, their success relying heavily on more defined culture conditions.
Feeder free methods require two main components: media broth and an attachment factor. Media broth contains nutrients to promote cell growth designed for a specific cell type. An attachment factor promotes adhesion to the culture well. Although a common misconception, stem cells cannot readily attach to surfaces on their own. Culture plates are coated with matrigel, Synthemax, laminin, fibronectin or other agents to promote attachment. It is important to know how the media and substrate affect your cells in order to optimize healthy and reproducible growth.
There are two common ways of passaging stem cells: clump passaging and single cell passaging. Clump passaging, as the name suggests, breaks large clumps of iPSCs into smaller clumps for seeding. The small clumps are transferred from the parent well into several offspring wells to continue to expand the cell line. To break up the parent culture, there are two methods, through the addition of enzymatic reagents or by manually selecting and transferring small colonies. The method you choose to passage depends on feeder or feeder-free culture and your cultures density.
Single cell passaging requires additional reagents and intermediate steps. ROCK inhibitor, washing solutions such as DMEM or PBS, and a detachment solution (such as Accutase), along with incubating and spinning down the cultures, are used to isolate a single iPSC that can be transferred into a new well for expansion. Single cell passaging has high scalability, rapid expansion, and high efficiency, but it also places the cells in stressful conditions if the proper reagents are not added following specific protocols.
To improve the quality of your iPSC cultures, it is important to understand the nuances of each method and how each of the factors involved impact your cells.
The reagents for each culture method are specific in how they are applied. Lengths of time, temperature, and proper concentration are key considerations and vary depending on the reagent. If improper precision is used when handling these materials, there is an increased likelihood that cell quality will be drastically impacted. To improve your iPSC cultures, it is critical to understand the specifications for each reagent.
It is important to understand how the culture methods employed impact cell growth. It is very useful to develop an expert eye to assess how your cells react to certain conditions. It is important to the morphology of high quality iPSC cultures. An image of a healthy-appearing iPSC colony is shown below for reference. The smooth borders and homogenous central cellular morphology indicates a high quality colony. This basic understanding of colony appearance provides for rapid assessment of how a particular culture condition impacts iPSC growth.
Figure 1: Left indicates a high quality iPSC culture. Note the tightly packed colonies. This morphology is indicative of high quality. Right shows a low quality culture. Note there are less clear and defined colonies. The arrows indicate unwanted spontaneous differentiation which can be a visual indication of quality.
Similarly, it is important to monitor the density of your iPSC cultures. If cells reach too high a density and level of confluency, cells begin to differentiate. It is recommended to passage your cells once they reach 70-80% confluency, which typically occurs after the cells have been in culture for one week. However, different cell lines, passaging methods, and culture conditions affect the speed of cell growth to impact when passaging is needed.
Likewise, it is important to remove cells that have differentiated. This can be done via manual scraping under a microscope. The removal process is tedious and serves as an incentive to maintain high quality cells throughout the culture process.
In order to understand and improve your cultures, it is important to have the correct tools to do so. Consider the following for the highest quality output possible:
Many methods require the scientist to replace cellular media on a daily basis. This ensures constant nutrient replenishment. However, even with daily feeding, the nutrients in cell culture media degrade over time. To overcome this, controlled-release growth factor technology can be used. StemCultures manufactures these products as StemBeads and DISC devices. With controlled-release technology, scientists can reduce feeds to only 2-3 times per week and maintain the quality of cell cultures produced. Making sure that the cells have the nutrients they need is one of the easiest and most beneficial methods to improve the quality of your cell cultures.
More information about controlled-release technology can be found on the StemBeads or DISC device page. Additional culturing tips and tricks can be found here. Overall, the greater control scientists have over their culture conditions, the more likely they are to produce high quality iPSC cultures.
