The growth rate and phenotype of stem cells is critically dependent on the substrate surface1. Use of an unsuitable surface leads to low cell attachment, slow growth or premature differentiation of stem cells. In a previous article, we discussed different types of growth surfaces, together with key benefits and drawbacks.
Another important decision is the method of applying coatings. Both ready-to-use coated surfaces and in-house coating approaches have important benefits that need to be considered when determining a suitable stem cell culture workflow – but when to choose which?
An in-house coating approach refers to any method whereby the culture surface (for example a cell culture plate or a T-flask) is purchased separately from the coating material. The process of coating the surface then takes place in the lab – usually shortly before cell culture. This means that, in most cases, preparation of new coated vessels needs to be planned in advance due to incubation time – something scientists need to keep in mind when planning experiments.
The key benefit of in-house coating is flexibility in terms of surface choice. If necessary, scientists can easily adjust the properties of coatings through small modifications to a protocol, which enables specific applications to be optimized without being dependent on standard specifications.
However, when carrying out coating of culture vessels in the lab, ensuring reproducibility is a challenge. When culturing stem cells over many successive passages, consistent culture conditions are a key factor for success. With in-house coating, the risk of variability due to inconsistencies is higher – especially with compounds that are difficult to handle. Several coatings are naturally unstable and often need to be stored in fridges or freezers. Checking the quality and consistency of coatings after application is often difficult which may affect data reproducibility.
In addition, the process of applying a coating – often in small batches – is highly time consuming. All coatings need to be applied under sterile conditions, which means that not just the researcher’s time is required, but also lab equipment such as laminar flow cabinets and suitable storage for coated cultureware (dependent on the type of coating material) – all of which can hamper lab efficiency.
In stem cell culture, ready-to-use surfaces are specially modified during production to support the proliferation of stem cells in the long term without causing premature differentiation.
A key reason for choosing ready-to-use coatings is consistency during manufacture. Furthermore, stringent quality control processes can confirm that the right coating is present at the right concentration and that sterility is maintained. This consistency helps to maintain cells in an undifferentiated state and reduces the need for discarding cultures – for example because of abnormal-shaped stem cell colonies.
Buying pre-coated cultureware also saves time and lab resources. An in-house coating protocol often involves applying the coating, incubation and washing steps – all under sterile conditions. When working with a large amount of cultureware these protocols require significant time and planning.
Many researchers that work with pluripotent stem cells still use either a feeder layer or other coatings directly derived from living cells2. These coatings are well suited to maintaining a pluripotent phenotype, but it is not possible or highly impractical to supply these coatings in a ready-to-use format. Storage of biologically coated ready-to-use cultureware is often also a challenge due to the amount of space required in fridges and freezers.
The ready-to-use format is, however, highly suitable for coatings that combine good stem cell attachment and proliferation with a long shelf life. These include for example synthetic surfaces pre-coated with short peptide sequences that mimic cell adhesion sites of extracellular matrix proteins3. These peptides can be used for xeno-free or even animal-free culture, making them well suited for ready-to-use surfaces.
The choice of whether to opt for a ready-to-use surface or to coat cultureware in the lab depends on many factors. These include the flexibility to use different coatings, the need for optimization and customization, as well as the available time, space, facilities and budget.
In general, in-house coating is preferred when working with non-chemically-defined coatings and when frequent modifications to coating parameters are necessary. In-house coating permits a high degree of flexibility and facilitates the use of short-lifecycle coatings such as biological coatings.
On the other hand, ready-to-use, pre-coated cultureware offers important benefits in consistency and lab efficiency. With the increased popularity of xeno-free or animal-free culture conditions, synthetic ready-to-use coatings can offer a reliable, user friendly and chemically-defined alternative to in-house coating.
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Continue to Part III - Scaling Up Stem Cell Culture
1. Peterson S. et al. Human Stem Cell Manual: A Laboratory Guide. Elsevier, 2011.
2. Villa-Diaz LG et al. The evolution of human pluripotent stem cell culture: from feeder cells to synthetic coatings. Stem Cells 2013;31(1): 1–7.