Coating (surface treatment) of cell culture plastic

Coating as an additional surface treatment stands for all additional modifications made to increase cell adhesion in addition to the standard plasma or corona treatment which is performed on all cell culture plastic by the manufacturer. Usually, coating is done with proteins or peptides.

Types of coating in cell culture

Why are coatings used ?

Coatings are more work or when pre-coated cell culture plastic is purchased more expensive as normal plastic. So, when should one use coated flasks or well plates ? For most basic experiments in 2D cell culture with cell lines, normal cell culture plastic is sufficient. However, there are several reasons that might make coating advisable or even absolutely necessary:

• Cells do not attach or do not well attach to normal surfaces (primary neurons, oligodendrocytes or hepatocytes as well as some cell lines as HEK293, OLN)
• The coating helps to avoid cell loss due to washing steps in assays (HEK293, OLN93, HepG2)
• The specific coating protein supports differentiation of cells (neurons, glia, hepatocytes, keratinocytes)
• Without coating the cells become senescent or dedifferentiate (MSC, hepatocytes)
• On specific surfaces, the cells develop a more complex morphology (glia, neurons, HepG2, hepatocytes, keratinocytes)
• Cells polarize only when cultured on a specific surface (HepG2, hepatocytes)
• Cells are culture in serum-free media and can't survive, proliferate or adhere without coating (s.u.) (glia, MSC, stem cells)
• The research focus is highly complex and can only be answered in a true 3D model (skin models, cartilage, bone, spheroids, angiogenesis)

In general, all cells would prefer to grow on a coated surface as this is more natural and allows for better adhesion. This means, normally the experimenter decides on the basis or the research focus and/or necessity. As for unpretentious cell lines there is no necessity to coat, cheaper non-coated plastic is used. For pretentious cell lines and especially primary cells, coating is helpful or even indispensable. No matter what we decide for, we always have to keep in mind that the closer the cell culture model is to the natural tissue situation, the easier it is to translate results for in vitro to to in situ.

Application examples for different coatings

Serum-free or xeno-free cell culture of MSCs (mesenchymal stem cells)

In the following image of denovoMATRIX, different cultures of mesenchymal stem cells (MSC) are shown after culture on normal and coated surfaces with either FCS-containing, serum-free or xeno-free medium. The two combination labelled in red clearly show that with the change from serum-containing to serum-free medium, the necessity of surface coating arises. Due to the lack of serum proteins the pretentious MSCs can not any more attach to the cell culture plastic surface unless it is coated either with purified fibronectin or a synthetic replacement fro fibronectin manufactured by denovoMATRIX (myMARTIX).

Culture of human fibroblasts on different surfaces with FCS-containing medium

The following images show normal human fibroblasts cultured in FCS-containing medium on different surfaces. The top row (normal cell culture plastic with or without additional coating) allows for good cell growth and the fibroblasts display a typical morphology. Only in the lower row (bacterial plastic surface) the effect of coating becomes obvious, as the fibroblasts can not well adhere to the non-treated, hydrophobic plastic. As a result one would judge that coating with FCS in the serum is unnecessary for human fibroblasts.

PLL-coating of 96-well plates help to avoid washing cell off in bioassays

In complex bioassays with many washing steps, it occurs quite often that the cells are partially or almost completely washed off while staining or during an ELISA. This might severely increase the standard deviation or in the worst case might make the whole assay non-measurable. In order to avoid these effects, well plates may be coated with PLL (easy, fast, cheap) for all cell lines or at least for such cell lines that are known to adhere not very well (HEK 293, OLN93). This helps to reduce cell loss at least partially or with the correct pipetting technique completely. The following photos show the effect of washing in an ELISA assay after identical cell numbers of HEK cells were plated on the same cell culture plastic but either with or without PLL-coating. Cells were visualized using a crystal violet staining protocol. The protective effect is easily seen on the right by the more intense color. The only exception is the very left row.

Extracellular matrix as a support for differentiation

For some cell types, the extracellular matrix helps the cells to orient themselves in which region or where in a tissue they are and helps them to detect who the neighboring cells are. These signals are detected by the binding of surface receptors (integrins) to the extracellular matrix. Intracellularly the binding elicits signalling cascades which then might contribute to proliferation, migration or differentiation. The images below show neonatal rat astrocytes (GFAP-stain) in a mixed glail culture on different coatings. Laminin triggers changes in the morphology of the astrocytes indication a different route of differentiation.

Market overview: Coatings

The following table summarizes the current coatings offered on the market in addition to the standard ECM proteins by different manufacturers.

Manufacturers and distributors
of coatings and pre-coated cell culture plastic ware

	denovoMATRIX
Production site	Germany
QMS	Quality management is based on ISO 9001.
Products	screenMATRIX: Screening tool for identification of chemical coating components for culturing specific cell types in specific scientific applications. myMATRIX: Standardized, coated cell culture plastic ware for the expansion and differentiation of special stem cell types.
Chemical components	The composition of the coatings is completely chemically defined. Glycosaminoglycans Polyethylenglycol (PEG) Polypeptides
Media	Dedicated serum-free media: StemPro MSC SFM (Thermo Fisher) PRIME-XV MSC Expansion SFM (Irvine Scientific) Mesenchymal Stem Cell Growth Medium DXF (PromoCell) Dedicated serum-containing media: DMEM + 10 % FCS αMEM + 10 % FCS Note: This coating is especially formulated for use with serum-free media and there are considerable advantages according to the manufacturer to use this combination.
Coated formats	96 Well plate screenMATRIX - Coated microtiter plate with 96 wells. Each well contains a variation of a chemically defined, modular composite coating. 6, 12, 24 Well plate myMATRIX - By default, these coated formats are offered. T25, T75, T225 Cell culture flasks myMATRIX - By default, these coated formats are offered. Special formats: myMATRIX - Two-layer cell factories to multi-layer cell factories are coated on demand.
Storage	Coated cell culture flasks are supplied dry. The product can be stored non-refrigerated. Refrigerated storage increases shelf life fourfold.
Shelf life	Shelf life at 37 ° C for 6 months. Shelf life at 4 ° C for 24 months.
Price range	Products are in the upper price segment.
Cell types	screenMATRIX Primary cells Stem cells myMATRIX Mesenchymal stromal cells /mesenchymal stem cells (MSC) Induced pluriopotent stem cells (iPSC)
GMP-compatibility	In general, use under GMP is possible as the products are fully chemically-defined.
Known advantages	Allows serum-free cell culture. According to the manufacturer, stabilization of growth factors in the medium extends its usability and extends the time until the media change. A high reproducibility through chemically defined coating compared to products of animal origin. Coating is 100% optically transparent and is therefore suitable for high-resolution microscopy, spectrophotometry and plate reader. Known disadvantages.
Known disadvantages	No 3D applications. There is currently no GMP certification. Additional information.
Additional information	The shelf life of the products is 6 months without refrigeration and extended to 24 months with refrigerated storage. Prolonged usability of the medium by stabilizing growth factors on the coating surface. The creation of tailor-made coatings is offered. Specific cell adhesion occurs via integrins and glycosaminoglycans. The products can be used with tested, serum-free media. GMP certification is basically possible due to chemical definition (GMP compatible).
Coating protocol	The products are completely protocol-free. The cell culture vessels are pre-coated and thus ready for use without further preparation.
Compatible analyzers	High Resolution Microscopes (Fluorescence, Phase Contrast, Brightfield) Plate Reader
Website	https://www.denovomatrix.com/products/screenmatrix https://www.denovomatrix.com/products/mymatrix