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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

As described here, all cell culture plastic (T-flasks, multiwells, dishes etc.) for culture of adherent cells is made from poly-styrene and is surface-treated by the manufacturer in a specific chemico-physical way to allow for cell adhesion. However, this basic treatment does not even come close to the natural "surfaces" or extracellular matrix cells are growing on in tissues. This results in the option to tailor a cell culture model from very primitive and cheap up to highly complex and histotypic:

  • Untreated poly-styrene (bacterial dishes), hydrophobic and only suited for rare cell types (e.g. thyroid cells, keratinocytes)
  • Surface-treated poly-styrene (normal cell culture plastic), polar and/or charges depensing on the manufacturer
  • Artificial, cheap coating (poly-amino acids)
        x  Poly-L-Lysine, Poly-D-Lysine
        x  Poly-Ornithine
  • Cheap, non-specific, proteineous coating
        x  Gelatine
  • Specific extracellular matrix coating with one purified protein (more expensive, mostly isolated from animal tissue)
        x  Collagen I, IV
        x  Fibronectin
        x  Laminin
        x  Vitronectin
        x  Osteopontin
  • Specific partial sequences of eytracellular matrix proteins (partially even more expensive, partially GMP-conform as synthetically produced)
        x  RGD-Motivs
        x  Fibronectin domains
  • Specific, komplex mixture of matrix proteins
         x  Matrigel (several components of the extracellular matrix with bound growth factors etc.)
  • 3D models in which cells are growing embedded into a matrix of gel
        x  Collagen gels
        x  Matrigel
        x  Alginate gels
        x  Lactate gels

PLL Struktur

 

 

Fibronektin Struktur

 

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, oligodendrycytes 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 adheseion. This means, normally the experimenter decides on the beasis 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, differents 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).

MSC auf DeNovo Matrix in verschiedenen Medien

© denovoMATRIX modified by InCelligence

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

The following images show normal human fibroblasts cultured in FCS-containg 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 suface) 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.

MSC auf DeNovo Matrix in verschiedenen Medien

© InCelligence from course bioassays

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 unmeasurable. 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 tecnique completely. The following fotos 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.

HEK Zellen mit und ohne PLL coating auf Greiner Plastik

© InCelligence from course bioassays

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 neighbouring cells are. These signals are detected by the binding of surface receptors (intergins) to the extracellular matrix. Intracellularly the binding elicits signalling cascades which then micht contribute to proliferation, migration or differentiation. The images below show neonatal rat astroctes (GFAP-stian) in a mixed glail culture on different coatings. Laminin triggers changes in the morphology of the astrocytes indication a different route of differentiation.

Ratten-Astrozyten auf PLL, Fibronektin und Laminin

© InCelligence from Cell culture QA course

 

Market overview: Coatings

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

 

 

Ease of use (protocol free)

Adhesion Ligand

Oligo-saccharide

Degradable

Cell recovery/ Trypsination

Chemical reaction free

Growth Factor storage/
release

Chemically-defined

Microscopy compatible

Suitable for Stem cell culture

Suitable for Cancer cell culture

Suitable for Primary cell culture

Suitable for screening

 

Fibronectin

           

ECM protein

Vitronectin

         

ECM protein

Osteopontin

               

ECM protein

Collagen (I,IV,etc)

            ▪  

ECM protein

Gelatin

             

ECM protein

Laminin

           

ECM protein

Biolamina, recombinant Laminin

     

ECM protein

3D MATRIX, puramatrix

               

Peptide nanofiber hydrogel

Pbiogelx, biogelx powder

               

Peptide nanofiber hydrogel

ESI-BIO, Hystem

           

Hyaluronate, collagen, Polyethylene glycol

Trevigen, cultrex

           

EHS tumor extract

East River BioSolutions, TissueSpec

           

Tissue-specific extracts

Ectica, 3DProSeed

               

Polymer

Corning, matrigel

           

EHS tumor extract

Corning, Synthemax

     

Vitronectin-derived peptide

Corning, Synthemax II

                 

Plastic microbeads coated with synthemax

Corning, Osteo-assay

               

Crystalline calcium phosphate

Corning, Ultra-low attachment

                 

hHdrophilic uncharged polymer surface

BRTI Life Sciences, Cell-Mate3D

           

Hyaluronate -Chitosan hydrogel

Pepgel, PGmatrix

               

Peptide hydrogel

Novamatrix, Novatach

             

RGD coupled alginate

Orla, OrlaExplorer-ECM

       

32 recombinant ECM proteins 

Cellendes, 3D-Life

             

Dextran PVA - PEG hydrogels

Amsbio, mimetix

               

PLLA electrospun fibres

Amsbio, iMatrix

       

E8 Fragment from laminin

Amsbio, MAPTrix

       

Recombinant chimeric ECM proteins

Amsbio, Alvetex

                   

Porous Polystyrene sheet

Noviocell, PIC hydrogel

               

Thermoreversible polyisocyanopeptide hydrogel

Organogenix, NanoCulture Plate

                 

Low adhesion patterned plates

Primorigen biosciences, StemAdhere

           

Recombinant protein, human origin

The well bioscience, Vitrogel 3D

             

Polysaccharide hydrogel, non-covalent crosslink

Thermofisher, Algimatrix

               

Alginate gels

Thermofisher, CELLstart

         

Human plasma extract

Thermofisher, Geltrex

         

EHS tumor extract

Lonza, RAFT

               

Collagen, RAFT (Real Architecture For 3D Tissue)

Global Cell Solutions, GEM

             

Alginate microcarrier ferromagnetic beads

InSphero, GravityPLUS

                   

Hanging drop ECM-free

DenovoMATRIX, screenMATRIX

 

Glycosaminoglycan PEG-Peptide biomatrix

 

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

Logo denonvo_MATRIX 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 unrefridgerated. Refridgerated 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 platereader.
  • 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 precoated 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