Chondrex, Inc. provides bovine type I collagen and porcine type I collagen for use in both 2-dimensional (2D) and 3-dimensional (3D) cell culture systems. Our Cell Culture Grade Collagen is tropocollagen that has been purified from skin tissue. This grade of collagen also contains other components native to the skin’s extracellular matrix (ECM) (e.g. integrins), making it slightly less pure than our other grades (>95% purity). However, the inclusion of minor ECM components allows for 2D and 3D scaffolds made using our Cell Culture Grade Collagen to more closely mimic natural cell environments, therefore promoting cell adhesion, proliferation, and differentiation. Our collagen has been sterile-filtered and contains less than 1 EU/ml, making it ideal for use in 2D or 3D cell culture systems.
While Chondrex, Inc. only offers Type I Collagen as Cell Culture Grade, our Immunization Grade Type II Collagen can also be used to create scaffolds for cell culture. Please visit our Immunization Grade Collagen page for more information.
In order to accommodate the variety of collagen studies available, Chondrex, Inc. offers different grades of collagen such as immunization grade, cell culture grade, ELISA grade, T-cell assay grade, and collagen CB fragments. If you need help deciding which grade of collagen is right for your study, please see our Understanding Chondrex, Inc.’s “Grades” of Purified Collagen article or contact us. For more information distinguishing between Type I and Type II collagen, please see our Type I & Type II Collagen: Structure Influences Staining Patterns article or contact us.
Cell Culture Grade Type I Collagen
|Product||Quantity||Catalog #||Price (USD)|
|Cell Culture Grade Bovine Type I Collagen||4 mg/ml x 12.5 ml||1202||121.00|
|Cell Culture Grade Porcine Type I Collagen||4 mg/ml x 12.5 ml||1203||121.00|
Traditionally, cell cultures have been performed using 2D systems where cells grow in a monolayer on plastic or glass surfaces (Figure 1A). 2D cell cultures have advanced our understanding of cell proliferation, cellular permeability, and cellular responses to drug treatments and viral infections, but they are not perfect. Cells grown using 2D systems can have an altered gene expression (1) and phenotype (2,3) compared to in vivo cells, therefore limiting the biological relevance of in vitro cell based assays from 2D cell cultures. For instance, adult human articular chondrocytes (AHAC) cultured in 2D systems (as a primary culture) tend to dedifferentiate towards a fibroblast-like morphology and produce type I and type III collagen, rather than type II collagen, during prolonged cell culture periods (4, 5). Changes like these ultimately affect how cells may respond to assays or treatments, reducing the applicability of cell-based assays from 2D cell cultures to clinical research (6,7).
To help improve the biological relevance of findings from in vitro cell-based assays, 3D cell culture systems are preferred. These 3D cell systems are generally categorized as scaffold-based cell cultures (Figure 1B) and scaffold-free (spheroid cultures, Figure 1C). In scaffold-based 3D cultures cells can grow, proliferate, and differentiate in a network environment formed by biological proteins and/or synthetic materials that mimic their natural ECM. These scaffolds come in different forms: hydrogels (hydrophilic polymer networks that absorb water but keep their 3D shape), sponges, electrospun scaffolds, or nano/microparticles. By providing cells with a more physiologically relevant environment, cells exhibit more natural gene expression profiles and morphologies, leading to more clinically relevant results (6-8).
When culturing stem cells for tissue engineering or cell-based assays, choosing an appropriate 3D culture scaffold is vital. Cell-cell and cell-ECM interactions drastically affect the differentiation of stem cells to specific cell types (9,10). Properties of a 3D cell culture scaffold (such as stiffness, pore size, and cell adhesion sites) can also affect the dispersion of growth and/or differentiation factors and therefore modulate cell differentiation and proliferation (11). Thus, when culturing stem cells, it is important to choose a scaffold material that best mimics the ECM of the tissue where the desired cell type is found naturally.
Materials Used for Cell Culture Scaffolds
3D scaffolds for tissue engineering and cell culture can be made using ex-vivo proteins (collagen, fibrin, fibronectin, etc.), polysaccharides (agarose, alginate, hyaluronan/hyaluronic acid) and/or by using synthetic polymers (poly(lactic-co-glycolic acid) (aka. PLGA), poly(ethylene glycol) (aka PEG), Polycaprolactone (aka PCL)), peptides, or ceramics. Choosing the appropriate scaffolding material depends on the desired qualities of the scaffold, the type of cells being cultured, and the application of the cultured cells (12).
Synthetic materials have a couple advantages: their chemical structure is well defined making the constructs more easily reproducible, the mechanical properties and shape of synthetic materials are easy to manipulate, and the biodegradability of the constructs can be modified (12). However, many synthetic materials do not contain the cell adhesion sites necessary to facilitate cell growth and proliferation. Therefore, synthetic scaffolds often contain biomaterials (i.e. Type I collagen) or undergo extensive modifications to promote cell adhesion (12).
Type I collagen, as the predominant collagen in connective tissues and a primary component of the interstitial matrix in many tissue types, is one of the most common natural materials used for 3D scaffolds (13-17). Collagen has many properties that make it a desirable scaffold: low antigenicity, low toxicity, high water solubility, and high biodegradability. Additionally, as type I collagen binds integrins on cell surface, type I collagen scaffolds can effectively promote cell migration, cellular adhesion, proliferation, and differentiation of numerous cell types, including: stem cells (13,14), chondrocytes (15), and fibroblasts (16). Type I collagen scaffolds are commonly used to culture articular chondrocytes and mesenchymal stem cells for cartilage tissue engineering applications (17).
Type II collagen, the main component of cartilage, is also a common choice as a scaffold biomaterial for culturing stem cells with articular cartilage tissue engineering applications in mind (17). Scaffolds made from type II collagen can promote differentiation of mesenchymal stem cells to chrondrogenic phenotypes, without the use of growth factors (18). While we do not offer Cell Culture Grade Type II collagen, our Immunization Grade Type II Collagen is suitable for use in cell culture. Please see our Understanding Chondrex, Inc.'s Grades of Collagen blog for more information on our Immunization Grade Collagen.
The mechanical properties of collagen scaffolds are not as customizable compared to synthetic materials, however preparation procedures can influence scaffold characteristics such the fiber diameter, pore size, tensile strength, and stiffness (12, 17, 19, 20). Ultraviolet (UV) irradiation, gamma radiation, dehydrothermal treatment (DHT) and Glutaraldehyde (GA) are the most commonly used treatments to increase the degree of cross-linking between collagen fibers in order to stabilize the collagen scaffold. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), a covalent cross-linking agent, is also used (with or without N-hydroxysuccinimide (NHS)) to increase cross-link stability (20). Furthermore, scaffolds can be prepared as a collagen/polymer blend scaffold by using a mixture of natural polymers (chitosan, silk, fibrin) and/or synthetic polymers (PCL, polylactic acid (PLA), PEG, polyglycolide (PGA), PLGA, and polyvinyl alcohol (PVA)) (21).
Ultimately, the proper scaffold material for your studies will depend on the cultured cell types and the intended application of the scaffold/cells. However, given the ubiquity of type I collagen throughout the body, type I collagen is an ideal scaffold for 3D cell culture of a variety of cell types.
Like all products derived from animal tissue, there is an increased risk of batch-to-batch variations in type I collagen scaffolds. If you have concerns regarding lot consistency of our Cell Culture Grade Collagen, please contact us. We can help ensure lot consistency for your study.
Native Collagen vs. Gelatin Scaffolds
Type I/II collagen, either in its native (undenatured) form containing the characteristic triple-helix motif or in its denatured form (gelatin), can be used for creating hydrogels for 3D cell culture. However, there are some key differences between them.
Native collagen is resistant against most proteinases produced by cultured cells, making the system beneficial for long-term culture. In contrast, gelatin is highly sensitive to many proteinases making it unreliable for long-term culture (22).
Denaturing collagen to make gelatin destroys the collagen triple-helix motif, which affects collagen fiber formation. Gelatin therefore has decreased tensile strength and stiffness compared to native collagen scaffolds (23). However, the loss of the triple-helix motif also decreases the antigenicity of the collagen (although native collagen is considered a weak antigen) (24) making gelatin scaffolds an ideal material for tissue engineering applications. Despite the loss of the triple-helix structure, gelatin still contains the characteristic Gly-X-Y amino acid sequence and promotes cell adhesion, migration, differentiation, and proliferation (20). Gelatin is often modified with methacryloyl, a photocrosslinking agent, to create gelatin methacryloyl (GelMa) scaffolds) (12, 25).
Our Cell Culture Grade Collagen is acid solubilized from bovine or porcine skin tissue and is provided in the native (undenatured) form. It is suitable to create both native collagen scaffolds and gelatin scaffolds (heat the collagen higher than 45°C to denature the collagen).
Collagen and ECM Component Analysis
Analyzing collagen, glycosaminoglycans (GAGs), and DNA can be an important metric for evaluating cell culture samples. Chondrex, Inc. provides several kits for quantifying both type I, type II, and total collagen content from samples. Please visit our Collagen Detection page for more information.
Hoechst, DAPI (4,6-diamidino-2-phenylindole), and other blue-fluorescents dyes can stain DNA and facilitate DNA quantification (26). For tissue analysis, Chondrex Inc. provides an alternative DNA Assay Kit employing the Hoechst 33258 fluorescent dye which specifically binds to Adenine-Thymine base pairs, resulting in fluorescence at excitation 360 nm/emission 460 nm.
In 2D culture systems, collagen production can be evaluated using Sirius Red/Fast Green Semi-Quantitative Assay Kit as well as Type I and Type II Collagen Imunostaining Kits. Glycosaminoglycans (GAGs) can be stained with Alcian blue, toluidine blue dyes, including the cationic dye, 1,9 dimethylmethylene blue (DMB) which binds to highly charged sulfated GAGs and is used in the Chondrex GAGs Detection Assay Kit (27).
If culturing chondrocytes or fibroblasts for cartilage tissue engineering purposes, evaluating the collagen production is important to monitor the culture progression. However, if using our Collagen Detection Kits for this purpose, sample preparation must be considered. Some artificial scaffolds require severe conditions (high heat, chemical treatment) for solubilization of the scaffold. These conditions may denature collagen, resulting in poor reactivity with our ELISA kits. Our Type I and Type II Collagen ELISA Kits are highly specific for native form collagen and therefore should not be used to detect denatured collagen. Our collagen sample preparation protocols are suitable for use with native collagen scaffolds. Please contact Chondrex, Inc's customer support for more assistance with your ECM analysis.