Product Description

VitroCol® collagen is the first widely available, naturally produced purified human collagen for research purposes. VitroCol® sets the standard for purity (>99% collagen content), functionality and represents the only native-like human collagen offered.

VitroCol® collagen is naturally secreted from human neo-natal fibroblast cells. The human fibroblasts are cultured in optimal conditions allowing the fibroblast to naturally and efficiently secret extracellular matrix. The extracellular matrix is then processed and purified to yield the naturally produced human collagen.

VitroCol® is approximately 97% Type I human collagen with the remainder being comprised of Type III collagen. VitroCol® is supplied at approximately 3 mg/ml concentration. The concentration for each specific lot is provided on a Certificate of Analysis that is available with the purchase of each product. VitroCol® is soluble atelocollagen in 0.01 N HCI, therefore, the pH is 2.

VitroCol® is especially ideal for human cell culture systems when coating of surfaces, providing preparations of thin layers of culturing cells, or use as a solid gel. VitroCol® human collagen is provided in user-friendly packaging for use and storage. VitroCol® is sterile filtered and is supplied as a ready to use solution.

Parameter, Testing, and Method VitroCol® Type I Collagen #5007
Sterilization Method Filtration
Extraction Method Enzyme - atelocollagen
Form Solution
Package Size 20 mL, 100 mL
Storage Temperature 2-10°C
Shelf Life Minimum of 6 months from date of receipt

Collagen Concentration - Biuret

2.9-3.2 mg/mL

Collagen Purity - Silver Staining

>99%
pH 1.9-2.1
Kinetic Gel Test (Minutes) <40
Gel Formation Tube Test (Minutes) <40

Fibrillogenesis (Absorbance Units)

>0.5

Electrophoretic Pattern - Coomassie Blue

Characteristic
Sterility - USP modified No growth
Endotoxin - LAL <5.0 EU/mL
Osmolality (mOsmo H2O/kg) <35
Cell Attachment Assay Pass
Source Human Neo-Natal Fibroblasts
Hydrogel Young's Modulus E (Pa) Characteristic



Directions for Use

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Coating Procedure

Note: Use these recommendations as guidelines to determine the optimal coating conditions for your culture system.

  1. Transfer desired volume of collagen solution from the bottle to a dilution vessel as required. Dilute to desired concentration using sterile 0.01 N HCl solution. A typical working concentration may range from 50 to 100 ug/ml.
  2. Add appropriate amount of diluted VitroCol® material to the culture surface.
  3. Incubate at room temperature, covered, for 1-2 hours. Aspirate any remaining material. Alternatively, incubate at room temperature until surface is dry.
  4. Rinse coated surfaces carefully with sterile medium or PBS, avoid scratching surfaces.
  5. Coated surfaces are ready for use. They may also be stored at 2-8°C damp or air dried if sterility is maintained.

3-D Gel Preparation Procedure

  1. Slowly add 1 part of chilled 10X PBS or 10X culture media to 8 parts of chilled collagen solution with gentle swirling.
  2. Adjust pH of mixture to 7.0-7.5 using sterile 0.1M NaOH.  Monitor pH adjustment carefully (pH meter, phenol red, or pH paper)
  3. Adjust final volume to a total of 10 parts with sterile water.
  4. To prevent gelation, maintain temperature of mixture at 2 – 10°C.
  5. To form gel, warm to 37°C. Allow approximately 90 to 120 minutes for gel formation.

Product Q & A

We completed a study to show that DNA is completely destroyed at pH 2, and demonstrated that our collagen products do not contain DNA.

The collagen is fully hydrolyzed. The amino acid analysis is done using the Waters AccQ-Tag derivatization method. During the acid hydrolysis step, asparagine (N) is converted to aspartic acid (D) and glutamine (Q) is converted to glutamic acid (E). Tryptophan (W), if present, is destroyed during acid hydrolysis. Experimentally, one can determine the picomoles (pmol) of each amino acid per injected detected using amino acid standards.For the concentration determination, the total number of pmol of each amino acid is summed to get the total pmol of the 18 amino acids detected. The total pmol amino acids is divided by the theoretical number of amino acid residues in collagen based on the published sequence. The result is the pmol of collagen injected. The result is then multiplied by the dilution and 300,000 is used as the collagen molecular weight to get to mg/mL. The molecular weight of collagen is not well agreed upon.

Diluting with 1X PBS (rather than water or 0.01 N HCl) would have an effect for coating purposes.   It would change the pH of the diluted collagen solution from acid to neutral pH. The pH change will transform the collagen molecules from a molecular form to a fibrillar form; and then the nature of coating surface will be changed from a monomeric coating to a fibrillar coating.

The major collagen molecular species in our Type I collagen products are monomers (approx. 70%), but there are dimers, trimers and a few percentages of oligomers too (approx. 30%) with some minor amounts of collagen fragments. The collagen monomer is a rod shaped molecule with 300 nm in length and 1.5 nm in diameter. The dimer, trimer and oligomer are 600 nm, 900nm and even longer in length respectively. According to the coating procedures, the collagen molecules are attached to the charged polystyrene surface randomly by charge or affinity in acid conditions during the 1-2 hrs incubation period at 37°C, and any unattached materials are removed by aspiration and rinsing. Therefore, the coated surface is a single layer of collagen monomer, dimer, trimer and oligomer mixtures. The thickness of the mono-molecular layer is dependent on how those molecules are attached on the surface. The coating density thickness would generally be characterized as a 1 molecule thickness which could be ranging from a few nanometers to a few hundred nanometers with the whole surface being covered by collagen.

The net charge of Type I collagen products’ (PureCol®, Bovine Collagen and VitroCol®, Human Collagen) molecule is directly related to the pH.  At an acidic pH, the amino acids (zwitterions) along the collagen molecule are positively charged, making the entire collagen molecule positive. At the isoelectric point (or zone) of collagen, around pH 7-8, the amino acids along the collagen molecule are positively and negatively charged, making the net charge of the collagen molecule close to zero. At a basic pH, the amino acids along the collagen molecule were negatively charged, making the entire collagen molecule negative.

Further, the nature of the charge of the collagen coating surface will be dependent on the type of coating applied. For a monomeric collagen coatings when the collagen is applied under an acidic pH condition, the surface is positively charged. If the surface is rinsed with pH neutral buffer or media then it will change the charge of the collagen surface net charge close to zero. For a 3D gel coating, the collagen prepared under neutral pH; the net charge of the collagen surface is close to zero.

Using rotary shadowing technique under transmission electron microscopy, it was found that our collagen, on average, consists of approximately 80% monomers, 13% dimers, trimers, and oligomers with the remaining 7% collagen fragments.

Yes. The collagen molecule in PureCol, Nutragen, VitroCol, and all of our other Atelo collagen products were prepared from native collagen matrix by pepsin treatment under controlled conditions to remove the non-helical portion, telo-peptides, only and the helical portion is intact. In this case, the enzymatic active sites for MMP (Matrix Metalloproteinase), such as for Mammalian Collagenase Matrix Metalloproteinase 8 (MMP-8), on the molecule was preserved.

These pepsin treated collagen products should behave as native intact collagen.

TGF beta would have been digested with the pepsin enzymatic digestion step. It was undetectable by SDS PAGE silver stain as well. We didn’t do any specific measurements by ELISA however but presences of TGF beta is not anticipated.

We primarily use the Biuret method, but we also use BCA, AAA, and hydroxyl-proline assays.

- Collagen solutions that are frozen tend to have issues forming 3D hydrogels, and will likely not work. The solutions should still be good for 2D coatings. 

- Collagen solutions that are left out at room temperature for extended periods of time may show signs of degradation, which will affect the formation of 3D hydrogels. It is likely still fine for 2D coatings. 

Our recommendation is this: If you are using the product directly for a publication, we highly suggest buying a new bottle if the one you have was compromised. 

Product References

References for VitroCol®:

Andrée, B. et al. Formation of three-dimensional tubular endothelial cell networks under defined serum-free cell culture conditions in human collagen hydrogels. Scientific Reports 9, (2019).

Shieh, Hester F., et al. "Comparisons of human amniotic mesenchymal stem cell viability in FDA-approved collagen-based scaffolds: Implications for engineered diaphragmatic replacement." Journal of pediatric surgery 52.6 (2017): 1010-1013.

van der Velden, Jos LJ, et al. "Transforming Growth Factor-ß Induces A Mesenchymal Profile In Human Nasal Epithelial Cells." D76. ALVEOLAR EPITHELIUM: NOVEL TOOLS AND PHENOTYPES. American Thoracic Society, 2012. A6322-A6322.

Tashima, Takumi, et al. "Osteomodulin regulates diameter and alters shape of collagen fibrils." Biochemical and biophysical research communications 463.3 (2015): 292-296.

Spiller, Kara L., Suzanne A. Maher, and Anthony M. Lowman. "Hydrogels for the repair of articular cartilage defects." Tissue engineering part B: reviews 17.4 (2011): 281-299.

Brilha, Sara, et al. "Monocyte adhesion, migration, and extracellular matrix breakdown are regulated by integrin αVβ3 in Mycobacterium tuberculosis infection." The Journal of Immunology 199.3 (2017): 982-991.

Jonsdottir, Hulda R., and Ronald Dijkman. "Characterization of human coronaviruses on well-differentiated human airway epithelial cell cultures." Coronaviruses. Humana Press, New York, NY, 2015. 73-87.

Sabbione, Florencia, et al. "Neutrophil extracellular traps stimulate proinflammatory responses in human airway epithelial cells." Journal of innate immunity 9.4 (2017): 387-402.

Dos Santos Brilha, S., et al. "Monocyte adhesion, migration and extracellular matrix breakdown is regulated by integrin αVβ3 in Mycobacterium tuberculosis infection."

Brilha, Sara, et al. "Monocyte Adhesion, Migration, and Extracellular Matrix Breakdown Is Regulated by Integrin aVb3 in Mycobacterium tuberculosis Infection." (2017).

Colace, T., et al. "Analysis of morphology of platelet aggregates formed on collagen under laminar blood flow." Annals of biomedical engineering 39.2 (2011): 922-929.

Muthard, Ryan W., and Scott L. Diamond. "Blood clots are rapidly assembled hemodynamic sensors: flow arrest triggers intraluminal thrombus contraction." Arteriosclerosis, thrombosis, and vascular biology 32.12 (2012): 2938-2945.

Maloney, S. F., Lawrence F. Brass, and S. L. Diamond. "P2Y12 or P2Y1inhibitors reduce platelet deposition in a microfluidic model of thrombosis while apyrase lacks efficacy under flow conditions." Integrative Biology 2.4 (2010): 183-192.

Bauer, Rebecca N., et al. "Interaction with epithelial cells modifies airway macrophage response to ozone." American journal of respiratory cell and molecular biology 52.3 (2015): 285-294.

Kawamura, Shunsuke, et al. "Identification of common monocyte progenitors, pre-monocytes, and granulocyte monocyte progenitors in human umbilical cord blood." Experimental Hematology 43.9 (2015): S72.

Product Certificate of Analysis

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Safety and Documentation

Safety Data Sheet

Certificate of Origin

Product Disclaimer

This product is for R&D use only and is not intended for human or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.