Product Focus — Rat Tail Collagen I, the Soft "Mattress" for Cells

Collagen is the main structural component of the extracellular matrix in connective tissues and internal organs, and is most widely distributed in the skin, tendons, and bones. Collagens are classified into many types based on genetics and structure.

Collagen Type I is a heteropolymer composed of 2 α1 chains and 1 α2 chain (Figure 1). It spontaneously forms a triple-helical skeleton at 37°C and neutral pH, making it an excellent matrix for cell culture. It is widely used for hepatocytes, fibroblasts, dorsal root ganglia, muscle cells, Schwann cells, embryonic lung cells, epithelial cells, and numerous other cell lines. It can also be used to study cell growth, differentiation, migration, and tissue morphogenesis during development.

Rat Tail Collagen I is prepared according to the Birkedal-Hansen method through steps such as acetic acid extraction, sodium chloride precipitation, and disodium hydrogen phosphate precipitation. It can be used to coat cell culture vessels to enhance cell surface adhesion, and is particularly suitable for cells that are not easily adherent to the surface of ordinary culture vessels, such as primary cells like fibroblasts and hepatocytes. It can also be used for the preparation of three-dimensional (3D) gels to simulate the natural growth environment, allowing cells to grow in a 3D environment.

This product is a sterile solution dissolved in 6mM HAc (Figure 2) with a concentration of 5mg/ml. Each batch of products has passed cell culture tests (including 3D culture) to ensure the reliability of quality. Next, let's learn about the two main applications of Rat Tail Collagen I.

I. Surface Coating of Cell Culture Vessels

The recommended coating concentration for tissue culture vessels is 1-5μg/cm², and the initial concentration can be preferably 5μg/cm². It is recommended to optimize according to the specific cell type.

1. Preparation of 6mM HAc Diluent

This product is provided as a sterile solution dissolved in 6mM HAc (=0.36g/L HAc) and is not soluble in neutral pH. It is recommended to use 6mM HAc solution for further dilution. Preparation method: Add 34.5μl of glacial acetic acid (17.4M) to 100ml of double-distilled water, mix thoroughly to obtain 6mM HAc solution, and filter and sterilize with a 0.22μm filter membrane for later use.

2. Coating Steps

1.       Calculate the amount of collagen required according to your own experimental system and the optimized coating concentration, and add it to the corresponding wells to ensure that the collagen solution completely covers the surface.

      a. For a coating concentration of 5μg/cm², first dilute the collagen (5mg/ml) to an appropriate intermediate concentration (e.g., 50μg/ml) with 6mM HAc diluent, then add the diluent to each well with reference to Table 1: Volume of Collagen Diluent Added to Different Culture Vessels.

      b. For a coating concentration of 2μg/cm², first dilute the collagen (5mg/ml) to an appropriate intermediate concentration (e.g., 12μg/ml) with 6mM HAc diluent, then add the diluent to each well with reference to Table 1: Volume of Collagen Diluent Added to Different Culture Vessels.

1.       Incubate at room temperature for 1h, carefully aspirate the excess liquid, wash 3-4 times with sterile PBS, and use directly. Alternatively, after adding the collagen solution, open the lid and dry overnight in a clean bench. Under sterile conditions, the coated vessels can be stored at 4-25°C for at least 3 months.

II. Preparation of Three-Dimensional (3D) Collagen Gels

When the concentration of Rat Tail Collagen I is >1mg/ml and the pH is around 7.0, a 3D gel with a certain strength can be formed. The recommended gel-forming concentration is 1-2mg/ml.

Since this product is provided as a sterile solution dissolved in 6mM HAc, 0.06 volumes of 0.1M NaOH need to be added for neutralization during the gel-forming process.

1. Preparation of Required Solutions (Sterile, Pre-cooled)

10× cell culture medium (depending on the type of cells being cultured), 0.1M NaOH, sterile water (abs9259)

2. Preparation of 3D Collagen Gels (Without Cells) (Taking the Preparation of 1ml, 1mg/ml 3D Gel as an Example):

1.       Add 200μl of collagen (5mg/ml) to a centrifuge tube placed on ice, and add 690μl of sterile water. Then add 12μl of 0.1M NaOH [Note: This step cannot be reversed. If 12μl of 0.1M NaOH is added to the collagen solution first, local collagen coagulation will occur because NaOH cannot be mixed quickly], and mix immediately. Add 100μl of 10× cell culture medium, mix well, and immediately add to the culture vessel. [Note: The pH after mixing is around 7.0. If phenol red is not added to the culture medium, it is necessary to test with pH test paper for the first use].

2.       Place the culture vessel at room temperature (around 25°C) for 20min to allow the gel to solidify, then transfer it to the incubator.

Preparation of 3D Collagen Gels (With Cells) (Taking the Preparation of 1ml, 1mg/ml 3D Gel as an Example):

1.       Prepare the cell suspension and place it on ice.

2.       Add 200μl of collagen (5mg/ml) to 12μl of 0.1M NaOH [Note: This step cannot be reversed. If 12μl of 0.1M NaOH is added to the collagen solution first, local collagen coagulation will occur because NaOH cannot be mixed quickly], and mix immediately. Add 23μl of 10× cell culture medium, mix immediately [Note: The pH after mixing is around 7.0. If phenol red is not added to the culture medium, it is necessary to test with pH test paper for the first use]. Then add 760μl of the cell suspension, mix well, and immediately add to the culture vessel.

3.       Place the culture vessel at room temperature (around 25°C) for 20min to allow the gel to solidify, add an appropriate volume of cell culture medium, and transfer to the incubator for culture.

That's all for this issue. If you have any cell culture questions, welcome to scan the QR code to join our WeChat group for communication.

Note: The images are from the Internet and customers, for reference and learning purposes only.

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Table 2 Recommended ANT BIO Cell Culture Products

Brand Mission

ANT BIO PTE. LTD. is dedicated to advancing life science research by providing high-quality, reliable reagents and comprehensive solutions. We recognize the critical challenges in cell culture experiments and the urgent need for standardized, specialized experimental tools. Through our specialized sub-brands (Absin, Starter, UA), we have developed a targeted product portfolio for cell culture research, covering core cell matrix products (Rat Tail Collagen I), various grades of fetal bovine serum, multiple types of cell culture media, buffers, digestive solutions, cryopreservation solutions, and supporting antibacterial and disinfectant products.

Our team adheres to stringent quality control standards throughout the product development and production process, ensuring the high purity, stability, and biological activity of each product. We are committed to providing professional technical support and customer-centric services, helping researchers overcome experimental challenges such as poor cell adhesion, difficult 3D culture model construction, and unstable cell growth environment, and accelerating the pace of scientific research breakthroughs in cell biology-related fields. ANT BIO PTE. LTD. strives to be a trusted partner for scientists worldwide, contributing to the advancement of cell culture technology and the development of innovative biological research methods.

Disclaimer

This article is AI-compiled and interpreted based on the original work related to Rat Tail Collagen I research. All intellectual property (e.g., experimental protocols, data, images) of the original publication shall belong to the relevant research team. For any infringement, please contact us promptly and we will take immediate action.

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At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of cell culture needs, from core cell matrix products (Rat Tail Collagen I) to various supporting cell culture reagents. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in overcoming cell culture challenges and driving progress in cell biology research. Explore our product portfolio today and elevate your research to new heights.