Matrix gels provide a biomimetic environment that supports cell adhesion, proliferation, and differentiation in traditional two-dimensional (2D) culture systems. When used as a coating substrate, matrix gels supply essential extracellular matrix (ECM) components—such as laminin, collagen, and entactin—that promote physiological cell behavior and morphology.

Compared to conventional plastic surfaces, matrix gel–coated culture plates allow cells to maintain better viability, more natural spreading, and enhanced responsiveness to growth factors. This approach is widely applied in stem cell maintenance, cancer cell line expansion, and primary cell culture studies, serving as an ideal platform for drug screening, gene expression research, and functional assays.

I. Protocol for 2D cell culture

1. Thawing and Storage of Ceturegel™ Matrix Gel

[Note]: Ceturegel™ Matrix Gel is highly temperature-sensitive. Avoid repeated freeze-thaw cycles. All steps involving dispensing or preparation prior to gelation must be performed on ice (4 °C), as slight temperature increases may cause premature gelation, leading to uneven distribution or impaired gel formation. All tubes and pipette tips used must be pre-chilled.

a) Upon receipt, if not used immediately, store the entire vial directly at –20 °C (do not use frost-free freezers).

b) For first use, place the entire vial on ice and transfer to 4 °C overnight for complete thawing. It is recommended to centrifuge at 14,000 × g for 20 min at 4 °C and carefully collect the supernatant.

2. Special Notes on Use of Ceturegel™ Matrix Gel

Matrix Gel rapidly gels at 22–35 °C. To ensure optimal gelation performance and stability, the final dilution concentration may vary by lot. Dilute with serum-free medium and use immediately after dilution.

A. Thin Gel Coating Method

1) After thawing, mix Matrix Gel thoroughly using a pre-chilled pipette tip.

2) Place the culture plate on ice and add Matrix Gel at a concentration of 50 μL/cm² of growth surface area.

3) Incubate at 37 °C for 30 min. The plate is then ready for use.

B. Thick Gel Method

1) After thawing, mix Matrix Gel thoroughly using a pre-chilled pipette tip.

2) Place the culture plate on ice. Mix cells with Matrix Gel and suspend evenly using a pre-chilled pipette tip. Add Matrix Gel at a concentration of 150–200 μL/cm² of growth surface area.

3) Incubate at 37 °C for 30 min, then add cell culture medium. Cells may also grow on top of the thick gel layer.

C. Thin Layer Coating Method

1) After thawing, mix Matrix Gel thoroughly using a pre-chilled pipette tip.

2) Dilute Matrix Gel with serum-free medium to the desired concentration. A gradient test is recommended to determine the optimal coating concentration for specific experiments.

3) Add diluted Matrix Gel to the culture vessel, ensuring complete coverage of the growth surface. Incubate at room temperature for 1 hour.

4) Remove unbound Matrix Gel and rinse gently with serum-free medium. The plate is now ready for use.

[Note]: Coated plates should ideally be used the same day, but may be stored depending on application. After adding culture medium, coated plates can be stored at 37 °C for up to 1 week.

D. Validation Procedure

1) After thawing, dilute Matrix Gel 10-fold with pre-chilled PBS and keep on ice.

2) Add 0.5 mL of diluted Matrix Gel to three wells of a non-tissue-culture-treated 6-well plate, ensuring even coverage of the bottom. Incubate at 37 °C for 30–60 min, then remove excess liquid.

3) Digest 293T cells using standard protocol and adjust cell suspension to 1 × 10⁵ cells/mL.

4) Slowly add 3 mL of cell suspension down the side of each well to avoid disturbing the coated layer. Seed the same number of cells in uncoated control wells.

5) Incubate the 6-well plate overnight (12–18 h) in a 37 °C, 5% CO₂ incubator.

6) Observe cell attachment and take photos. Results are shown below (a: untreated, b: coated with 10× diluted Matrix Gel):

II. Experimental Results

Figure 1. Support of 2D Cell Culture

III.Troubleshooting

Q1. How should I prepare matrix gel for 2D coating?

A1. Thaw the matrix gel on ice overnight at 4°C. Dilute it with cold serum-free medium to the desired concentration, then evenly coat the culture surface. Incubate at 37 °C for 30–60 min before cell seeding.

Q2. What cell types can benefit from matrix gel coating?

A2. Matrix gel supports a wide range of adherent cells, including stem cells (ESCs, iPSCs, MSCs), epithelial cells, endothelial cells, hepatocytes, and cancer cell lines.

Q3. Can matrix gel coating improve reproducibility in experiments?

A3. Yes. By providing a consistent ECM-like surface, matrix gel reduces variability caused by differences in plastic surface properties, improving data reproducibility across batches and laboratories.

Q4. Is the matrix gel suitable for serum-free or defined medium systems?

A4. Yes. Matrix gel is compatible with both serum-containing and serum-free culture systems, making it ideal for defined, xeno-free, or feeder-free workflows.

Q5. How does it differ from 3D culture applications?

A5. In 2D culture, matrix gel acts as a coating substrate to enhance cell adhesion and morphology, while in 3D culture, it serves as a structural scaffold that supports cell–cell and cell–matrix interactions in all dimensions.

Q6: How to ensure sterility during culture?

A6: Add 1% antibiotics and an appropriate amount of gentamicin sulfate to the culture medium.

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