Three-dimensional (3D) cell culture provides a physiologically relevant environment that closely mimics the in vivo cellular microenvironment. Matrix Gels serve as a biomimetic scaffold composed of extracellular matrix proteins such as laminin, collagen IV, and entactin, allowing cells to grow, differentiate, and self-organize into tissue-like structures. Compared to traditional 2D systems, 3D cultures using Matrix Gel enable more accurate modeling of tissue architecture, signal transduction, and drug responses.

I. Protocol for 3D cell culture

1.Cell Suspension Preparation

Pre-chill 24-well plates and pipette tips on ice. Keep Matrix Gel on ice or thaw slowly at 4 °C. Harvest HepG2 cells in logarithmic growth phase, gently rinse with PBS, and digest with trypsin. After digestion, prepare and collect a single-cell suspension. Centrifuge at 1500 rpm for 3 min, discard supernatant, and resuspend in complete DMEM medium. Count cells using a hemocytometer and adjust concentration to 1.5 × 10⁵ cells/mL.

2.Cell Seeding

Mix thawed Matrix Gel and adjusted single-cell suspension at a 1:1 ratio on ice with gentle mixing. Using a pre-chilled 200 μL pipette tip, transfer 40–50 μL of the mixture and dispense vertically into the center of pre-chilled 24-well plates to form dome-shaped droplets.

3.Data Analysis

After incubating in a 37 °C, 5% CO₂, humidified incubator for 30 min to stabilize, add 500 μL to 2 mL of complete DMEM medium per well and continue culture. Observe and photograph daily.

II. Experimental Results

Figure 1. Results of 3D cell culture after 4 days

Figure 2 MDKC Vesicle Growth Assay

MDCK cells were induced with 10 μmol/L FSK for 4 days. Specifically, the resuspended MDCK cells were mixed 1:1 on ice with Yeasen Matrigel (thawed overnight on ice). The mixture was then vertically plated as a hanging drop in the center of each well of a Corning ultra-low attachment 24-well plate. The plate was placed flat in a 37°C incubator for 45 minutes to allow solidification and adhesion. After this, the corresponding complete culture medium was added for further intervention.

Figure 3 In Vitro Culture of Dorsal Root Ganglia (DRG)

Isolate spinal DRG from postnatal pups. Pre-coat dish with Yeasen Matrigel (3:5 dilution). Embed DRG in gel. After polymerization, add DMEM culture medium.

III.Troubleshooting

Q1: What is the recommended Matrix Gel concentration for 3D culture?

A1: Typically, 8–12 mg/mL (standard concentration) is suitable for most organoid and 3D culture applications.

Q2: Can the gel stiffness be adjusted?

A2: Yes. Gel stiffness can be modulated by varying the protein concentration or blending with reduced growth factor Matrix Gel.

Q3: How should the gel be handled to maintain consistency?

A3: Always work on ice and pre-cool pipette tips and plates to prevent premature gelation.

Related Products

Category	Name	Cat. No.	Size	Note
Basic concentration of Matrigel 8-12 mg/mL	Ceturegel™ Matrix LDEV-Free	40183ES08/10	5 mL / 10 mL	Suitable for 2D/3D culture, invasion and migration assays, and in vivo tumor formation studies.
	Ceturegel™ Matrix Phenol Red-Free，LDEV-Free	40184ES08/10	5 mL / 10 mL
High concentration 18-20 mg/mL	Ceturegel™ Matrix High Concentration，LDEV-Free	40187ES08/10	5 mL / 10 mL	High-concentration gel is viscous and gels quickly, ideal for in vivo tumor formation with hard-to-grow cell lines.
	Ceturegel™ Matrix High Concentration，GFR，LDEV-Free	40189ES08/10	5 mL / 10 mL
	Ceturegel™ Matrix High Concentration，Phenol Red-Free，LDEV-Fre	40188ES08/10	5 mL / 10 mL
Low Growth Factor	Ceturegel™ Matrix GFR, LDEV-Free	40185ES08/10	5 mL / 10 mL	Minimizes growth factor interference in signaling pathway studies.
	Ceturegel™ Matrix GFR, Phenol Red-Free, LDEV-Free	40186ES08/10	5 mL / 10 mL
Stem cell	Ceturegel™ Matrix hESC-Qualified，LDEV-Free	40190ES08/10	5 mL / 10 mL	Mainly used for hESC/iPSC stem cell culture
Organoid-Specific	Ceturegel™ Matrix for Organoid culture, Phenol Red-Free, LDEV-Free	40192ES08/10	5 mL / 10 mL	Upgraded organoid matrix gel for normal and tumor tissues with improved culture performance.