Cell invasion refers to the process by which invading cells (such as cancer cells) secrete proteases to degrade the extracellular matrix (ECM) or basement membrane (BME), allowing them to penetrate the matrix or tissue and enter blood vessels or lymphatic vessels. This enables the cells to migrate from one area to another. Cell invasion is commonly observed during processes like cancer metastasis, which is considered a specialized form of cell migration^[2].

Cell invasion is the cumulative result of various changes in tumor cells and their microenvironment, which enable the cells to migrate and invade healthy host tissues. When proliferating tumor cells attempt to escape the primary tumor site, local cell adhesion and invasion of surrounding tissues must occur. Before penetrating the endothelial lining of blood vessels and entering the bloodstream, cancer cells must invade local tissues by degrading ECM proteins and eventually breach the basement membrane. Once in the bloodstream, these cells can form metastatic colonies at secondary sites.

Both cell migration and invasion involve the movement of cells within their environment, relying on the reorganization of the cytoskeleton and dynamic changes in the cell membrane. They are regulated by extracellular signals (such as chemokines, cytokines, etc.) and are closely associated with interactions with the ECM. However, they differ in terms of their mechanisms, functions, and significance.

The cell scratch assay is a classic method used to assess cell migration and repair capabilities. Principle: a monolayer of adherent cells cultured in vitro on a dish or plate is scraped across the central region using a pipette tip or other rigid object, removing the cells in the center. The cells are then cultured for a predetermined period, and the ability of the cells to migrate into the vacant area, as observed by the gradual closure of the 'scratch' at the edges, is used to evaluate their migratory and growth capacity^[5]. The scratch assay simulates the wound healing process and is used to assess the migration ability of cells at the wound site.

Preparation before the experiment: cells, culture medium, transwell chambers or culture dishes, scratching tools (sterile pipette tips, scratch spatula, or cell scratcher), microscope and appropriate staining reagents (such as crystal violet or DAPI).

(1) Cell Culture: Seed cells into culture dishes or chambers at an appropriate density and incubate until cells reach 80%-90% confluence.

(2) Scratch Preparation: Using a sterile pipette tip or cell scratcher, gently scratch a straight line or grid-like wound on the cell monolayer, ensuring a uniform scratch. Take care to avoid damaging surrounding cells to minimize data interference.

(3) Cell Washing: Gently wash the cells twice with PBS to remove any unattached or floating cells.

(4) Culture Medium Replacement: Replace with fresh culture medium to maintain consistent experimental conditions.

(5) Observation of Scratch Healing: Capture images of the scratch using a microscope. Record the initial scratch state and subsequently take images at different time points (e.g., 0, 12, 24, 48 hours) to observe the width of the scratch or the number of migrating cells.

(6) Data Analysis: Use image analysis software (e.g., ImageJ) to analyze the captured images, measure the scratch width, and calculate the healing area.

Scratch: Ensure that the scratch width is consistent and the shape is as regular as possible.

Control Group: Set an appropriate control group (e.g., untreated group) for comparison with experimental results.

Microscopic Observation: Regularly capture images, ensuring consistent angles for accurate comparison during later analysis.

The Transwell migration assay is a widely used experimental technique for studying cell migration.

Principle: a chamber is placed into a culture plate, where the upper chamber (inside the Transwell) is separated from the lower chamber (within the culture plate) by a polycarbonate membrane. The upper chamber contains the upper culture medium, while the lower chamber is filled with the lower culture medium. Cells are seeded into the upper chamber. Since the membrane is permeable, components in the lower medium can influence the cells in the upper chamber. This setup allows for the study of the effects of the components in the lower medium on cell growth, migration, and other behaviors^[9].

Preparation before Experiment: Transwell chambers (typically with an 8 μm pore size), cells, culture medium, chemotactic factors, PBS, methanol or 4% paraformaldehyde, staining reagents (such as crystal violet, DAPI or Giemsa stain), pipettes and tips, microscope.

(1) Cell Culture: Seed cells into the upper chamber of the Transwell at an appropriate density and incubate until cells reach 70%-80% confluence.

(2) Preparation of the Lower Chamber: Add the appropriate concentration of chemotactic factors (such as growth factors or cytokines) and culture medium to the lower chamber to establish a chemical gradient.

(3) Experimental Setup: Set up a control group (without chemotactic factors) and an experimental group (with chemotactic factors) to compare changes in cell migration ability.

(4) Migration Assay: Place the Transwell chamber in an incubator, typically for 24-48 hours (depending on the cell line and experimental goals), to allow cells to migrate to the lower chamber.

(5) Cell Washing: Gently wash the upper chamber of the Transwell with PBS to remove any non-migrated cells. This wash is usually performed 2-3 times to reduce background noise.

(6) Cell Fixation: Fix the cells with methanol or 4% paraformaldehyde, typically for 10-15 minutes. After fixation, wash the cells twice with PBS to remove excess fixative.

(7) Cell Staining: Add the staining solution (such as 0.1% crystal violet solution) to the upper chamber and stain for 20-30 minutes. After staining, gently wash with PBS 2-3 times to remove excess dye.

(8) Observation and Counting: Observe the Transwell membrane under a microscope and capture images. Randomly select areas to count the cells that have migrated to the bottom of the membrane. Compare the number of migrated cells in each group and calculate the migration rate.

Transwell System: Choose the appropriate pore size (typically 8 or 12 μm) or membrane material.

Cell Condition: Ensure cells are in the logarithmic growth phase to maintain high viability suitable for migration.

Gentle Handling: Handle cells gently during washing and fixation to avoid damaging the migrated cells.

Time Selection: Migration ability varies across different cell lines, so experimental timing should be adjusted according to the specific characteristics of the cells.

The Transwell invasion assay is a widely used in vitro technique for evaluating the invasive capacity of cells.

Principle: similar to the Transwell migration assay, but with a critical distinction: the upper surface of the polycarbonate membrane is coated with a matrix-like material (e.g., Matrigel or collagen) to mimic the ECM environment. Cells secrete enzymes (e.g., matrix metalloproteinases, MMPs) to degrade the matrix gel before migrating into the lower chamber. The invasive ability of cells is quantified by counting the number of cells that have entered the lower chamber^[10]. The assay helps elucidate the regulatory mechanisms of cell invasion, providing a scientific basis for the treatment of related diseases.

Preparation before the Experiment: Transwell chambers (typically with a pore size of 5 or 8 μm), matrix gels (such as Matrigel or collagen), cell lines, culture medium, chemotactic factors, PBS, methanol or 4% paraformaldehyde, staining reagents (e.g., crystal violet, DAPI or Giemsa stain), pipettes and tips, microscope.

(1) Cell Culture: Seed cells into a culture dish at an appropriate concentration and incubate until cells reach 70%-80% confluence.

(2) Matrix Gel Preparation: Dissolve the required amount of matrix gel (Matrigel or collagen) on ice according to the manufacturer's instructions. Apply an appropriate thickness of matrix gel (usually 50-100 μL) to the bottom of the upper chamber of the Transwell. Incubate at 4°C for approximately 30 minutes to 1 hour to ensure the gel solidifies.

(3) Preparation of the Lower Chamber: Add an appropriate concentration of chemotactic factors (such as growth factors, cytokines) and culture medium to the lower chamber of the Transwell.

(4) Invasion Assay: Suspend cells (usually 1-2×10⁵ cells) in suitable culture medium and add them to the upper chamber of the Transwell. Incubate in an incubator for 24-48 hours. Cells migrate and invade towards the lower chamber.

(5) Cell Washing: Gently wash the upper chamber of the Transwell with PBS to remove non-invaded cells. This wash is usually performed 2-3 times to reduce background noise.

(6) Cell Fixation: Fix the cells in the upper chamber of the Transwell with methanol or 4% paraformaldehyde for 10-15 minutes. After fixation, wash the cells twice with PBS to remove excess fixative.

(7) Cell Staining: Add the staining solution (such as 0.1% crystal violet solution) to the upper chamber of the Transwell and stain for 20-30 minutes. Then, gently wash 2-3 times with PBS to remove excess dye.

(8) Observation and Counting: Observe the Transwell membrane using a microscope and capture images. Randomly select areas to count the cells that have invaded to the bottom of the membrane. Compare the number of invaded cells in each group, and statistically analyze and calculate the invasion rate.

Matrigel Gel Handling: Matrigel gel needs to be handled on ice to maintain its biological activity; when coating, try to add it vertically to the center of the bottom of the chamber to avoid bubbles.

Cell Condition: Ensure cells are in the logarithmic growth phase to maintain high viability suitable for invasion.

Gentle Handling: Handle cells gently during washing and fixation to avoid damaging invaded cells.

Observation Time Selection: Invasion ability varies across different cell lines, so experimental timing should be adjusted according to the specific characteristics of the cells and the expected effect of the experiment

What could cause uneven cell distribution after staining?

Possible reasons include: the Transwell chamber is not placed evenly in the well plate; the cell suspension is not properly mixed; the chamber is tilted or shaken during handling; the chamber membrane is uneven.

What is the correct order of adding liquids when seeding cells into a Transwell chamber?

First, add complete culture medium to the well plate (lower chamber). Then gently place the Transwell chamber into the lower chamber. It is recommended to tilt the chamber slightly so that one side touches the liquid surface first—this helps avoid bubble formation caused by vertical placement. Afterward, add the well-mixed cell suspension into the upper chamber.