Cytotoxicity Testing for Medical Devices: Which Method is Right for You?

What is Cytotoxicity in Medical Devices?

Cytotoxicity refers to the potential of a substance or material to cause damage to living cells. In the context of medical devices, cytotoxicity testing is a critical part of the biocompatibility evaluation process, helping manufacturers determine whether their products may cause adverse cellular reactions when in contact with the human body.

Why is Cytotoxicity Testing Important in Biocompatibility?

Cytotoxicity testing is often the first step in the biological evaluation of a medical device under the ISO 10993 series. It provides a sensitive screening tool to detect harmful leachables or residues before proceeding to more complex and costly animal-based tests. A positive cytotoxicity result (i.e., showing toxicity) may indicate the presence of substances that could lead to irritation, sensitisation, or even carcinogenic effects, and should prompt further investigation.

Which Cytotoxicity Test Method Should You Choose?

Cytotoxicity testing can be performed using qualitative or quantitative methods, each with its own strengths and applications. 

What are the Different Qualitative Cytotoxicity Test Methods?

Qualitative cytotoxicity test methods rely on microscopic visual grading of cell morphology and lysis. The test material is placed in direct or indirect contact with cultured cells (typically L929 mouse fibroblasts), and the cells are observed for signs of damage such as rounding, detachment, or death. 

Grading Scale (0–4): 

• 0 – No reactivity 
• 1 – Slight reactivity (≤20% cell damage) 
• 2 – Mild reactivity (≤50% cell damage) 
• 3 – Moderate reactivity (≤70% cell damage) 
• 4 – Severe reactivity (near-complete cell destruction) 

Common Qualitative Tests: 

• Direct Contact Test 
• Agar Overlay (Diffusion) Test 
• MEM Elution Test 

These methods are useful for screening and are relatively simple, but they can be subjective, depending on the operator’s interpretation. 

How do Quantitative Cytotoxicity Assays Work?

Quantitative cytotoxicity tests measure cell viability using biochemical assays, reducing operator bias and increasing reproducibility. 

• MTT Assay: A widely used method where a yellow dye (MTT) is metabolised by viable cells into a purple formazan product. The intensity of the colour correlates with the number of living cells and is measured using a plate reader. 

• Passing Criteria: 

• ≥70% viability = Non-cytotoxic 
• <70% viability = Cytotoxic 

Quantitative methods are preferred for regulatory submissions and provide more robust data for risk assessment. 

When Should You Use the Agar Overlay Method?

The Agar Overlay (Diffusion) Test is an indirect contact method ideal for medical devices where only one surface contacts the patient, such as catheters or wound dressings. 

• A layer of agarose is placed over the cell monolayer. 

• The test material is placed on top of the agar. 

• Cytotoxic substances diffuse through the agar and affect the cells below. 

• The zone of cell destruction is measured and scored. 

This method helps avoid overestimation of cytotoxicity that might occur if all surfaces of a medical device were extracted, rather than just the patient-contacting surface. 

How are Cytotoxicity Extraction Times Determined?

Extraction conditions are defined by ISO 10993-12 and vary depending on the intended duration of contact: 

• Transient Contact (<1 minute): e.g., hypodermic needles:

Extraction Time: 4 hours at 37°C 

• Short-Term Contact (≤24 hours): 

Extraction Time: 4–24 hours at 37°C 

• Prolonged or Long-Term Contact (>24 hours): 

Extraction Time: Up to 72 hours at 37°C 

These durations ensure that the test reflects worst-case exposure without degrading the material. 

What Factors Influence the Choice of Cytotoxicity Testing Method?

Choosing the right cytotoxicity test method for medical device depends on: 

• The type of device 

• Contact duration 

• Regulatory requirements 

• The level of precision needed 

For screening, qualitative methods may suffice. For regulatory submissions or detailed risk assessments, quantitative methods like the MTT assay are preferred. The Agar Overlay method is particularly useful for surface-contact devices, ensuring accurate and relevant results.