Autoclaving techniques and microfluidics: a review

With the increase of cell culture chips and organs-on-chips, sterilization is essential in microfluidics for any equipment in contact with sensitive fluids. Many processes exist, but the autoclave (or steam sterilizer) remains the most common technique thanks to its low cost, effectiveness, easiness to process and non-producing of toxic compounds.

In this review, we will introduce the different types of autoclaves and identify the materials that are the most compatible with this sterilization method.

How does autoclave for sterilization work?

Heat sterilization or autoclaving is a relatively simple process that exposes the device to saturated steam at 121°C for 20 min, at a pressure of 115 kPa. Protein denaturation occurs and microbial activity is interrupted. It is possible that the activity is only stopped momentarily and resumed as soon as the element reaches room temperature.

For complete germ destruction, the steam must be homogeneous and saturated, and the temperature very high, which will lead to a pressure according to Regnault's law*. Water for steam should be demineralised to avoid any suspended substances.

The autoclave starts with successive injections of steam to increase the temperature and pressure. This step may require more time, as the sterilization chamber must not contain air. Steam injections can be alternated with vacuum injections. The next step is true sterilization. For this, the chamber is kept at a bearing temperature for a certain time. After a few minutes, the chamber cools down and the pressure decreases with the temperature to reach atmospheric pressure.

A standard sterilization cycle is represented on the figure 1. 

Different types of autoclaves

There are three different types of autoclaves: the class N autoclave, the class B autoclave and the class S autoclave.

Class N autoclave

Class N autoclaves are small machines used to sterilize simple materials. The letter N stands for "naked solid products" and refers to dissolved solid products. These autoclaves are therefore not suitable for sterilizing textiles, porous objects, hollow objects or products in bags. Another potential disadvantage of these tools is that they do not guarantee proper steam penetration, which depends mainly on the initial vacuum, not required on these types of devices.

Class B autoclave

The letter B here stands for "big small sterilizers": small, but efficient. Any type of object can be sterilized with a class B autoclave: porous materials, but also wrapped material, textiles and hollow bodies, such as glass syringes, luer or tips. The reference standard for this equipment is EN 13060, dedicated to small steam autoclaves (those with a sterilization chamber smaller than the sterilization unit). This standard indicates the different sterilization cycles which differ according to the characteristics of the equipment to be treated.

Class S autoclave

Finally, class S autoclaves include all the other machines: actually, this is the intermediate class between class N autoclaves and class B autoclaves. Their characteristics are not defined by any standard, as they depend on the way they are manufactured. This is why their performance is only specified and defined by the manufacturer according to specific tests.

What is the best autoclave for microfluidics?

The most suitable autoclave for microfluidics is therefore the class B autoclave, guaranteeing superior versatility and use adapted to each situation. In spite of its small size, it provides excellent performance while complying with the latest safety standards.

Routine tests in autoclaves

Routine tests are similar to simple checks (tyre pressure, etc.) carried out on a vehicle, whereas qualifications are similar to technical inspections. They essentially include vapour penetration tests and the vacuum tests.

 Vapour penetration tests

The Bowie-Dick test has been developed to evaluate the penetration of steam within loads of textile. This type of load is currently no longer frequently used.

The Helix test, on the other hand, mimics a hollow body using a hollow plastic device. In addition, the design of this test allows for the reuse of the plastic holder which is changed every 250 to 350 tests.

Vacuum tests

The second routine test, corresponding to the vacuum, is used to demonstrate that air leakage into the sterilizer chamber does not exceed a level that will prevent steam penetration into the sterilizer load and will not constitute a risk of recontamination of the load during drying.

The most used autoclavable materials in microfluidics

In microfluidics, the most common problem is the sterilization of microfluidic chips: steam sterilization operates at 121°C so heat-sensitive materials will be damaged or destroyed. It is therefore necessary to know which materials can be autoclavable or not.

Microfluidic chips made in PDMS (polydimethylsiloxane), in PMMA (polymethylmethacrylate), in COC (cyclic olefin co-polymer) or in PC (polycarbonate) are autoclavable [A Pfreundt et al 2015 J. Micromech. Microeng.]. It is also important to note that connectors such as Luer or Nanoport (before to be irreversibly glued or bonded to the devices) [A Pfreundt et al 2015 J. Micromech. Microeng.], tubing made in PTFE, glass slides and some glass syringes are autoclavable.

It is nevertheless advisable to refer to the manufacturer's instructions for each element before any sterilization operation.

Sterilization of microfluidic chips is an essential step prior to customer use in biomedical and biological applications. Steam sterilization seems to be the most indicated in microfluidics but the respect of a whole protocol (easily available on the Internet) is necessary for the good realization of this indispensable step.

*Law of pressure states that for a fixed mass of gas, at constant volume, the pressure (P) of the gas varies directly with absolute temperature (T) of gas.