Maintaining IVF Culture Conditions: IVF Lab 101 with Dr. Barry Behr

Last time, we discussed the importance of maintaining the pH of the culture medium within the physiological range of 7.25 to 7.30. But how exactly are pH and other variables maintained in the lab during the IVF process?

There are three main factors involved in maintenance of the culture:

1. Buffers

Buffers are compounds within the IVF culture media whose primary functions are the maintenance and regulation of pH. There are two main buffers used in IVF culture and handling media: Sodium Bicarbonate and HEPES.

Sodium Bicarbonate is essentially baking soda. When used in conjunction with a high Carbon Dioxide environment, it provides an excellent way to maintain pH in the desired range. This is accomplished using a specialized incubator. HEPES does the same thing that Sodium Bicarbonate does, but is designed for use when handling eggs, sperm, and embryos outside of the specialized incubator, in normal ambient air.

2. Specialized Incubators

Fertilization and subsequent embryo culture for up to 7 days happen inside of the above-mentioned specialized incubators. There are huge variations in the physical footprint of IVF incubators – some, for example have cameras inside – but they all do the same thing. They maintain a set temperature, with or without humidity, of 37 degrees Celsius (98.5 F) to replicate our own body temperature, as well as specific concentrations of Oxygen, Nitrogen, and Carbon Dioxide gases.

The gas concentrations required for optimal embryo culture and pH maintenance is 6-7% Carbon Dioxide (20x more than ambient air), 5% Oxygen (75% less than ambient air) and 89% Nitrogen (10% more than ambient air). The Carbon Dioxide concentration is especially critical here, as it works with the buffers in the culture medium to maintain the ideal pH.

It is not difficult to understand the need for specialized incubators when one considers the differences between ambient air and that required for human fertilization and embryo growth. Along with this comes the requirement for robust quality control programs to ensure these parameters are met and maintained.

3. IVF Culture Dish Setup

Much like incubators, there is a wide variety in the shape and size of embryo culture (petri) dishes. Embryos may be cultured in drops or in wells, individually or in groups; however, all dishes serve the same functional purpose of containing sterile culture media, which is overlaid with sterile mineral oil.

The mineral oil is key to insulating the culture media in two ways. First, as oil is not water soluble, it prevents any evaporation from the culture media over the 7-day period, even in non-humidified incubators. Second, and equally important, the oil overlay dramatically reduces the gas exchange with the ambient environment, allowing for short excursions out of the incubator without negatively impacting the culture conditions and the embryo’s development.

Each of these factors are critical on their own and interplay with each other to help maintain the precise environment for optimum embryo culture. Next time, we’ll discuss how we monitor embryo development in the IVF Lab.