Boundary conditions

The seven geometries described above are exposed to the environment(s) within the laboratory. Figure 3.8 displays some of the equipment which provides the various environments surrounding the embryos.

3.3.2.1 Incubators

For the majority of the culture process, dishes are kept in an incubator. The majority of incubators conform to two types, each of which may utilise different ways of controlling temperature and gas composition.

(i) Open volume incubators (Figure 3.8B), which typically range from 50-250 litres, maintain a large cavity at the required temperature, humidity and gas concentrations. Culture vessels, such as Petri dishes, sit on perforated metal trays within the incubation chamber. Normally larger volume incubators will have several trays stacked vertically to maximise utilisable space. Temperature within the chamber is

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maintained by convection from heating elements within the chamber floor, walls and door. Many contain fans, not to provide air flow within the chamber, but to thoroughly mix gases within the vicinity of gas sampling ports.

Figure 3.8: Boundary Conditions for dish systems in an embryo laboratory. A) One of the two chambers of the MINC Benchtop Incubator (Cook Medical, Cook Group Inc., IN USA) incubator which may hold four dishes. B) An open volume SANYO incubator (SANYO Biomedical, IL USA). C) A glass heated microscope stage (Thermoplate, Tokai Hit, Japan).

Open volume incubators control gas composition by injecting pure gas directly into the chamber. The gas is mixed with the existing incubator atmosphere and samples of the gas pass over feedback sensors. Injection of the pure gas will continue until the required concentration has been achieved. The minimum requirement of any incubator is to maintain a concentration of CO2 higher than atmospheric levels by injecting pure CO2. A concentration of 5-6% is required in the chamber to maintain the correct pH within a bicarbonate buffered culture medium. Other incubators are also capable of maintaining a chamber with a low oxygen atmosphere. In such incubators, the O2 level is lowered by displacing atmospheric air within the chamber with pure N2. Absolute levels will vary between laboratories.

55 (ii) The second type of incubator, the MINC (Figure 3.8A), has very low internal volume. The culture vessels sit directly on a heated plate, and are covered by a heated lid. A pre-mix of humidified gas (at appropriate CO2 and O2 concentrations) is either perfused through the chamber, or may purge and soak the chamber.

3.3.2.2 Laboratory temperature

The ambient laboratory air surrounds all geometry systems at some stage throughout the process and is invariably cooler (typically 23-27 °C) than the generally targeted embryo temperature of 37 °C. Optimally, laboratories would be maintained at a constant temperature allowing accurate calibration of equipment while trying to maintain a balance between minimising temperature fluctuations (for embryos while they are out of the incubator) and maintaining a comfortable working environment. Culture critical gasses such as CO2 and O2 are sub-optimal at atmospheric concentrations. Air flow is likely to be minimal in a lab environment but it is worth noting the position of equipment – such as a heated stage – with respect to any air conditioning unit as increased air flow will affect both heat and gaseous mass transfer.

3.3.2.3 Heated surfaces

Dishes are removed from the incubator for embryo manipulation and observation under the microscope. Heated stages provide the base boundary of dishes outside an incubator. There are many types of heated microscope stages available (different materials and makes). Two main types of heated stage are used at Fertility Associates Auckland and they provide significantly different boundaries. With the first, a metal heating plate provides a heated surface. The advantage of metal is that the rate of heat transfer within the metal itself is high and so the temperature throughout the body of

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the stage will remain fairly uniform. The disadvantage is that metals are opaque and as embryo observations require transmitted light, a transparent aperture must be provided. The aperture may be a physical space or be filled with a transparent material such as plastic or glass. The rate of heat transfer within either material is significantly reduced in comparison to the surrounding metal which increases the difficulty of maintaining a uniform temperature across the entire working area. In contrast, glass heated stages (Figure 3.8C) are transparent so the entire surface is homogenous. However, the properties of glass do not facilitate heat transfer as fast as in metal, so a localized heat loss / gain will not be as quickly dispersed across the stage, slowing any response by the temperature controller.

Heating blocks are used to hold test tubes and for temporary placement of dishes. These are deep metal blocks held within a heated channel and provide a uniformly heated surface. Test tubes sit within cylindrical cut outs of the heating block; these holders are dry so air gaps exist between the test tube and the wall. Depending on the make of heating block and the volume of test tube, a proportion of the test tube will stand proud of the top surface of the block. Thermocoins (aluminium disks) sit under dishes on the heating block replacing the air gap beneath the dish and therefore provide direct contact between the heated surface and the dish floor.

The way in which the temperatures of heated microscope stages or heated blocks are set is laboratory dependent. Several methods used are listed here:

• setting the temperature of the control system for the heated microscope stage to read 37 °C

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• by placing a thermistor (or other temperature measuring device) onto the surface of a stage, and adjusting the set point of the control system to read either 37 °C or another value which has proved, by trial and error to give better pregnancy rates

• by placing a thermistor (or other temperature measuring device) into a dish (with its lid on and with or without liquid in it) on the surface of a stage, and adjusting the set point of the control system to read either 37 °C (or another value which has proved, by trial and error, to give better pregnancy rates) at steady state.