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Cultural problems, Part II.

Development of an incubator for mammalian embryology

Mission unaccomplished

Large box-type or household refrigerator-style incubators are the most common models in a biological laboratory, developed for various purposes, including cell and tissue culture. They were successfully used for culturing mammalian oocytes and embryos during the first decades of IVF, too. Today, most embryology laboratories use them as "workhorse incubators" for pre-equilibration of solutions and dishes prepared for various purposes or short-term incubation of samples held in bicarbonate-buffered media. 

Considering the sample holding capacity, these incubators are relatively inexpensive. Their various heating mechanism ensures a fairly consistent temperature, although the difference between the upper and lower shelves may reach an unacceptable 1 degree (not shown on their single display). To decrease gas consumption and to provide versatility, most of these incubators use mixers to add carbon dioxide (and, if required, nitrogen) to the external air. Humidity is usually provided by a large tray filled with distilled water. Formerly copper trays, copper wires or copper sulphate solution was used to prevent contamination. Today,  purpose-developed disinfectants are available - however, most incubator manuals suggest just distilled water, monitored frequently and changed every week. Unfortunately, this procedure may disturb the environment of all cultures inside.

Lack of consistency in the three major parameters - temperature, gas composition, humidity - is a major problem of these incubators for other reasons, too. A single door opening may disturb the environment of all cultures for hours. Semi-divided areas with multiple doors were introduced to minimise this effect, but even if this structure may decrease the degree of the changes, it also slows down recovery rates, with minimal or no net benefit. In an old-fashioned IVF program, the culture of embryos of a single patient required at least six openings in five days; accordingly, samples from five patients cultured in the same incubator resulted in a suboptimal environment for at least half of the incubation period. Embryos did their best to accommodate, but the price was high: compromised quality and high mortality.

From the early '90s, when commercialisation replaced experimentation, bovine and human IVF laboratories started to recognise this problem, and various attempts were made to eliminate it. The first obvious solution - to run one incubator for every cycle - was unaffordable for most IVF laboratories because of spatial and financial limits.

The initial homemade solutions included placing small airtight containers in the large incubator, including desiccator jars or plastic lunchboxes. These containers were usually filled with a short injection by the appropriate gas mixture (humidified before or after loading), ensuring a consistent environment for cultures inside and decreasing the running costs considerably, as the continuous gas supply of the large incubator could be disarmed.



Left: The desiccator jar that hosted Louise Brown in her early days. Right: A lunchbox with gas inlet/outlet holes, covered by insulating tapes

However, the temperature recovery rate in these containers was compromised. Moreover, desiccator jars were far too big -  incubators could only store one or two  -, and lunch boxes were unprofessional, unreliable and smelled of onions and bacon. Also,  handling these devices was inconvenient and boring for many embryologists, so alternative solutions were needed.

The second obvious solution was to build small gassed box-type incubators, one for 1 or 2 patients. These incubators could be placed in two or three rows covering a laboratory wall and providing a safe and consistent environment for each culture. Unfortunately, the price of these incubators did not proportionally decrease with the size, and the cumulative running costs were also high, so only a few IVF units could afford this arrangement.

(I have to mention that a crossbreed from the mésalliance of small gassed incubators and lunchboxes was born in the workshop of Henning Knudsen Engineering, the ancestor of K-Systems, in the early '90s. Simple water-jacketed small (10 x 15 x 20 cm) metal boxes were connected to a circulating water bath. The gas mixture was manually injected into the box through small holes, closed with insulating tapes afterwards. No displays, no sensors (except for the water bath thermometer). The system had great recovery rates in all parameters; it was extremely cost-efficient, safe and highly efficient (Avery and Greve, 1992). In a few years, it was completely forgotten and never used again.)

So, we entered the 21st century and started a new era of incubators, too - with the introduction of the first bechtop toploaders, namely the MINC of Cook.


The era of benchtops and TLs -

- lasts up till today, with all the benefits and disadvantages. The benefits are the possibility to culture the embryos of all patients individually in a more-or-less standard environment (more or less, depending on types and makes, but in general, better than in the previous incubators) for more-or-less affordable price and running costs.

However, - retrospectively -  the greatest disappointment was the fact that these benchtops focused on controlling two crucial parameters out of the three. And the third, humidity, was not just disregarded. Manufacturers and distributors proudly declared - these are DRY incubators! and listed the benefits of this new approach, including

- lower risk of infection,

- less corrosion,

- more accurate sensors.

These points seem to be reasonable at first glance. At second glance, however... infection? In a well-established IVF lab, bacterial and fungal infection happens once in a blue moon, and the source is almost never the incubator - unless the maintenance is seriously compromised. Less corrosion? from the vapour of distilled water? While submarines spend months deep in highly corrosive saltwater? And for the sensors - we can make proper sensors for the Moon, for the space, is humid air an unsurmountable issue?

On the other hand  - do we really need a humid atmosphere? All common embryo culture systems use oil overlay that - obviously -  prevents evaporation! Almost everybody shared this opinion and did not deal much with the problem. And, in the initial years of benchtops, they were used for communal cultures, large drop volumes, and D3 embryo transfer systems. Results seemed to be OK, everybody was happy.

However, around 2010, three changes have happened.

- culture to the blastocyst stage was getting more and more popular,

- some companies started to produce single media again, and after some hesitation, suggested uninterrupted cultures from Day 1 to 5,

- new quality assessment methods required individual culture, and to compensate for the lack of communal effect, many labs minimised the volume of drops.

Theoretically, all these changes were positive and should have a considerable positive effect on the overall efficiency. However, the outcome was variable. In some labs, the system worked beautifully; in others, no improvement was detected, and some units - or even countries - never really adopted this system based on their controversial experience.

Many factors might have contributed to these problems, including the higher sensitivity of a long-term culture to chemical contaminations, the lack of application of low-oxygen atmosphere, or methodical -  technical issues. Finally, but very slowly, the attention of scientists started to focus on the well-known parameter of osmolality - and the possible effect of evaporation, despite the oil overlay.

An increasing amount of data showed that a 20 to 30 mOsm elevation might occur in oil-covered drops during the uninterrupted culture to the blastocyst stage. It may compromise the outcome both in vitro and in vivo - resulting in an estimated 15% loss in the overall efficiency, i.e., take-home baby/fertilised oocyte rate. Many factors - drop size and shape, type and thickness of the oil, the technique used for drop preparation, the actual humidity of the "dry" incubators may influence the outcome. However, the risk is considerable. 15% may mean tens of thousands of unsuccessful cycles worldwide every year. Just because we use dry incubators.

One would suppose that these data created panic at manufacturers; in a rush, they replaced their dry models with humidified ones by adding reliable humidifying systems and sensors to monitor humidity continuously. I have to disappoint you. According to a recent survey,  out of the 22 most popular benchtops and TLs, only 4 offers humidity as a standard feature. In 5, humidity is "optional" (??? optional for a vital parameter? can you imagine a car with an "optional" brake?), and 13 is proudly advertised as dry. Unfortunately, these dry models are the largest ones, with many chambers; accordingly, most of the IVF cycles around the globe are run now in dry incubators.

What is even more disappointing - out of the 22 models, only 2 has a humidity sensor; and an independent investigation found 60% humidity in a "humidified" TL chamber, either (the required safe humidity level in cell-tissue culture incubator is above 90, preferably 95%).

The situation seems to be similar to the low-oxygen incubators - despite the strong evidence of their benefit starting from the '90s in cattle and confirmed twenty years later in humans, models with atmospheric oxygen levels are still used in many labs. We have good reason to think that replacing dry incubators with humid models everywhere in the world will also require a decade or more.

Unfortunately, we cannot afford to wait for so long. We cannot afford inefficient systems, unsuccessful attempts, disappointed childless couples because of a known issue that we fail to eliminate.

An alternative solution is needed. Not in ten years, not even in a year.


(to be continued - fortunately)