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Reproductive Health

Q&A: Osmolality rise in nonhumidified incubators

18新利手机app下载Team Lead, Senior Scientist Steven Mullen对未经讨论的孵化器发生的渗透性的变化进行了研究人类繁殖2021年,他的研究还形成了下面列出的演示和研讨会的基础。





这easiest way is with an osmometer. We use a Wescor Vapro®5600 vapor pressure osmometer. Another common method involves the use of a freeze point machine. Others estimate evaporation and then extrapolate the osmolality change by measuring volume or weight changes, but that method may not be very accurate.

Would changing drops (dishes) on day 3 in a dry incubator with one-step culture media mitigate the evaporation?

Changing media on day 3 would avoid excessive evaporation, because the media would experience only 3 days of evaporation instead of 5 or 6 days. This is one way to avoid the issue, but there are others, such as using more oil, using denser oil, etc. Although changing media on day 3 will reduce the amount of evaporation, the osmolality will still increase over the 3 days. Thus, under certain conditions, the osmolality rise after 3 days may be great enough to be detrimental.


If evaporation is occurring under the culture conditions that are used, then yes, osmolality would increase each day over time. It depends on the variables of the culture system: humidity, volume, oil volume, oil density, etc.


在整个研究中,特定时间或因子组合的测量数量变化。当我们执行此类测量时,该值几乎始终在2 MOSM / kg彼此内。最常见的测量是初始测量的+/-1,并且再次获得相同的结果并不罕见(即,当测量两次培养基时相同的渗透性)。


It shouldn’t affect the rate of change. The absolute values at any time will differ, but the rate of change shouldn’t differ.

Is there any relationship between osmolality and the amount of fungus present, especially in a nonhumidified incubator?


Do direct and indirect heat have different effects on media osmolality?




这slides you presented show the difference in osmolality change between dry and humidified incubators. Did you evaluate more significant endpoints, like fertilization rate, embryo quality, cumulative pregnancy rate, and live birth rate?

不,在我们进行的研究中,仅在不同的条件和培养设置中研究了渗透压(MOSM / kg)。有关受精率,裂解和胚泡率的临床证据,以及临床妊娠率在着名的期刊上提供。

What is the relationship between pH and osmolality?




While most data is from a research setting with no embryos present, there is little reason to think that the evaporation rate would be different if embryos were present in the media. The type of media wouldn’t really affect the evaporation rate (i.e., there should be no difference between single-step and sequential media). If single-step medium isn’t changed over the 5–6 days of culture, it will likely see more evaporation and a higher osmolality increase than a sequential medium that was changed from step 1 to step 2. Changing to a single-step medium is one possible solution for addressing this osmolality change. Another possible solution is adjusting other culture parameters to mitigate the rate of evaporation (adding more oil, changing the type of oil, etc.).


胚胎确实具有用其无机离子或有机渗透物(氨基酸,主要在早期切割胚胎发育的氨基酸,主要甘氨酸和谷氨酰胺)调节细胞体积的机制。Postcompaction胚胎(第3天和第4天)具有具有不同渗透和运输蛋白的更强壮的机制。但是,我们必须记住,这种机制只能在狭窄范围内工作。当培养基中的渗透压培养基超过300-310个MOSM / kg时,它可能会影响胚胎的活力并抑制IVF培养中的胚胎发育。



不是我知道的。Mineral oil does help reduce evaporation. There are different types of oil, and some appear to reduce evaporation more effectively than others, such as higher density mineral oils. If the density isn’t listed on the commercial product, it can be difficult to discern which oil is more dense than another, because the nomenclature used to describe commercial oils—light vs. paraffin, etc.—is inconsistent.




Using too much oil won’t have a negative effect, really, other than creating a risk of possible spillage in the lab when you’re walking with the dish. Excessive oil usage may prolong the amount of time required for pH equlibration, but it shouldn’t have a negative effect on osmolality.

How do you recommend that the embryo transfer be performed in order to avoid evaporation of the transfer media? Do you use oil on it?


Do you prefer mineral oil over paraffin oil?

This is a hard question to answer, because it’s not always clear as to what the actual difference is. As long as the oil is nontoxic, we don’t have a strong preference and have used both. Nomenclature seems to vary by manufacturer.

We keep a 100 mL bottle of oil in the incubator. Is it harmful for the oil?





Yes, but you should limit how long it is kept in the incubator, because it can accumulate VOCs or other toxins over time. Keeping oil in the incubator should have no effect on evaporation or osmolality.

Is there is any significant benefit to using washed and humidified oil?

也许毒性控制,但蒸发and osmolality control, our data indicate that it provides no benefit.


No. It would be interesting to investigate. I suspect that the chemical structure of the molecules affects the density of the oil and consequently the rate of evaporation that occurs when oils of different densities are used.

Is it advisable to use oil from one company and culture media from a different company? Will it affect the efficiency of oil?

I see no issues with using supplies from different companies.

Is it beneficial to let oil stabilize in a humid incubator for later use in a dry incubator?



It is difficult to say which oil is ideal. Higher viscosity oils tend to reduce the rate of evaporation, but only in nonhumidified incubation conditions. Lower viscosity oils tend to be easier to work with, in my opinion, but allow for faster rates of osmolality rise in nonhumidified incubation. For many situations, the value of one variable in a culture system depends on other variables.

Can you give us some brand names of high-density culture oils?

Oils marketed as “heavy” have higher density, generally speaking. I do not wish to name brands, because I don’t want the study to appear biased toward any particular brand or manufacturer.

Did you use washed or unwashed oil, and could this have influenced your outcomes?

I used unwashed oil. Other studies have shown that using washed oil has no effect on the rate of osmolality change.

Which is better to use for culture, paraffin oil or mineral oil?



How do osmolarity, pH, and temperature differ with regard to the use of an oil overlay in closed vs. open culture systems?




What is the lowest culture media volume recommended for a drop culture system?


What is your recommendation for proper media droplet size, amount of oil overlay, and type of oil?




Did you prepare the droplets with the overlay method? How do you avoid the change of osmolality from exposure of the media to air during dish preparation?

I did not use an oil overlay method. I prepared the media on a room-temperature surface and usually outside the hood to avoid air-flow-related evaporation. We also use an electronic repeat pipettor to prepare microdrops, which is very fast. Having said this, I am not concerned about small changes in osmolality during media preparation, simply because that should not affect the rate of change in osmolality, although it will affect the actual value at any given time. My reasoning for designing the study as I did was so that the results can be broadly applicable and not restricted to any specific factor that I chose, such as the medium’s starting osmolality.

Does 1 mL of oil completely cover the microdroplets of medium in a 35 mm dish?


How did you make the droplets in the 35 mm dish to ensure that the surface area would always be the same?

I was very careful to make the drops consistently. In the study where I measured the drop surface area, I used the same technique that I do for making drops for the rest of the experiment. I used the average value for the drop surface areas that I directly measured when I estimated the surface-area-to-volume ratios for the drops that were not directly measured. It is important to note that I when I developed the model for the experiments, I used 96 well plates that have a cylindrical geometry in the well so that the surface area would be consistent for each treatment. There are variations in the drop size for the same volume of media, but the variability is pretty small relative to the size.


When I used dishes with wells, the medium was not in the form of a drop. I ensured that the medium covered the entire surface of the well bottom so it would have a cylindrical geometry. The results seemed to suggest that whether the medium is in a cylindrical geometry, as it is in a well, or in a drop configuration, as is common in 35 mm dishes, the osmolality change will be the same for a given surface-area-to-volume ratio.

How should dishes be prepared to minimize changes in osmolality?



This would depend on the specific protocol for the noninvasive preimplantation genetic testing (NiPGT) system that the lab is using. I would comment only to say that they should follow their test protocol for that system.








If humidified incubators yield better results than dry incubators, and this applies to the nonhumidified time-lapse incubators as well, is it advisable to culture in nonhumidified timelapse incubators?

To be clear, it’s not that dry culture cannot work. In fact, many labs are very successful with dry incubators. Nonhumidified incubators can also help reduce issues of contamination. They can have improved temperature stability and environmental recovery. However, culture conditions should take into account the dry nature of the incubator and ensure that evaporation isn’t an issue. Steps can be taken to address it, like using more oil, using a denser oil, altering the media volume, doing media exchanges, etc.






这embryo is bathed in 100% moisture when in the microdrop, so I’m not sure that the humidity per se is a concern to embryo health. It is debatable whether the embryo is submerged in liquid in vivo like it is in vitro, and this is likely another topic unto itself (i.e., microfluidics, novel surface coatings to culture on, etc.). Although the embryo may experience a more moist environment in the female reproductive tract, the amount of humidity in an incubator does appear to impact evaporation, which can alter the chemical properties and formulation of the culture media, which in turn can impact media efficacy and embryo development.

有人的经济学新蒂姆相比吗e-lapse culture systems with the traditional culture conditions?



A hygrometer can measure the relative humidity of the environment.

What is your advice for protecting against osmolality increase in dry incubator?



Based on theSteve Mullen完成的研究,渗透压的升高是使用干燥培养箱,小培养基下降和薄层培养油的后果。所有上述单步培养基的组合将增加培养中渗透性渗透性甚至加速的风险。





我们在实验室中交换媒体,不使用不间断的文化。We didn’t adopt our paradigm because of evaporation concerns—we have always used a sequential system that is based on the research done at CCRM and on improved outcomes—but it has helped us avoid this issue of evaporation, since we do use dry incubators.

How can you avoid evaporation of HEPES media in a center well for oocyte retrieval? Cover it with oil?


Do you think preparing culture media in a cold plate or on top of an ice pack would be a good practice to avoid evaporation?

I don’t think this is needed. Preparing on a room-temperature surface and doing so quickly by limiting the number of dishes prepped at one time before adding oil is sufficient to avoid excessive evaporation during dish prep.

Since there is a change in osmolality with culture time, is it recommended to switch to sequential media rather than single-step media?

Not necessarily. Other methods can be used: humified incubation, increasing the amount of oil, adjusting the media volume, etc. Also, a single-step media can be changed during use.

Do you prefer to subculture on day 3 even after using single-step medium?


Single-step culture is not a very old technique. Is any data available on the safety of this culture system and the development of babies born as a result of it?



This may help. However, when you’re working in a humidified incubator, the osmolality change over time is quite small. It is probably a good idea to incorporate many processes into your protocol in order to reduce osmolality change.


No, we didn’t. During the experiments we used Cook Medical’s Sydney IVF Cleavage Medium, which contains human serum albumin.


18新利手机app下载库克医疗的顺序文化介质是以胚胎生长的每个阶段支持胚胎发育的方式。制剂设计用于特定的显影阶段,并添加所有组分的组合效应。这些组分中最重要的 - 支持胚胎代谢的那些是碳水化合物和氨基酸。

With the emergence of time-lapse culture systems, the current trend is toward the use of single-step medium for extended uninterrupted culture. Why is Cook Medical not developing a single-step medium to address this market need?

Our belief is that the most important need to meet is that of the developing embryo. The more recent trend toward single-step systems is driven largely by static culture systems (time lapse) and the efficiency and convenience of the lab. We believe that a sequential media system is the most appropriate system for supporting embryo development, because sequential media systems mimic nature. Sequential media are based on the natural changes that occur in the oviduct and the uterus during embryo development.

How often should one change to fresh culture media to maintain a normal osmolality?

在顺序培养基的情况下,培养基和培养皿仅制备2-3天,然后在此之后,将改变盘子,并更新培养基。在单步介质的情况下,盘将保持6-7天。这Steve Mullen完成的实验shows a rise of 6.96 mOsm per day in a microdrop setup. Depending on the culture medium’s osmolality, the amount of culture medium used, the amount of oil used, and the type of incubator, you might be able to predict the time when the osmolality will be exceed the set limit. To avoid an osmolality rise above 30 mOsm/kg, changing the media after 2–3 days of culturing should help maintain a safe range.