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Reproductive Health
4月14日,2021年

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。

对于那些无法参加原始演示的人,我们将与会者的问题和演示者的答案收集并将其分组成分类。

Sources

osmolality基础知识

如何测量渗透性次数?

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.

渗透性是否随着每天的文化而增加?

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.

您为每个时间点执行多少测量值?你的测量有多一致?

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)。

不同种类媒体的起始渗透性变化。这个初始差异如何影响变化率?

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?

真菌的存在可能影响渗透压测量的唯一方法是如果真菌在用于渗透测量的培养基中生长。

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

唯一可能导致效果的是培养基对温暖所需要的时间,然后渗透性变化的速率应该是温度的影响。

如果我们将培养培养基(裂缝,胚泡)放入培养箱中而不在封闭管中油,渗透压多少变化?

在紧密封闭的管子的情况下,蒸发是最小的或没有,因此在这种情况下,渗透性的上升不会是一个问题。但是,我们必须记住,培养培养基(裂缝和胚泡介质)需要平衡并保持在气体交换环境中。这对于维持适当的pH是重要的。

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?

没有直接关系。如果发生蒸发,渗透性会增加。pH值的增加也可能是由于蒸发。

渗透压和胚胎文化

您对胚胎发育过程中渗透性变化的观察是什么,尤其是单步培养基?

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.).

您认为胚胎可以在一定程度上适应渗透性吗?

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。

CULTURE OIL

是否有替代品可以在胚胎培养期间用作抗抗原剂?

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.

在没有水夹克的情况下保持干燥培养箱中适当的渗透性,哪种油是最佳的?

答案根据盘的类型,介质的体积,微滴的大小,油的类型等而异。

使用太少的培养油可能会产生负面影响,但可以使用太多的油也有负面影响?

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?

我们使用加湿室或钟罩覆盖盘子,同时我们等待然后快速加载导管。我们不使用石油。我们迅速工作并使用更大量的媒体来试图减轻风险。

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?

也许。保持油太长可以导致挥发性有机化合物(VOC)或过氧化积累的潜在问题。最好是遵守石油的短属保质期,而不是储存它长期。

油中的小气泡是蒸发的指示吗?

不是我知道的。石油中非常小的气泡有时似乎是CO的结果2当盘子从培养箱中渗出几分钟后切出。这可能是你所看到的。

将油保存在培养箱中是否可以接受,以便它准备使用?

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.

您是否调查了油的化学结构如何影响其疏水性和亲水性的适当?

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?

不是我们在尝试加湿或洗涤油时看到的。饱和油是不可能的。蒸发仍然以相同的速度发生。

这是理想的油,一种高粘度或低粘度?

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?

两个名称之间真的没有区别。公司销售一个或另一个的主要原因是美国药典(USP)将物质定义为矿物油,而英国药典(BP)将物质定义为石蜡油。我们符合两者的规格。基本上,您正在讨论亚替代石油蒸馏的副产品,该副产品主要由烷烃和环状聚碳链组成。18新利手机app下载库克医疗的培养油是矿物油。它是由符合USP和全国美容专着为轻质矿物油的所有要求的石油制造。此外,厨师的培养油对欧洲药典石蜡的欧洲药典专着进行了测试。

DISH PREPARATION

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

这些在在适当条件下实现的设定值方面,这些真的不应与开放或封闭的文化不同。平衡定时可能不同,并且在pH值方面具有较好的用油覆盖物。蒸发,温度损失和pH升起将在封闭系统中覆盖比在开放系统中较低。均衡值应该是相同的。油覆盖物将增加系统需要与周围环境达到平衡的时间,两者都将盘放入培养箱并将其取出时。

是由盘子疏水影响的滴形状吗?不会使用亲水性的菜肴与疏水盘不同的液滴形状吗?

下降的几何形状可能受到各种因素的影响。下降顶部和表面积到体积比的油量可能是关键因素。所以,制作和形状的下降可能是重要的。我用于我的实验的媒体和菜肴是漂亮的标准,因此我确定的表面区域的值应该接近其他人在其系统中获得的东西。但是,有可能是这样的,就像你指出的那样,这可能会改变这个。

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

不可能的问题回答。具有非常小的卷的问题是通过蒸发等容易地无意中改变媒体组成。

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

这取决于培养系统。最好衡量和看看有什么作用。它也可能取决于胚胎的数量等。

50μl培养基的足够大量的油是什么?

取决于滴形状,也是盘的尺寸。基于我的研究,我将使用足够的油使油层的高度至少为4毫米。

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?

当我使用35毫米菜肴时,我总是用3毫升的油。在96孔盘中使用较小的油量,我总是使用足够的油来覆盖培养基的表面。

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.

如果你比较一个井中的表面区域的差异与平坦的菜肴中的一滴液滴,你认为带有良好设计的文化菜更好吗?

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?

盘子和文化媒体准备应该尽可能短的时间,一次一道菜。应使用IVF柜的室温。培养介质在洗碗准备前从冰箱取出,因此不需要加热的表面。事实上,加热的表面将增加培养基的蒸发。如果您在干燥培养箱中培养,则Microdrops应更大(50μL)。如果您使用的是使用潮湿的培养箱,滴剂的尺寸无关紧要,因为蒸发不会像干燥培养箱一样快,所以可以使用25μL滴剂,并且仍然保持低渗透压直到培养结束。油层应尽可能厚。6毫米的油层将比2mm的油层更有效地降低蒸发。当您准备盘时,底层方法优于覆盖方法,主要是因为落叶,所以表面积到体积比可能更小,这再次会降低蒸发风险。

对于非稀倍数(PGT-A)的非侵入性预体遗传检测,推荐的液滴体积是多少?

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.

每个Microdroplet都放置了多少胚胎?

我们没有在我们的研究中使用胚胎。如果我们有,我会每次下降使用一个胚胎。

培养胚胎后,如果我覆盖盘子以减少对渗透压的任何变化?

是的,我建议使用盖子并覆盖菜。盖子提供防止蒸发并减慢渗透性的升高。

培养皿的类型是否会影响培养基的渗透性?

市场上有许多不同的菜肴。菜肴类型不会直接影响渗透压。培养基和放置在盘子上的油量可能会影响渗透性,如演示文稿所示。

INCUBATORS

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.

将孵化器分类为干燥或加湿是典型的,但都是潮湿的孵化器同样有效吗?我注意到湿度可能有所不同,并且在不同的时期,没有石油的相同媒体将具有不同的蒸发速率。

在同一盘中,在相同的潮湿培养箱内,除非微透镜是不同的尺寸,否则在微量滴水之间的蒸发速率方面真的不应该有任何显着差异,并且没有用相同量的油覆盖。在油下,蒸发在潮湿的培养箱中是最小的。您可以使用湿度计来测量孵化器中的湿度。根据其设计,不同的孵化器可以实现不同的湿度水平。有相对便宜的仪器可用于测量湿度和温度。挑战是它们是否与您的特定孵化器中的使用兼容。这些仪器需要在孵化器环境中平衡,这可能需要几个小时,因此如果您打算使用它们来测量孵化器中的湿度水平,则应验证其在系统中的使用。

有什么我可以安全地为干燥的台式孵化器添加湿度吗?

试图加湿通常干燥的培养箱是有风险的,因为它可能会损坏内部电子器件,并且也可能会影响气体传感器等。我相信这是在许多时间间隔孵化器中消除湿度的主要原因。可提供潮湿的台式孵化器。如果培养箱旨在使用干燥,则调整介质体积,油,油种量等更安全,以减少蒸发,而不是试图加湿培养箱。我会建议试图加湿培养箱,该孵化器旨在没有湿度的情况。

38年前,我们不仅在盒培养箱中添加了一个带有三蒸水的平底锅,而且在我们的实验室中也使用加热器为胚胎创造潮湿的环境。我们认识到输卵管不干燥,为什么任何人都会在干燥的环境中孵育?

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?

有些人这样做了。我没有个人。延时孵化器可以很好地工作。实际优势可能是使用胚胎选择的时间流逝数据。虽然这些是良好的孵化器,但其他孵化器在环境回收和稳定性方面也非常好。

在IVF实验室中使用的是湿度计?

A hygrometer can measure the relative humidity of the environment.

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

我建议您使用可用于评估在您的环境中感到舒适的不同场景的影响的信息,并使用因素的任何组合导致最小的渗透速度变化。

在非吸入的Benchtop孵化器中,一步媒体的渗透变化会在第0天到第6天覆盖有油吗?

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。

CULTURE MEDIA

培养胚胎3天的理想培养基的理想体积是多少?

我无法真正回答这个问题。有多个变量需要考虑。在蒸发方面,媒体的体积只是一个变量。使用的油量和其他因素会影响蒸发。

您是否每天对细胞进行批量评估?

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?

这将是一种方式,是的。

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?

我们在实验室中交换媒体,不使用不间断的文化。

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?

它可以很好地工作,并且没有明确共识,对一个媒体类型的优势相比另一个(顺序与单步)。

如果我们在准备期间使用较高的培养基避免渗透性的变化,只要我们不在时间流逝?

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.

你看过蛋白质补充剂对渗透性的影响吗?

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

影响胚胎发育的媒体中最重要的组成部分是什么?

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.