Gamete, Fertilization and Embryo Culture Media FAQ



Q. Is it possible to add protein, such as Serum Substitute Supplement™ (SSS™) or Human Serum Albumin (HSA) to complete media?

A. Our complete media contain the concentration of protein indicated below; it is possible to add more protein if needed, as long as you consider that the following has already been added.


Q. What is the advantage of using Continuous Single Culture®–NX (CSCM-NX) and Continuous Single Culture®–NX Complete (CSCM-NXC)?

A. CSCM-NX and CSCM-NXC are clinically-proven, low lactate; single-step culture media that help improve blastocyst development. CSCM-NX and CSCM-NXC are designed for fertilization and embryo culture through day 5/6 of embryo development. These media are optimized to be used in an uninterrupted culture system, without dish change or medium renewal.


Q. Does Irvine Scientific provide culture media without antibiotics?

A. No, all Irvine Scientific culture media contain gentamicin as an antibiotic. All the Continuous Single Culture media (CSCM, CSCM-C, CSCM-NX, and CSCM-NXC) are formulated with 10 μg/mL gentamicin.


Q. If the expiration date of the culture media falls in the middle of the culture period can it still be used?

A. No, our expiration dates are based on our stability studies and we cannot guarantee product performance past the date of expiration. We recommend the customer to count back from the final day of culture and not use the product if the expiration date falls before the culture period is complete.


Q. Can we use Continuous Single Culture® (CSCM), Continuous Single Culture® Complete (CSCM-C), Continuous Single Culture®–NX (CSCM-NX), and Continuous Single Culture®–NX Complete (CSCM-NXC) even though my protocol requires dish changes?

A. Yes, you can use your lab’s protocol with all the Continuous Single Culture media (CSCM, CSCM-C, CSCM-NX, and CSCM-NXC); however, our data supports improved implantation and blastocyst utilization rates with an uninterrupted culture system which reduces embryo stress by eliminating the need for embryos to be exposed to varying media.

Protocols

Brochures


Q. What is the recommended amount of protein supplementation to be used with Continuous Single Culture® (CSCM) and Continuous Single Culture®–NX (CSCM-NX)?

A. When using Irvine Scientific HSA, a 100 mg/mL protein solution, we recommend 5% v/v for a final concentration of 5 mg/mL. For example, to prepare 10 mL of medium, add 0.5 mL of HSA solution to 9.5 mL of the medium. When using Irvine Scientific SSS™, a 60 mg/mL protein solution, we recommend 10% v/v for a final concentration of 6 mg/mL. For example, to prepare 10 mL of medium, add 1.0 mL SSS™ to 9.0 mL of medium.


Q. Is each lot of Irvine Scientific culture media tested by the MEGA assay?

A. No, currently we do not use the MEGA assay as a final release test of Irvine culture media. The MEGA assay is currently only used for critical raw materials, such as Human Serum Albumin (HSA).


Q. What is the pH range for Continuous Single Culture® (CSCM), Continuous Single Culture® Complete (CSCM-C), Continuous Single Culture®–NX (CSCM-NX), and Continuous Single Culture®–NX Complete (CSCM-NXC)?

A. The working pH range for all the Continuous Single Culture media (CSCM, CSCM-C, CSCM-NX, and CSCM-NXC) is 7.25-7.40 with a targeted pH of 7.26-7.35.


Q. Why doesn't Continuous Single Culture®–NX (CSCM-NX) and Continuous Single Culture®–NX Complete (CSCM-NXC) contain phenol red?

A. Phenol red does not contribute to the performance of the culture medium and serves only as a visual indicator of pH (1). We recommend that CSCM, CSCM-C, and CSCM-NX, and CSCM-NXC be equilibrated in a CO2 incubator with verified pH measurements prior to use. We will continue to offer CSCM and CSCM-C with phenol red.


Q. How will I know if pH is out of range without phenol red?

A. Like CSCM and CSCM-C, CSCM-NX and CSCM-NXC are formulated using a bicarbonate buffering system that maintains a neutral system by balancing the presence of both a weak acid and its conjugate base. Moreover, phenol red is not a sensitive indicator of pH and does not participate in controlling the pH of the culture medium. Accordingly, pH is maintained in the recommended working range under the proper CO2 concentration.


Q. Are there different protocols for Continuous Single Culture®–NX (CSCM-NX), and Continuous Single Culture®–NX Complete (CSCM-NXC), and Continuous Single Culture® (CSCM), and Continuous Single Culture® Complete (CSCM-C)?

A. No, all the Continuous Single Culture media (CSCM-NX, CSCM-NXC, CSCM, and CSCM-C) use the same recommended uninterrupted culture media protocol. Depending on a laboratory’s preference, a microdrop or well-based protocol can be used.

Protocols


Q. Why do Continuous Single Culture®–NX (CSCM-NX) and Continuous Single Culture®–NX Complete (CSCM-NXC) have a shorter shelf life after opening of 4 weeks compared to 8 weeks for Continuous Single Culture® (CSCM) and Continuous Single Culture® Complete (CSCM-C)?

A. The shelf life of CSCM-NX and CSCM-NXC was reduced to four (4) weeks based on the maximum length of time product is used in a laboratory after opening under aseptic conditions.


Q. What is the concentration of lactate in Continuous Single Culture®–NX (CSCM-NX), and Continuous Single Culture®–NX Complete (CSCM-NXC)?

A. The lactate concentration of CSCM-NX and CSCM-NXC is 1 mM.


Q. Why was the lactate concentration reduced?

A. The reduction of lactate concentration in CSCM-NX and CSCM-NXC is a result of both MEA and BEA assays that support optimal blastocyst development in the presence of lower lactate concentrations. Lactate is a natural by-product of glucose metabolism produced by the embryo. Lowering the lactate concentration of CSCM-NX effectively reduces metabolic stress on the embryo and promotes efficient used of energy sources by the embryo (2,3).


Q. We occasionally observe small particles under the microscope when culturing with protein supplemented media. What is the source of this particulate matter?

A. Protein supplements contain human source material which can develop particulate matter during storage. When using protein-supplemented media, it is not uncommon to have particles form over time as a result of multiple warming and cooling cycles during their shelf life. This type of particulate matter does not adversely affect the performance of the media or embryo development.


Q. What pH do you recommend when using your culture media?

A. These are the recommended target pH ranges for media with protein during use in a CO2 incubator. Typically the addition of protein will lower the pH by 0.02 – 0.05 units, but it should be within the ranges indicated as follows at 37°C and in a CO2 incubator.

Protocols


Q. What amino acids are present in Irvine Scientific media?

A. Our P-1® Medium, a modification of HTF, includes taurine as the only amino acid shown to provide equivalent early cleavage embryo development to Day 3 in animal models when compared to (1) glutamine alone, or (2) the presence of non-essential amino acids. Our ECM® (for early cleavage development) includes both taurine and alanyl-glutamine. Multiblast Medium®, CSCM and CSCM-NX contain both essential and non-essential amino acids.


Q. Why are the complete media expiration dates longer than that of the culture media without supplementation?

A. Our culture media products that require protein supplementation were originally designed with the philosophy that “fresh is best”. This was largely due to the presence of the unstable amino acid L-glutamine in many initial culture media formulations. The development of more recent products which include stable components such as alanyl-glutamine dipeptide (to replace the labile amino acid, L-glutamine), antioxidants (EDTA, citrate) and stabilizers in the protein supplements have enhanced the stability of the products to allow for extended shelf lives. Stability studies were conducted on the full culture media range of products to determine functional performance and confirm the current expiration dates on all protein-free and supplemented culture media.


Q. Is it okay to use culture medium if it has been frozen?

A. No, as indicated in the product insert the storage recommendation is between 2°C to 8°C. The product is stable and ready to use until the date shown on the bottle when stored unopened within that temperature range. The media should not be exposed to extreme temperatures at any time, and if frozen we would recommend discontinuing the use of the product. Please refer to individual product inserts for recommended storage and stability information.


Q. Does Irvine Scientific provide culture medium with protein?

A. Yes, culture media is offered with protein (complete media) or without protein.

Catalog # Culture Medium Protein Source Protein Concentration
90142 Complete Early Cleavage Medium® DSS 5 mg/mL
90143 Complete MultiBlast Medium® DSS 5 mg/mL
90165 Continuous Single Culture® Complete HSA 5 mg/mL
90166 Multipurpose Handling Media®-Complete HSA 5 mg/mL
90168 Continuous Single Culture®-NX Complete HSA 6 mg/mL



Q. Why is EDTA added to your culture media?

A. The addition of EDTA has been shown to enhance the rate of early cleavage in embryos in culture and also serves as an antioxidant to stabilize the media.


 

References

  1. Berthois, Y., Katzenellenbogen, J. A., & Katzenellenbogen, B. S. (1986). Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture. Proceedings of the National Academy of Sciences,83(8), 2496-2500. doi:10.1073/pnas.83.8.2496
  2. Guerif, F., Mckeegan, P., Leese, H. J., & Sturmey, R. G. (2013). A Simple Approach for COnsumption and RElease (CORE) Analysis of Metabolic Activity in Single Mammalian Embryos. PLoS ONE,8(8). doi:10.1371/journal.pone.0067834
  3. Zander-Fox, D. & Lane, M. The future of human embryo culture media – or have we reached the ceiling? in The Human Embryo (ed. Yamada, S. & Takakuwa, T.) 73-98 (Intech, 2012).