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.

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).
Q.

What is the density and density gradient of ISolate, ISolate Stock Solution and ISolate Concentrate?

A.

Here are the density and density gradients of ISolate.

Product Catalog # Kit Components Density (g/mL) Density Gradient (%)
ISolate Kit 99264 99257 (Upper Layer – 50%) 1.06 50%
99258 (Lower Layer – 90%) 1.11 90%
ISolate Stock Solution (90%) 99275 N/A 1.11 90%
ISolate Concentrate (100%) 99306 N/A 1.13 100%
Q.

How should I prepare a 40% upper layer of ISolate from 50% ISolate Upper Layer?

A.

To obtain a 40% upper layer from our 50% ISolate Upper Layer, mix 8.0 mL of (upper layer) with 2.0 mL of Modified HTF Medium with Gentamicin - HEPES.

Q.

How should I prepare an 80% lower layer of ISolate from 90% ISolate Lower Layer?

A.

To obtain an 80% lower layer from our 90% ISolate Lower Layer, mix 8.9 mL of lower layer with 1.1 mL of Modified HTF Medium with Gentamicin - HEPES.

Q.

Once ISolate gradient is prepared, how long can I leave it in the incubator prior to use?

A.

We recommend using prepared ISolate gradient within two hours of preparation to avoid the mixing of the gradient prior to use.

Q.

What are the diameter of cells that can pass through ISolate and the diameter of cells that cannot pass through ISolate?

A.

ISolate uses a density gradient centrifugation technique as the method of separation. The gradients increase in density from the top to the bottom of the preparation, the sample is gently placed on top of the preparation, and centrifuged. During the process highly motile sperm cells actively move in the direction of the sedimentation gradient. This is in contrast to poorly motile/ immotile sperm cells that do not penetrate the boundaries of the gradient. Upon completion of the centrifugation highly motile sperm cells are enriched in a pellet at the bottom of the preparation1.

Q.

Why does FUJIFILM Irvine Scientific provide sperm washing media without antibiotics when all the other companies do?

A.

The addition of antibiotics to sperm washing media is at the discretion of the end user. Please reference your own clinic’s protocols when adding antibiotics. For additional details, each laboratory should consult its own laboratory procedures and protocols which have been specifically developed and optimized for your individual medical program. Please reference table below for a complete list of sperm processing products and gentamicin content.

Product Catalog # Gentamicin (Yes/No)
Sperm Washing Medium 9983 No
Freezing Medium - TYB with Glycerol & Gentamicin 90128 Yes
Refrigeration Medium - TYB with Gentamicin 90129 Yes
Multipurpose Handling Medium (MHM) with Gentamicin 90163 Yes
Multipurpose Handling Medium - Complete 90166 Yes
Sperm Maintenance Medium 99176 No
ISolate Kit 99264 No
ISolate Stock Solution 99275 No
ISolate Concentrate 99306 No
Q.

How do you thaw semen or sperm frozen in Freezing Medium - TYB with Glycerol & Gentamicin or Sperm Maintenance Medium?

A.

FUJIFILM Irvine Scientific recommends the following protocol for thawing raw semen and processed sperm.

Q.

Can ISolate, ISolate Stock Solution, or ISolate Concentrate be diluted?

A.

Yes, ISolate can be diluted with Sperm Washing Medium, or with Modified HTF Medium with Gentamicin - HEPES.

Q.

What are the differences between your sperm preservation media?

A.

This table details the cryoprotectant, intended use, and use ratio of our sperm preparation media.

Product Catalog # Cryoprotectant Intended Use Use Ratio
Freezing Medium - TYB with Glycerol & Gentamicin 90128 20% egg yolk Freezing sperm 1 to 1
12% glycerol
Refrigeration Medium - TYB with Gentamicin 90129 20% egg yolk Short term storage of sperm up to 96 hrs at 2-8°C 1 to 1
Short term storage/capacitation at room temp
Arctic Sperm Cryopreservation Medium 90170 28% glycerol Freezing sperm 3 to 1
Sperm Maintenance Medium 99176 28% glycerol Freezing sperm 3 to 1


Capacitation at room temperature for approximately two hours.

Q.

What are the storage recommendations for UNOPENED sperm cryopreservation media?

A.

These are the storage instructions for UNOPENED items.

Product Catalog # Packaging Size Storage Instructions (unopened product)
Freezing Medium - TYB with Glycerol & Gentamicin 90128 20 x 5 mL Below -10°C
100 mL
Refrigeration Medium - TYB with Gentamicin 90129 20 x 5 mL Below -10°C
Arctic Sperm Cryopreservation Medium 90170 12 x 5 mL 2-8°C
Sperm Maintenance Medium 99176 100 mL Below -10°C
Q.

What are the storage recommendations for OPENED sperm cryopreservation media?

A.

These are the storage instructions for OPENED items.

Product Catalog # Storage (temp. and duration) after Opening Shelf life after Opening Can product be aliquoted?*
Freezing Medium - TYB with Glycerol & Gentamicin 90128 N/A (single use only) N/A (single use only) Yes
Refrigeration Medium - TYB with Gentamicin 90129 N/A (single use only) N/A (single use only) Yes
Arctic Sperm Cryopreservation Medium 90170 N/A (single use only) N/A (single use only) No
Sperm Maintenance Medium 99176 2-8°C Eight weeks from opening when stored at 2-8°C N/A


After opening

*If smaller aliquots are desired, the end user may thaw the product and using aseptic technique aliquot into sterile labeled containers and freeze until time of use. Aliquots should be single use and product should not be exposed to repeated freeze thaw cycles.

Q.

Can I freeze sperm with Refrigeration Medium - TYB with Gentamicin?

A.

No, Refrigeration Medium - TYB is intended for short term refrigerated sperm storage. Refrigeration Medium- TYB is typically used to store sperm for up to 96 hours between 2-8°C, transporting semen (e.g. home collection then transported to clinic), and as an aid in sperm capacitation for conventional fertilization.

For freezing sperm we recommend using Freezing Medium - TYB with Glycerol & Gentamicin or Arctic Sperm Cryopreservation Medium.

Q.

How long can I use Freezing Medium - TYB with Glycerol & Gentamicin and Refrigeration Medium - TYB with Gentamicin after they are thawed?

A.

Both products are intended to be single use and we recommend discarding any excess medium that remains after the procedures are completed. To optimize usage of your product, we recommend thawing the bottle/vial and aseptically aliquoting the media into smaller sterile bottles/vials and stored as directed on the product insert. Do not use aliquots that show a shift in color, particulate matter or any evidence of contamination.

Q.

How is Arctic Sperm Cryopreservation Medium different from Freezing Medium - TYB with Glycerol & Gentamicin?

A.

This product is for customers who prefer to freeze sperm without the use of test yolk buffer for long term storage. The product should be used in a ratio of 3:1 (sperm:Arctic).

Q.

What is the approximate recovery rate of motile spermatozoa after freeze/thaw using sperm cryopreservation Freezing Medium - TYB with Glycerol & Gentamicin?

A.

In general, the recovery rate is around 50% post-thaw when using Freezing Medium - TYB with Glycerol & Gentamicin.

  Fresh Post-Thaw
Raw Semen Freezing Medium TYB Commercial Medium, Glycerol Only
Healthy semen (n=25) Normal morphology (%) 26.3 ± 7.5 22.2 ± 6.4 19.4 ± 6.5
Condense chromatin (%) 92.4 ± 8.5 88.7 ± 11.2 85.2 ± 12.5
Motility (%) 52.8 ± 27.3 32.7 ± 19.1 29.1 ± 17.1
Poor quality semen (n=35) Normal morphology (%) 11.7 ± 6.1 9.3 ± 5.6 7.8 ± 4.2
Condense chromatin (%) 78.9 ± 10.3 70.7 ± 10.8 64.6 ± 13.0
Motility (%) 28.3 ± 22.5 13.3 ± 9.9 10.7 ± 7.5
Q.

Is it acceptable to put TYB in the uterus at insemination? Should TYB be washed prior to IUI?

A.

We recommend that the TYB freezing medium should be removed prior to IUI either by simple washing with Sperm Washing Medium or Multipurpose Handling Medium - Complete, or if washing thawed raw semen, by density gradient centrifugation with ISolate2,3,4.

Q.

Sperm were frozen using another vendor's kit and protocol. Can they be thawed using FUJIFILM Irvine Scientific reagents and protocols?

A.

FUJIFILM Irvine Scientific has not performed studies to confirm this usage and does not recommend the combined usage of its reagents with those from other vendors. However, independent recent studies report usage of MHM-C with other vitrification reagents and protocols5.

Q.

Is the dual-buffered Multipurpose Handling Medium - Complete better for handling sperm than single buffered media?

A.

A study by Bormann and Curchoe demonstrated the superior performance of dual-buffer solution of HEPES and MOPS containing Multipurpose Handling Medium - Complete (MHM-C) for key sperm parameters over single buffer controls6.

Q.

Can oocytes or embryos be vitrified using Arctic Sperm Cryopreservation Medium?

A.

Components of cryopreservation media are different for sperms and oocytes/embryos. FUJIFILM Irvine Scientific offers specific media for this application. Please consider Vit Kit-Freeze and Vit Kit-Thaw for oocytes and embryos.


 

References

  1. Henkel, R. R., & Schill, W. B. (2003). Sperm preparation for ART. Reprod Biol Endocrinol. Nov;14;1:108.
  2. Hammadeh, M.E., Greiner, P., Rosenbaum, P., Schmidt, W. (2001). Comparison Between Human Sperm Preservation Medium and TEST-Yolk Buffer on Protecting Chromatin and Morphology Integrity of Human Spermatozoa in Fertile and Subfertile Men After Freeze-Thawing Procedure. J Androl. Nov-Dec;22(6):1012-8.
  3. Wolf, D. P., Patton, P. E., Burry, K. A., & Kaplan, P. F. (2001). Intrauterine insemination–ready versus conventional semen cryopreservation for donor insemination: a comparison of retrospective results and a prospective, randomized trial. Fertil Steril. Jul;76(1):181-5.
  4. Larson, J. M., McKinney, K. A., Mixon, B. A., Burry, K. A., & Wolf, D. P. (1997). An intrauterine insemination-ready cryopreservation method compared with sperm recovery after conventional freezing and post-thaw processing*. Fertil Steril. July; 68(1), 143–148.
  5. Medranoa L., Encisob M., Gomez-Torresc M.J., Aizpuruaa J. (2019). First birth of a healthy infant following intra-cytoplasmic sperm injection using a new permeable cryoprotectant-free sperm vitrification protocol. Cryobiology, 2019 Apr;87:117-119.
  6. Optimization of sperm culture conditions for human ART. Charles L. Bormann and Carol L. Curchoe. http://www.irvinesci.com/uploads/technical-documentations/10775ART_Optimization-of-sperm-culture-Rev.0.pdf.
Q.

All Products — How long after opening bottles, can we use the products?

A.

When stored as directed, products are stable until the expiration date shown on the bottle. Please refer to each item's product insert for the recommended use time after opening.

Q.

HSA - Human Serum Albumin — Is HSA azide-free?

A.

Yes it is. Azide is commonly used as a preservative for diagnostic grade albumins.

Q.

HSA - Human Serum Albumin — Is HSA a blood-based product?

A.

Yes. HSA consists of human serum albumin from therapeutic-grade source material (100mg/mL) in a saline solution.

Q.

Oil — What kind of oil do you produce?

A.

We provide a light mineral oil that is intended for use with embryo culture as an overlay.

Q.

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

A.

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 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 below at 37C˚ and in CO2.


- P-1®: 7.27 - 7.32
- ECM®: 7.20 - 7.25
- Multiblast Medium®: 7.30 - 7.40
- HTF: 7.20 - 7.30
- CSCM™: 7.26 - 7.35

There are no FAQs in this category at this time.
Q.

How do I pick which Makler® Counting Chamber to use in my laboratory?

A.

The Makler® Counting Chamber comes in different configurations depending on your microscope set up. IS-363 is for use with a standard microscope. IS-365 is for use with an inverted microscope. IS-373 is for use with a CASA system.

Q.

Part of my Makler® Counting Chamber is broken. Do I have to replace it?

A.

If the Makler® Counting Chamber cover glass is broken, then a replacement can be purchased. IS-364 is the replacement cover glass for IS-363 (standard microscope). IS-366 is the replacement cover glass for IS-365 (inverted microscope). IS-375 is the replacement cover glass for IS-374 (CASA). If there is damage to the chamber grip or it was lost, IS-363CG can be used to replace in any of the Makler Counting Chambers. If there is damage to any other part of the chamber, including the main body of the chamber, you may need to replace it.

Q.

All Products — How long after opening bottles can we use the products?

A.

When stored as directed, products are stable until the expiration date shown on the bottle. Upon opening the bottles, refer to the product insert for the recommended usage time.

Q.

Vitrification – Can blastocysts cultured in other culture media (other than Irvine Scientific’s) be vitrified with your kits?

A.

Irvine Scientific vitrification media can be used to vitrify blastocysts that have been cultured in any culture media.

Q.

Embryo Thaw Media Kit — What is the percentage of HSA in the Embryo Thaw media?

A.

There is 1.2% (12mg/mL) HSA in the Embryo Thaw media.

Q.

Vit Kits® — What is the shelf life of products after they have been opened?

A.

We have confirmed the shelf life using unopened and properly stored vials of solutions. Once opened, it is recommended that you use solutions within 8 weeks, assuming they are used under aseptic conditions and do not exhibit any of the characteristics of contamination indicated in the "precautions" section of the product inserts.

Q.

Freezing Medium-TYB — How many times can you freeze & thaw the Test Yolk Buffer freezing Medium?

A.

It is best to aliquot the media aseptically into the right vial sizes and store frozen for further use. When stored as directed, Test Yolk Buffer will be stable until the expiration date shown on the bottle.

Q.

All Products — How long after opening bottles can we use the products?

A.

When stored as directed, products are stable until the expiration date shown on the bottle. Upon opening the bottles, refer to the product insert for the recommended usage time.

Q.

All Products — Is it ok to aliquot into smaller vials?

A.

As long as the clinic works in an aseptic, sterile environment, the products can be aliquoted into smaller bottles or vials and stored per the directions. Please note that we cannot guarantee that the aliquots are of the same high quality as products manufactured and bottled at Irvine Scientific. For this very reason, we offer multiple configurations and sizes. To stay in-line with manufacturing protocol, it is recommended that if you are aliquoting from a plastic bottle, then use a plastic bottle for the aliquot. As a general recommendation, do not use products that have been opened that show a change in color, contain particulate matter, or show any evidence of contamination.

Q.

CryoTip® — If you open the package, how can you maintain the sterility of the tips in order to use the others later?

A.

The CryoTips® are packaged at 5 per pack and the best way to maintain their sterility is to always open and remove tips from the pack in a sterile environment (laminar flow hood). Before removing the pack containing any remaining (unused) tips from the sterile environment, the pack should be taped closed (fold over the open end and tape closed as is typically done with sleeves of culture dishes), in order to maintain sterility of the contents for future use. Alternatively, the open pack of CryoTips® can be stored in the laminar flow hood until later use if space allows.

Q.

CryoTip® — Do CryoTips® need to be inspected prior to use?

A.

As an added precaution during the preparation procedure, we recommend that each CryoTip® is carefully examined when taken out of the package. Prior to use, all CryoTips® should be examined under a suitable magnification (40x power) for possible damage (such as tip breakages or cracks) that may have occurred during transport.