Equipment and Culture Media1

A well-planned laboratory is the foundation of a successful in vitro fertilization (IVF) practice. There are several factors that should be taken into consideration when designing the laboratory. The equipment you choose is as important as the laboratory space itself. The success of an IVF lab is dependent on the conditions in the laboratory. The media you use should be conducive to the environment and the working culture of the laboratory.

These are virtual fallopian tubes and high performing incubators are critical for the success of an IVF lab. Reliability and consistency are absolutely necessary.

These are the new generation incubators and are compact with a very small chamber volume. They can easily fit over the workstation. They reduce transit time between the incubator and the workstation, besides low gas consumption.

The laminar flow hood provides an aseptic work area while allowing the containment of infectious splashes or aerosols generated by many microbiological procedures. Three kinds of laminar flow hoods, designated as Classes I, II and III, have been developed to meet varying research and clinical needs.

Class I laminar flow hoods offer significant levels of protection to laboratory personnel and to the environment when used with various microbiological techniques but they do not provide cultures protection from contamination. They are similar in design and air flow characteristics to chemical fume hoods.

Class II laminar flow hoods are designed for work involving BSL-1, 2 and 3 materials, and they also provide an aseptic environment necessary for cell culture experiments. A Class II biosafety cabinet should be used for handling potentially hazardous materials (e.g. primate-derived cultures, virally infected cultures, radioisotopes, carcinogenic or toxic reagents).

Class III biosafety cabinets are gas-tight and they provide the highest attainable level of protection to personnel and the environment. A Class III biosafety cabinet is required for work involving known human pathogens and other BSL-4 material.

Horizontal laminar flow or vertical laminar flow “clean benches” are not biosafety cabinets; these pieces of equipment discharge HEPA-filtered air from the back of the cabinet across the work surface toward the user, and they may expose the user to potentially hazardous materials. These devices only provide product protection. Clean benches can be used for certain clean activities, such as the dust-free assembly of sterile equipment or electronic devices, and they should never be used when handling cell culture materials or drug formulations, or when manipulating potentially infectious materials. Recent guidelines suggest that vertical flow cabinets should be used to handle samples from patients with HIV or hepatitis B or C. They should be kept clean at all times and the number of items inside the flow hood should be minimized to ensure that the air flow is not disrupted. The flow cabinets should be serviced annually with filter changes every six months.

Awareness about air quality has increased in the last few years. Air contaminants include: volatile organic compounds (VOCs) such as aldehydes; small organic molecules such as nitric oxide, sulphur dioxide and carbon monoxide (from exhaust fumes); and liquids such as floor wax that contains heavy metals. The degree of pollution depends on the location of the IVF unit. Urban laboratories are more likely to have higher pollution. Contaminants in the ambient air will also circulate through the incubators. Cohen et al. (1997) showed that certain adhesives arrested > 90% of mouse embryos at the 2-cell stage with very low blastocyst formation. Brand new incubators emit>100 times higher concentrations of VOCs than used ones (Cohen et al. 1997). Many laboratories have a positive pressure HEPA filtration system to extract inorganic contaminants (e.g. dust, bacteria). Additional absorption systems containing activated carbon and potassium permanganate are required to remove VOCs. Airconditioning systems should recirculate HEPAa-filtered air rather than draw air from the outside.

This is a controlled environmental chamber which is mobile and is specifically designed to maintain ideal temperature and pH during the handling of gametes and embryos. The working chamber is maintained at a temperature of 37°C with 5 to 6% CO₂ in air. Temperature is monitored by a thermostat and CO₂ levels by an infrared sensor. A dissecting microscope is needed for egg collections inseminations, fertilization checks, denuding eggs for ICSI (intracytoplasmic sperm injection), normal evaluation and grading of embryos and embryo transfers.

Good microscopes are required for routine embryological and andrological procedures. A dissecting microscope is essential to score oocyte cumulii complex, pronuclei and embryos for transfer. The range of its magnification should be between 20 and 160x.

A micromanipulator is necessary for ICSI and embryo biopsy for preimplantation genetic diagnosis. In addition, a good inverted microscope with a heated stage and a magnification of up to 400x is needed. The controls can be either pneumatic or hydraulic. Although hydraulic 7syringes have more effective control and movement, they can be fairly time-consuming and messy to prime the tubing with oil every time there is trapped air within the system. The micromanipulation system should be placed in a vibration-free environment. Hydraulically controlled antivibration tables are commercially available. If there is limited air filtration in the laboratory, the micromanipulator system can be placed within a class II flow hood. When choosing the manipulators, it is very important to consider the ease of manoeuvrability.

It creates a negative pressure and is used for ovum pickups. It has foot operated switch with vacuum gauze variable from 0 to 500 mm Hg. Fluid trap prevents aspiration of fluid into vacuum unit.

CryoMed Freezers for IVF are designed for human invitro fertilization with precise control, ease of setup and operation, and convenience. Chamber, sample temperature and operation status are continuously displayed on the control panel. These IVF models provide convenient front and top access for easy sample “seeding”.

Centrifuge is used for density gradient semen preparation. Nowadays good centrifuges are available which can be programmed to give accurate g force. REMI centrifuges are used by our lab.8

It is used to store culture media and other media used for embryology and andrology. Culture media are very temperature sensitive. Certain amino acids and proteins in the media can be inactivated by extreme temperatures and hence should be kept at 2 to 8°C.

It is used for drying and sterilizing glass pipettes and other nondisposable items such as tweezers, spatulas and media-making glassware.

These are inescapable requirement for andrology lab and you should go for the best available. Indian equipment is equally good and we have used them for package.

Saturn 3 by RI or XY Clone by Hamilton Throne are available and can be integrated into the ICSI micromanipulator. The software and accuracy of Saturn system is slightly better than that of Hamilton.

All plasticware that is used in the laboratory should be proven to be nonembryotoxic. The most commonly used plasticware in IVF laboratories are supplied by equipment companies. All the plastic ware should be disposable and not re-used under any circumstances. All new batches should undergo in-house quality control testing before use. Batches that fail in-house checks should be returned. If possible, new packs of dishes should be opened for each patient or each working day to keep the contents sterile. Resealing opened packs should be kept to a minimum. It is strongly recommended to use individually wrapped sterile pipette tips and plastic pipettes for inseminations and sperm preparation. This will minimize the potential risk of any cross-contamination.

Ovum pickup needle is used for ovum aspiration with help of TV probe and aspiration pump. It is designed for precise recovery with minimal trauma. It is available with single and double lumen systems with a choice of 16G or 17G SS needle. Ecomarking maximizes ultrasound visibility.

This catheter is used for transvaginal transfer of embryos into uterus along with tissue culture medium with ultrasound guided marking and available in different sizes. Being designed to avoid embryo damage, the inner catheter is made of medical grade polyurethane which is known to be non embryotoxic. The hand finished round tip prevents traumatic introduction and graduation on the inner catheter allows alignment with the outer sheath, producing a smooth radius for minimal trauma during insertion.

It is used for the transfer of the oocyte or embryos from dish to dish. The tip is well rounded by fire polishing. Available in different IDs as per application.

It is used for the denudation of the oocyte before the microinjection by ICSI. Cumulus mass and corona tissues are removed by repeated aspiration and expiration of the oocyte. Available in different ID as per application.

Used for ICSI, assisted hatching and blastomere biopsy. The well shaped fire polished tip allows secure fixation of oocyte and avoids damage of cell. Maximal contact surface, 95 micron or 120 micron OD, bend angle available from 5 to 35 degree.10

Used to perform intracytoplasmic sperm injection into oocytes. It is available with/without heat formed spike having different angles, spiked or hypodermic bevel. Four to six micron ID, bend angles available 5 to 35 degree.

Culture media for embryo development must meet the metabolic needs of preimplantation embryos by addressing amino acid and energy requirements based on the specific developmental stage of the embryo.

Culture media containing a phosphate buffer or Hepes organic buffer are used for procedures that involve handling of gametes outside of the incubator, flushing of follicles and micromanipulation.

Most culture media utilize a bicarbonate/CO₂ buffer system to keep pH in the range of 7.2 to 7.4. The osmolarity of the culture medium must be in the range of 275 to 290 mosmol/kg.

The human oocyte is temperature-sensitive and a humidified incubator with a temperature setting of 37.0 to 37.5°C must be used for oocyte fertilization and embryo culture.12

Embryos should be cultured under paraffin oil, which prevents evaporation of the medium preserving a constant osmolarity. The oil also minimizes fluctuations of pH and temperature when embryos are taken out of the incubator for microscopic assessment. Paraffin oil can be toxic to gametes and embryos; therefore, batches of oil must be screened and tested on mouse embryos before use in culture of human embryos.

The medium is composed of 99% water. Purity of the water is crucial, and is achieved by ultrafiltration.

Albumin or synthetic serum are added in concentrations of 5 to 20% (w/v or v/v, respectively). Today, the commercial media includes synthetic serum in which the composition is well known.

NaCl, KCl, KH₂PO₄, CaCl₂. 2H₂O, Mgso₄. 7H₂O, NaHCO₃

Carbohydrates are present in the female reproductive tract. Their concentrations vary throughout the length of the oviduct and in the uterus, and are also dependent on the time of the cycle.

Supplement of the culture medium with amino acids is necessary for embryo development. Media that support the development of zygotes up to 8-cells are supplemented with non essential amino acids. Proline, serine, alanine, aspargine, aspartate, glycine, glutamate.

Their role in the culture medium is unclear.

The majority of ART laboratories use culture media containing antibiotics to minimize the risks of microbial growth. The most commonly used antibiotics being Penicillin (β-lactam Gram-positive bacteria disturbs cell wall integrity) and Streptomycin (Aminoglycoside Gram-negative bacteria disturbs protein synthesis). The antibacterial effect of penicillin is attributed to its ability to inhibit the synthesis of peptidoglycan, unique glycoproteins of the bacterial cell wall. Streptomycin and gentamycin belong to the aminoglycoside group of antibiotics which exert their antibacterial effect by inhibiting bacterial protein synthesis. The use of gentamicin is still controversial and it is not being used by every laboratory.

EDTA is used as a chelator in medium that supports the embryo from the zygote stage to 8-cells and prevents abnormal glycolysis.

Designed for oocyte retrieval and follical flushing. It is modified HTF formulated with EDTA and alanyl glutamine buffered with bicarbonate and hepes. It contains needed antibiotics.

Designed for in vitro procedure involving the culture of early cleavage stage embryo. Modified HTF formulated with EDTA, low phosphate, alanyl glutamine.

Designed for culture of embryo from day 1 through the morula stage, day 3 or 4. It is glucose and phosphate free with the addition of EDTA and alanyl glutathione.

Designed for culture of embryo for day 3 transfers or for Blastocyst culture. It is low glucose and phosphate free media formulated with EDTA, taurine, glutathime and alanyl glutamine.14

Designed for culture of embryo to blastocyst and day 5 embryo transfers. It is phosphate free and having elevated levels of glucose supplemented with vitamins, essential and non essential amino acids formulated with glutathione and alanyl glutamine.

It is the protein of choice for use as a tissue culture supplement in those applications requiring protein supplementation. It contains 100 mg/mL total protein (wt/volume) in normal saline.

It is the recommended oil for overlaying during fertilization of embryo culture and micromanipulation.

It is the recommended media to decrease sperm motility for ICSI procedure prevent sperm sticking to the glass pipette.

It is the recommended medium for removal of cumulas cells from the oocyte after egg retrieval and prior to ICSI.