Culture of embryos

Laser Assisted Embryo Hatching

Preparation of embryos for hESC derivation Frozen 2PN and day 3 embryos are rapidly thawed and cultured in blastocyst culture media until they develop into blastocysts Figure 21.1 . Unless they are to be manually dissected see below , the blastocysts are allowed to hatch, or induced to hatch by applying Acid Tyrode's solution with a micropipette on day 5 or 6. Alternatively, laser-assisted hatching can be used. Embryos that were frozen at the blastocyst stage are thawed and cultured overnight...

Oocyte insemination

Oocyte Insemination

Inseminate the oocytes approximately 3-5 h post-retrieval. 2. Each oocyte is inseminated with 5 X 103 motile sperm. The volume of sperm added to each oocyte is dependent on the concentration. For example, if the Figure 24.2 Oocyte insemination via intracytoplasmic sperm injection. Figure 24.2 Oocyte insemination via intracytoplasmic sperm injection. patient has a concentration of 45 X 106 motile sperm mL, each drop will be inseminated with approximately 1.1 pL of the prepared sperm sample....

MEF cells from commercial sources

Fibroblast Feeder Layer

There are several commercial sources for MEFs from various mouse strains and containing various selectable drug-resistant markers. Depending on the level of use, this can be a convenient and economical alternative to the de novo preparation of MEF feeder layers. Figure 3.1 Low A and high B magnification photomicrographs showing morphology of MEF feeder layers. MEFs do not form the whorls of cells that are typical of other fibroblasts used as feeder layers, such as human foreskin fibroblasts. C...

How to rid ESC cultures of contaminating feeder cells

Having hESC cultures contaminated with mitotically active feeders should be avoidable if care is taken to inactivate them thoroughly. But should valuable cultures Table 3.1 Example of a plating guide mouse embryonic fibroblast feeder layer for hESC culture density 1.2 X 105 cells cm2 medium normally suspension Culture container Container Total no. of MEFs used in the at 5.0 X type area cm2 per container container mL 105 mL mL a medium normally suspension Culture container Container Total no. of...

Culture medium supplement B27

Concentration of stock B27 is modification of serum-free Neurobasal medium. Its exact composition is proprietary, but a list of ingredients is given below. The reference to its composition is Brewer et al. 1993 . Components of Neurobasal B27 added Antioxidants B27 added Other Linoleic acid Linolenic acid Progesterone Albumin, bovine Putrescine Selenium Insulin Transferrin Superoxide dismutase DL-Tocopherol acetate DL-Tocopherol Glutathione reduced Modified from Podratz et...