Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Culture medium – per se
Reexamination Certificate
2001-06-08
2004-07-13
Nguyen, Dave T. (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Culture medium, per se
C435S325000, C435S404000
Reexamination Certificate
active
06762053
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to culture media which provide useful environments for cellular development. More particularly this invention relates to defined culture media supplement containing constituents produced from non-traditional sources that, when added to culture media, avoid the problems of prior culture media.
BACKGROUND OF THE INVENTION
For years media supplements for culturing mammalian embryonic cells have been derived from animal fluids, and in particular blood serum. While serum based media supplements have been somewhat effective for culturing certain types of cells and tissues, these media supplements have been found to be undesirable. One of the main reasons that these serum based media supplements are unattractive candidates for culturing cells is because of the possibility that the resulting media will contaminated with impurities, toxins, and infective agents found in the fluid from which the media is derived. Additionally, because the animals from which the blood is collected are different and live in differing environments, the fluids produced by these animals have different components at differing concentrations. One important aspect of serum based medium that has been recognized is the requirement of macromolecules in the medium. In an attempt to mimic serum based products, researchers have attempted to add synthetic macromolecules, such as polyvinyl alcohol, to replace the macromolecules in the serum, such as albumin. However, because serum is largely undefined chemically, removing the serum from culture media and attempting to replace only the larger molecules has produced culture media which are less than ideal or ineffective for many purposes because the media are missing essential components.
Accordingly, there is a need for culture media supplements which are as effective as culture media supplements based on blood products while at the same eliminating potential sources of contamination. Additionally there is a need for standardized culture media.
SUMMARY OF THE INVENTION
The present invention describes a novel, physiologically based completely defined supplement to culture media for mammalian embryonic cells and gametes. This medium supplement may be used with in vitro fertilization media, embryo transfer media and embryo cryopreservation media for the mammalian preimplantation embryo, as well as a supplement to media for the development of embryonic stem cells, or any other similar media well known in the art. This supplement contains recombinant human albumin (rHA), fermented hyaluronan (HYN) and/or citrate, and combinations thereof. Addition of this supplement to the culture medium results in equivalent development compared to media supplemented with serum albumin purified from blood.
DETAILED DESCRIPTION OF THE INVENTION
The supplement may comprise recombinant human albumin (rHA) at any appropriate concentrations for the media in which it is to be used. As further described herein, the use of rHA rather than naturally occurring human serum albumin (HSA) has numerous advantages.
Typically, when the supplement comprises rHA, the supplement comprises between about 0.1 mg/ml to about 20.0 mg/ml of rHA based on the total volume of the medium to which the supplement is added. In one embodiment, about 0.5 mg/ml to about 5.0 mg/ml of rHA is added based on the total volume of the medium.
The efficacy of the supplement can be enhanced by adding fermented hyaluronan (HYN) to the supplement. The addition of the fermented HYN to the media supplement demonstrates positive results.
The phrase “increases the viability of gametes or embryonic cells” as used herein is defined as including the increased development of the embryos to the blastocyst stage in the culture, the ability to hatch from the zona pellucida is increased in vitro, and/or an increase in the overall viability of the embryo in embryo cultures when embryos are cultured in a medium containing the supplement of the present invention as compared to being cultured in the same medium without the supplement.
Furthermore, the addition of fermented HYN to the appropriate medium significantly affects the ability of the blastocysts to survive freezing. The use of fermented HYN has several advantages over the use of HYN from a naturally occurring warm blooded vertebrate source such as purified from rooster comb or umbilical cord. By utilizing fermented HYN rather than HYN from a warm blooded vertebrate source, the ability to control the safety and stability of the HYN from different sources and batches is greatly increased.
When present, the amount of fermented HYN will generally be at concentrations between about 0.1 mg/ml to about 5.0 mg/ml based on the total volume of the medium. In one embodiment the fermented HYN will be added to the medium at concentrations between about 0.125 mg/ml to about 1.0 mg/ml based on the total volume of the medium.
The supplement can be further augmented by the addition of citrate. In one embodiment, citrate and rHA are both added to the medium supplement, as it has been surprisingly and unexpectedly discovered that the addition of citrate to a medium supplement containing rHA allows the rHA to closely duplicate the properties of HSA or bovine serum albumin (BSA). The addition of the citrate has a further enhancing effect on the development of the cultured cellular material. Any citrate used for media that is well known in the art may be used, including, but not limited to, choline citrate, calcium citrate, citric acid, sodium citrate, and combinations thereof. In one embodiment, sodium citrate is used. The citrate is generally added at concentrations between about 0.1 mM and about 5.0 mM, based on the total volume of the medium. In one embodiment, the citrate is added at concentrations between about 0.1 mM and about 1.0 mM, based on the total volume of the medium.
The medium supplement of the present invention comprises rHA, fermented HYN and/or citrate in any useful combination. In one embodiment, the medium supplement, and the medium to which the medium supplement is added, is free from non-recombinant macromolecules or macromolecules purified from an animal source. In another embodiment, the medium supplement, and the medium to which the medium supplement is added, is free of non-recombinant HSA and/or non-fermented HYN.
This invention is directed to the medium supplement described above, media containing the medium supplement, a method of making the medium supplement, kits containing the medium supplement, and a method of growing embryonic material employing the medium supplement described herein.
The present invention includes a method of growing cellular material, in one embodiment embryos, employing the medium supplement described herein such that they can be included in medium at the start of culture, or can be added in a fed-batch or in a continuous manner. Moreover, the components of the medium supplement may be added together, or separately, at different stages of the media production.
This supplement can be added to any appropriate mammalian cellular material culture media well known in the art, including but not limited to, embryo culture media, embryo transfer media and embryo cryopreservation media (to include both freezing and vitrification procedures) for embryos from any mammalian species, and stem cell media. Any media that can support embryo or cell development could be used, which includes, by way of example only, bicarbonate buffered medium, Hepes-buffered or MOPS buffered medium or phosphate buffered saline. Examples of media are G1.2/G2.2, KSOM/KSOMaa, M16, SOF/SOFaa, MTF, P1, Earle's, Hams F-10, M2, Hepes-G1.2, PBS and/or Whitten's. (Gardner and Lane, 1999; Embryo Culture Systems; Handbook of In Vitro Fertilization, CRC Press, Editors: Trounson A O and Gardner D K, 2
nd
edition, Boca Raton, pp 205-264.)
The production of rHA is well known in the art. In one embodiment, rHA is obtained from genetically modified yeast which produce a human albumin protein. One such methodology for the production of rHA from ye
Gardner David K.
Lane Michelle T.
Angell Jon Eric
Foley & Lardner
Nguyen Dave T.
Vitrolife, Inc.
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