Mammalian cell culture media which comprise supernatant from cohn fractionation stages and use thereof
View Patent ↗The present invention relates to mammalian cell culture media which comprise supernatant from some of the fractions of human plasma fractionation according to the Cohn method, more specifically, the supernatant of fractions I and II+III. When said supernatant is added as a culture medium supplement it provides various nutrients and factors for the effective maintenance and/or proliferation of the cultured mammalian cells. In addition, the present invention relates to the preparation process and use of said medium in the culture of mammalian cells.
1. A cell culture medium for the culture of mammalian cells, comprising basal culture media for mammalian cell culture and supernatant of fraction I of human plasma fractionation or supernatant of fraction II+III of human plasma fractionation,
the supernatant of fraction 1 being prepared by
precipitation with ethanol by adding ethanol to plasma up to a concentration of approximately 8% (volume/volume) and reducing temperature to approximately −2° C.,
separation of the supernatant,
freezing or drying of the supernatant, and
subsequent addition of the resulting supernatant to the basal cell culture,
or the supernatant of fraction II+III being prepared by
precipitation with ethanol by adding ethanol to a concentration of about 20% to about 25% (volume/volume) and reducing temperature to approximately −5° C.,
separation of the supernatant,
freezing or drying,
and subsequent addition of the resulting supernatant to the basal cell culture,
wherein these supernatants have an albumin content between 60% and 78% and a gammaglobulins content between 1.5% to 12.5%.
2. The medium according to claim 1 , wherein the supernatants are obtained from mixtures (pools) from at least 1000 human donors.
3. The medium according to claim 1 , wherein the supernatant is dried.
4. The medium according to claim 1 , wherein the supernatant is frozen.
5. A process of preparing a mammalian cell culture medium comprising obtaining a supernatant of fraction I of human plasma fractionation by
precipitation with ethanol by adding ethanol to plasma up to a concentration of approximately 8% (volume/volume) and reducing temperature to approximately −2° C.,
separation of the supernatant,
freezing or drying of the supernatant, and
subsequent addition of the resulting supernatant to the basal cell culture, or obtaining a supernatant of fraction II+III of human plasma fractionation by
precipitation with ethanol by adding ethanol to a concentration of about 20% to about 25% (volume/volume) and reducing temperature to approximately −5° C.,
separation of the supernatant,
freezing or drying, and
subsequent addition of the resulting supernatant to the basal cell culture.
6. The process of claim 5 , wherein the dried supernatant is reconstituted in the basal culture medium, in distilled water or deionised and apyrogenic water, or in saline solutions or buffers commonly used in cell culture.
7. The process of claim 5 , wherein the dried supernatant is reconstituted in the basal culture medium.
8. The process of claim 5 , wherein the human plasma is obtained from mixtures (pools) from at least 1000 human donors.
9. A method of culturing mammalian cells comprising the steps of preparing a culture medium including supernatant of fraction I of human plasma fractionation or supernatant of fraction II+III of human plasma fractionation, the supernatant of fraction I being prepared by
precipitation with ethanol by adding ethanol to plasma up to a concentration of approximately 8% (volume/volume) and reducing temperature to approximately −2° C.,
separation of the supernatant,
freezing or drying of the supernatant, and
subsequent addition of the resulting supernatant to the basal cell culture, or the supernatant of fraction II+III being prepared by
precipitation with ethanol by adding ethanol to a concentration of about 20% to about 25% (volume/volume) and reducing temperature to approximately −5° C.,
separation of the supernatant,
freezing or drying, and
subsequent addition of the resulting supernatant to the basal cell culture.