Preparation of a blood platelet lysate for use in a cell culture medium for hybridoma cells

• US 5,198,357 A
• Filed: 12/26/1990
• Issued: 03/30/1993
• Est. Priority Date: 04/26/1988
• Status: Expired due to Fees

• Current Assignee: Ellco Food AB (The Lauridsen Group, Inc.)
• Original Assignee: Ellco Food AB (The Lauridsen Group, Inc.)
• Inventors: Westermark, Bengt, Holmovist, Olov
• Primary Examiner(s): Naff, David M.
• Assistant Examiner(s): Ware, Deborah K.
• Application Number:
  US07/634,113
• Time in Patent Office: 825 Days
• Field of Search: 435/240.1, 435/240.3, 435/243, 435/7.21, 435/240.26
• US Class Current: 435/408
• CPC Class Codes: C12N 2500/84 from mammals
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 5/163 one of the fusion partners being a B or a T lymphocyte

Abstract

A platelet lysate is produced from plasma derived from animal whole blood that contains an added citrate to prevent coagulation of the blood during storage. The lysate is prepared by centrifuging plasma to produce a platelet rich paste, adding calcium to the paste to lyse the platelets therein and coagulate fibrinogen to produce a clear liquid containing lysed platelets, sterile filtering the liquid and collecting a liquid filtrate containing the lysed platelets. The obtained lysate of platelets can be used to wholly or partly replace foetal calf serum in cell culture, such as in culture of hybridoma cells for preparing monoclonal antibodies.

Claims

1. A method for preparing a blood platelet lysate from plasma derived from animal whole blood to which a citrate has been added to avoid coagulation during storage of the blood, which method comprises:

a) centrifuging said plasma so as to obtain a platelet rich paste,

b) adding Ca²⁺ to said paste, in equimolar amounts in relation to the citrate content in the centrifuged plasma to lyse the platelets in said paste and to coagulate fibrinogen to produce a clear, light red liquid consists essentially of lysed platelets,

c) sterile-filtering said clear, light red liquid to produce a liquid filtrate, and

d) collecting the liquid filtrate which consists essentially of lysed platelets.

2. The method according to claim 1, wherein said liquid filtrate contains about 90-100% by volume of lysed platelets.

3. The method according to claim 1 or 2, wherein said plasma is of food quality and has a hemolysis figure of 1-3, preferably 1-2.

4. The method according to claim 1 wherein in step a), said plasma is centrifuged in a bactofuge to yield a concentrate of about 100-fold concentration, and the resulting concentrate then being centrifuged a second time to yield a further concentration to said paste.

5. The method of claim 1 wherein the platelet lysate produced is combined with a nutrient substrate to produce a cell culture medium.

6. The method of claim 5 wherein the cell culture medium is used to culture hybridoma cells by contacting hybridoma cells with the culture medium.

7. A cell culture medium produced by the method of claim 5.

8. The cell culture medium of claim 7 wherein said medium also contains foetal calf serum.