Cell culture medium
The present invention relates to a cell culture medium for culturing human cells comprising an anti-coagulated total blood material wherein the hemoglobin level is from about 8 to about 16 g/dl. More particularly, the invention provides a cell culture medium in which cells present in the blood are disrupted and the insoluble remnants of the lysated cells are removed. Further, the invention provides a method for the preparation of a cell culture medium for culturing human cells, according to the invention.
1. A method for culturing human cells, said method comprising
preparing an anti-coagulated total blood material that lacks the membrane fraction of red blood cells and has a hemoglobin level from 8 to 16 g/dl by
(a) obtaining mammalian blood having an extracellular fraction, as starting material;
(b) adding an anticoagulant to the mammalian blood thereby providing anti-coagulated mammalian blood;
(c) mechanically disrupting the cells present in the anti-coagulated mammalian blood;
(d) removing insoluble remnants of the disrupted cells to produce a particle-free extract;
(e) modifying the ion content of the particle-free extract to match the extracellular fraction of the starting material or to be similar to that of a medium selected form the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute (RPMI) medium and Isocove's Modified Dulbecco's Medium (IMDM), to produce a cell culture medium; and
(f) culturing the human cells in the cell culture medium.
2. The method according to claim 1 wherein the mechanical disruption of the cells is achieved by freezing the total blood or by ultrasound, electric blade homogenizer or French press.
3. The method according to claim 1 , wherein removal of the insoluble remnants of the disrupted cells is achieved by ultracentrifugation or mechanical filtration.
4. The method according to claim 1 , wherein the composition of the nutrients with a molecular weight of less than 3000 Dalton in the cell culture medium is adjusted by dialysis against a medium selected form the group consisting of DMEM, RPMI medium and IMDM.
5. The method according to claim 1 , wherein the reducing capacity of the cell culture medium is restored by addition of reduced glutathione.
6. The method of claim 1 , wherein the blood is of human origin.
7. The method of claim 1 , further comprising carrying out high throughput microscopic imaging on the cell culture medium.
8. The method of claim 1 , wherein the growth and survival factor flora of the starting material is preserved in the cell culture medium.
9. The method of claim 1 , wherein the anticoagulant is heparin.
10. The method of claim 1 , wherein the disrupting of the cells achieves an even disruption of cell membranes of the cells.
11. The method of claim 1 , wherein the cell culture medium contains all macromolecular components of the mammalian blood.
12. The method according to claim 1 wherein the mechanical disruption of the cells is achieved by freezing the anti-coagulated mammalian blood.
13. The method of claim 1 , wherein the ion content of the particle-free extract is restored to match that of the extracellular fraction of the starting material.
14. A method for culturing human cells, said method comprising
preparing an anti-coagulated total blood material that lacks the membrane fraction of red blood cells by
(a) obtaining mammalian blood having an extracellular fraction, as starting material;
(b) adding an anticoagulant to the mammalian blood thereby providing anti-coagulated mammalian blood;
(c) lysing the cells present in the anti-coagulated mammalian blood to produce lysed anti-coagulated mammalian blood;
(d) removing insoluble remnants of the lysed cells from the lysed anti-coagulated mammalian blood; and
(e) modifying the ion content of the lysed anti-coagulated mammalian blood to match that of the extracellular fraction of the starting material or to be similar to that of a medium selected form the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute (RPMI) medium and Isocove's Modified Dulbecco's Medium (IMDM),
to produce a cell culture medium having a hemoglobin level from 8 to 16 g/d; and
culturing the human cells in the cell culture medium.
15. The method according to claim 14 , wherein removal of the insoluble remnants of the lysed cells is achieved by ultracentrifugation or mechanical filtration.
16. The method according to claim 14 wherein the lysing of the cells is achieved by freezing the anti-coagulated mammalian blood.