Method for the cryopreservation of cells for therapeutic purposes
Disclosed is a composition including, in a physiologically acceptable medium: a) human albumin; b) at least one saccharide; c) DMSO and L-cysteine or coenzyme Q10; and d) cells for therapeutic purposes, with the exception of specific tumour-infiltrating lymphocytes.
1. A composition consisting of a medium consisting of:
a) human albumin,
b) at least one saccharide selected from disaccharides and trisaccharides,
c) DMSO and L-cysteine, and
d) cells for therapeutic purposes, with the exception of tumor-infiltrating lymphocytes,
said tumor-infiltrating lymphocytes being obtained by in vitro culture of tumor-infiltrating lymphocytes from a sample taken of in-transit, lymph-node or metastatic cutaneous nodules from a patient suffering from a stage 3 or 4 melanoma, said culture comprising the emergence of said tumor-infiltrating lymphocytes contained in the sample taken of in-transit, lymph-node or metastatic cutaneous nodules, then the stimulation of the tumor-infiltrating lymphocytes resulting from the emergence step, then finally the amplification of the stimulated tumor-infiltrating lymphocytes.
2. The composition as claimed in claim 1 , wherein the saccharide is disaccharides.
3. The composition as claimed in claim 1 , wherein the saccharide is a disaccharide selected from trehalose and sucrose.
4. The composition as claimed in claim 1 , wherein the human albumin is present in an amount of between 2 and 10% by weight relative to the total weight of composition.
5. The composition as claimed in claim 1 , wherein the saccharide is present at a concentration of between 0.05 M and 1 M.
6. The composition as claimed in claim 1 , wherein the cells for therapeutic purposes are chosen from immune cells, human myoblasts, hematopoietic stem cells, mesenchymal stem cells, cardiac cells, fibroblasts and all other natural or genetically modified cells.
7. The composition as claimed in claim 1 , wherein the saccharide is
trehalose.
8. A process for the cryopreservation of at least one sample of cells for therapeutic purposes, with the exception of tumor-infiltrating lymphocytes, comprising the following steps:
i) mixing the sample of cells for therapeutic purposes with a medium consisting of:
a) human albumin,
b) at least one saccharide selected from disaccharides and trisaccharides, and
c) DMSO and L-cysteine, then
ii) freezing the mixture obtained in step i),
said tumor-infiltrating lymphocytes being obtained by in vitro culture of tumor-infiltrating lymphocytes from a sample taken of in-transit, lymph-node or metastatic cutaneous nodules from a patient suffering from a stage 3 or 4 melanoma, said culture comprising the emergence of said tumor-infiltrating lymphocytes contained in the sample taken of in-transit, lymph-node or metastatic cutaneous nodules, then the stimulation of the tumor-infiltrating lymphocytes resulting from the emergence step, then finally the amplification of the stimulated tumor-infiltrating lymphocytes.
9. The cryopreservation process as claimed in claim 8 , wherein the freezing ii) is carried out down to a temperature of between −100° C. and −180° C.
10. The cryopreservation process as claimed in claim 8 , wherein the freezing ii) is carried out by placing the mixture obtained in i) in a container submerged in a mixture of isopropyl alcohol at +4° C., everything being brought to a temperature of between −70° C. and −100° C.
11. The cryopreservation process as claimed in claim 8 , wherein the freezing ii) is carried out by means of a programmed freezer.
12. A method for the cryopreservation of at least one sample of cells for therapeutic purposes, with the exception of tumor-infiltrating lymphocytes, the method comprising:
providing a composition consisting of a medium consisting of:
a) human albumin,
b) at least one saccharide selected from disaccharides and trisaccharides, and
c) DMSO and L-cysteine,
said tumor-infiltrating lymphocytes being obtained by in vitro culture of tumor-infiltrating lymphocytes from a sample taken of in-transit, lymph-node or metastatic cutaneous nodules from a patient suffering from a stage 3 or 4 melanoma, said culture comprising the emergence of said tumor-infiltrating lymphocytes contained in the sample taken of in-transit, lymph-node or metastatic cutaneous nodules;
stimulating the tumor-infiltrating lymphocytes resulting from the emergence step; and
amplifying the stimulated tumor-infiltrating lymphocytes.
13. The composition of claim 4 , wherein the human albumin is present in an amount of between 2.5 and 6% by weight relative to the total weight of composition.
14. The composition of claim 5 , wherein the saccharide is present at a concentration of between 0.07 M and 0.5 M.
15. The composition of claim 7 , wherein the human albumin is present in an amount of between 2.5 and 6% by weight.
16. The composition of claim 15 , wherein the trehalose is present at a concentration of between 0.05 M and 0.5 M.
17. The composition of claim 16 , wherein the DMSO and L-cysteine are present, respectively, in an amount of between 2 and 15% by weight and in a concentration of between 0.5 mM and 2 mM.
18. The cryopreservation process of claim 9 , wherein the freezing ii) is carried out down to a temperature of between −140° C. and −160° C.
19. The composition as claimed in claim 2 , comprising L-cysteine.
20. The composition as claimed in claim 6 , wherein the immune cells are chosen from NK cells, monocytes, B lymphocytes, T lymphocytes, which are natural or genetically modified.
21. The composition as claimed in claim 20 , wherein the T lymphocytes are chosen from regulatory T lymphocytes, cytotoxic T lymphocytes, helper T lymphocytes, and chimeric antigen receptor (CAR) T lymphocytes.