AQUEOUS SOLUTION FOR CELL PRESERVATION

Abstract

The invention relates to an aqueous solution for cell preservation of preferably mammalian cells, which can be used for cell preservation as a cryoprotectant or as a pharmaceutical product or excipient. Furthermore, the invention relates to a method for preserving cells using the aqueous solution for cell preservation, and to a method for defrosting cells which are frozen in the aqueous solution.

Claims

1. Aqueous solution for cell preservation, comprising potassium chloride, potassium phosphate, preferably monobasic anhydrous potassium phosphate sodium chloride, sodium phosphate, preferably dibasic anhydrous sodium phosphate, glucose, preferably anhydrous D(+)- glucose, ascorbic acid, preferably magnesium-L-ascorbate-2- phosphate, cysteine, preferably L-cysteine HCl x H.sub.2O, glutathione, preferably reduced glutathione, methyl cellulose, and DMSO.

2. Aqueous solution for cell preservation according to claim 1, comprising 0.0185% w/v potassium chloride, 0.0185% w/v potassium phosphate, preferably monobasic anhydrous potassium phosphate 0.8695% w/v sodium chloride, 0.1064% w/v sodium phosphate, preferably dibasic anhydrous sodium phosphate, 0.0925% w/v glucose, preferably anhydrous D(+) glucose anhydrous, 0.1203% w/v ascorbic acid, preferably magnesium-L-ascorbate-2-phosphate, 0.0028% w/v cysteine, preferably L-cysteine HCl x H.sub.2O, 0.0925% w/v glutathione, preferably reduced glutathione, 0.1013% w/v methyl cellulose, and 8.25% w/v DMSO.

3. Aqueous solution for cell preservation according to claim 1 or 2, further comprising phenol red, preferably phenol red sodium salt.

4. Aqueous solution for cell preservation according to any one of claims 1 to 3, further comprising cells that are to be preserved.

5. Aqueous solution for cell preservation according to claim 4, wherein the cells to be preserved are mammalian cells.

6. Aqueous solution for cell preservation according to claim 5, wherein the mammalian cells are human induced pluripotent stem cells (hipSC), human mesenchymal stem cells from bone marrow (hMSC-BM), human mononuclear cells from peripheral blood (hMNC-PB/PBMC), human dermal fibroblasts from adult skin (NHDF-a), human dermal melanocytes from adult skin (NHEM-a) or human smooth muscle cells from the aorta (HAoSMC) or human cancer cell lines, e.g., the human breast cancer cell line MCF-7.

7. Use of the aqueous solution for cell preservation according to any one of claims 1 to 6 as a cryoprotectant.

8. Aqueous solution for cell preservation according to any one of claims 1 to 6 for use as a pharmaceutical product or excipient.

9. Method for preserving cells comprising (a) dispersing cells in the aqueous solution for cell preservation according to any one of claims 1 to 6 in a container; and (c) feeding the container to cryopreservation.

10. Method of defrosting cells dispersed and frozen in the aqueous solution for cell preservation according to any one of claims 1 to 6, comprising (a) thawing a container in which the frozen cells in the aqueous solution for cell preservation are dispersed and frozen according to any one of claims 1 to 6; and (b) adding the thawed cells to a culture medium suitable for the cells.

Description

OBJECT OF THE INVENTION

[0014] The underlying invention relates to an aqueous solution for cell preservation, comprising [0015] potassium chloride, [0016] potassium phosphate, preferably monobasic anhydrous potassium phosphate [0017] sodium chloride, [0018] sodium phosphate, preferably dibasic anhydrous sodium phosphate, [0019] glucose, preferably anhydrous D(+)- glucose, [0020] ascorbic acid, preferably magnesium-L-ascorbate-2- phosphate, [0021] cysteine, preferably L-cysteine HCl x H.sub.2O, [0022] glutathione, preferably reduced glutathione, [0023] methyl cellulose, and [0024] dimethylsulfoxide (DMSO).

[0025] “Cell preservation” or “preservation of cells” preferably involves cell cryopreservation or cryopreservation of cells. Cryopreservation is understood to mean a usually long-term storage of cells below 0° C, in particular at −20° C to −200° C. The freezing is generally carried out at 1° C/min.

[0026] The present invention preferably relates to an aqueous solution according to the invention for cell preservation, comprising [0027] 0.0185% w/v potassium chloride, [0028] 0.0185% w/v potassium phosphate, preferably monobasic anhydrous potassium phosphate [0029] 0.8695% w/v sodium chloride, [0030] 0.1064% w/v sodium phosphate, preferably dibasic anhydrous sodium phosphate, [0031] 0.0925% w/v glucose, preferably anhydrous D(+) glucose anhydrous, [0032] 0.1203% w/v ascorbic acid, preferably magnesium-L-ascorbate 2-phosphate, 0.0028% w/v cysteine, preferably L-cysteine HCl x H.sub.2O, [0033] 0.0925% w/v glutathione, preferably reduced glutathione, [0034] 0.1013% w/v methyl cellulose, and 8.25% w/v DMSO.

[0035] In a particularly preferred embodiment, the aqueous solution according to the invention for preserving cells is prepared as follows:

TABLE-US-00001 [mg/L] Potassium chloride 185 Potassium phosphate, monobasic anhydrous 185 Sodium chloride 8695 Sodium phosphate, dibasic anhydrous 1064 D(+) glucose, anhydrous 925 Magnesium-L-ascorbate-2-phosphate 1203 L-cysteine HCl × H.sub.2O 28 Glutathione, reduced 925 Methyl cellulose 1013 DMSO 82500

[0036] The aqueous solution for cell preservation according to the invention is preferably used for “slow freezing” methods for preserving cells. Preferably, the aqueous solution for cell preservation according to the invention is not used for methods that apply vitrification.

[0037] Preferably, the aqueous solution for cell preservation according to the invention can further comprise phenol red, preferably phenol red sodium salt.

[0038] The aqueous solution for cell preservation according to the invention preferably further comprises cells that are to be preserved. These cells to be preserved are preferably mammalian cells. The term “cell” or “cells” also comprises cell aggregates.

[0039] Preferred mammalian cells are lymphocytes, spleen cells, thymocytes, animal cells, somatic stem cells, mesenchymal stem cells, non-human embryonic stem cells, induced pluripotent stem cells, or cancer stem cells.

[0040] Particularly preferred mammalian cells are human induced pluripotent stem cells (hipSC), human mesenchymal stem cells from bone marrow (hMSC-BM), human mononuclear cells from peripheral blood (hMNC-PB/PBMC), human dermal fibroblasts from adult skin (NHDF-a), human dermal melanocytes from adult skin (NHEM-a) or human smooth muscle cells from the aorta (HAoSMC).

[0041] Further particularly preferred mammalian cells are human cancer cell lines, for example the human breast cancer cell line MCF-7.

[0042] The cells to be preserved can be both cells which are freshly isolated from tissue or cells which have already been cultured or expanded in vitro.

[0043] The cell count per milliliter of cell suspension that is to be preserved is preferably between 100,000 and 100 million cells.

[0044] Preferably, cells are separated from their culture or isolation medium/buffer before they are preserved with the aid of the aqueous solution according to the invention for preserving cells. All work is preferably carried out under sterile conditions.

[0045] The present invention further relates to the use of the aqueous solution according to the invention for cell preservation as a cryoprotectant.

[0046] The present invention likewise relates to an aqueous solution according to the invention for cell preservation for use as a medicament or excipient.

[0047] Furthermore, the present invention relates to a method for preserving cells, comprising [0048] (a) dispersing cells in the aqueous solution according to the invention for cell preservation in a container; and [0049] (b) feeding the container to cryopreservation.

[0050] Further, the present invention relates to a method for defrosting cells that are dispersed and frozen in the aqueous solution according to the invention for cell preservation, comprising [0051] (a) thawing a container in which the frozen cells are dispersed and frozen in the aqueous solution of the invention for cell preservation; and [0052] (b) adding the thawed cells to a culture medium suitable for the cells.

EXAMPLES

Materials Used

[0053]

TABLE-US-00002 Order No. Name Supplier M7140 Methyl cellulose Sigma D4540 DMSO Sigma A8960 L-Ascorbic Acid 2-Phosphate Mg Sigma C6852 L-Cysteine × HCl × H2O Sigma G6013 Glutathione (reduced) Sigma G7021 D(+)-Glucose anhydrous Sigma 207790250 Sodium Chloride Acros/ Fisher P5530 Phenolic Sodium Salt Sigma optional for version with Phenol Red Dulbecco's Phosphate Buffered Saline w/o Ca/Mg P0750- (powder) Biowest N10L Potassium Chloride Potassium Phosphate Monobasic Anhydrous Sodium Chloride Sodium Phosphate Dibasic Anhydrous

Example 1: Separating the Cells to be Frozen From Their Culture Medium

[0054] Both adherent growing cells and non-adherent cells growing in suspension can be frozen. The term “cells” here refers to individual cells and cell aggregates from up to several hundred cells.

Example 1A: Detaching Adherent Growing Cells

[0055] In order to detach adherent growing cells, the culture medium is first drawn off by suction. Then the cell layer is washed twice with a generous amount of PBS buffer (without Ca.sup.2+/ Mg.sup.2+) and also drawn off by suction again. After addition of a suitable release agent, e.g., Accutase (https://www.accutase.com/accutase.html) or Trypsin-EDTA/TNS, the cells are detached according to the manufacturer's specifications. The detachment process is monitored microscopically. As soon as the cells begin to round off, they are detached completely by knocking lightly on the cell culture container. The detached cells are transferred into a sample tube and the concentration and total number of cells are determined by means of cell counting After pelleting the cells by centrifugation (e.g. for 3 minutes at 300×g and room temperature), the supernatant is cautiously drawn off by suction. Remaining in the sample tube is the cell pellet, which is subjected to further treatment as described in Example 2.

Example 1B: Harvesting Non-adherent Suspension Cells

[0056] Cells present or growing in suspension are transferred together with medium into a tube. The concentration and total number of cells are then determined by means of cell counting. After pelleting the cells by centrifugation (e.g. for 3 minutes at 300×g and room temperature), the supernatant is cautiously drawn off by suction. Remaining in the sample tube is the cell pellet, which is subjected to further treatment as described in Example 2.

Example 2: Freezing Cells With the Aqueous Solution According to the Invention for Cell Preservation

[0057] The cell pellet produced in Example 1 is absorbed at room temperature in a corresponding amount (see below) of the aqueous solution according to the invention for cell preservation and resuspended by means of careful pipetting up and down using a serological pipette. The cell count per milliliter of cell suspension should be between 100,000 and 100 million cells. 1 ml of the cell suspension is rapidly distributed to freezing tubes in each case.

[0058] The tubes are then transferred without delay into a computer-controlled freezer (IceCube 14M). This has two reference temperature probes (a sample probe and one for the freezing chamber of the machine) which are positioned accordingly before the start of the freezing process. Thereafter, the controlled automatic freezing process is started with a suitable freezing protocol. The freezing process is complete when the samples have reached a temperature of ≤−80° C.

[0059] Alternatively, the cells can be cryopreserved on dry ice for at least 4 hours in freezing containers filled with 2-propanol, e.g. “Mr. Frosty”.

[0060] The tubes with the frozen cells are subsequently removed from the freezer or the freezer container. They are transported on dry ice at −80° C to the storage location, without interruption of the cooling chain, and are temporarily stored in liquid nitrogen. They can be kept there indefinitely.

[0061] The aqueous solution according to the invention for preserving cells is prepared as follows:

TABLE-US-00003 [mg/L] Potassium chloride 185 Potassium phosphate, monobasic anhydrous 185 Sodium chloride 8695 Sodium phosphate, dibasic anhydrous 1064 D(+) glucose, anhydrous 925 Magnesium-L-ascorbate-2-phosphate 1203 L-cysteine HCl × H.sub.2O 28 Glutathione, reduced 925 Methyl cellulose 1013 Dimethylsulfoxide (DMSO) 82500

Example 3: Thawing and Seeding Cells

[0062] A tube is initially charged with 9 ml of culture medium (room temperature). Furthermore, a suitable culture vessel is filled with a corresponding amount of culture medium (e. g., 0.2 to 0.3 ml/cm of heparin in the culture area) and this culture vessel is pre-equilibrated in the incubator at 37° C and 5% CO.sub.2 for at least 30 minutes.

[0063] The tube with the cryopreserved cells is removed from the liquid nitrogen and transported on dry ice. Thawing takes place for 2 minutes by means of a continuous pivoting movement in a temperature-controlled water bath at 37° C. The tube is disinfected with 70% (v/v) ethanol and transferred under the sterile bench. There, the tube with the thawed cells is opened and the cells are rapidly transferred into the 9 ml culture medium placed in a tube. Optionally, the viable cell count can be determined at this point in order to determine the exact cell count required for the optimal seeding density.

[0064] Thereafter, the cells are pelleted by means of centrifugation (for example for 3 minutes at 300×g and room temperature) and the supernatant is drawn off by suction. The cell pellet is absorbed in a suitable amount of fresh culture medium and the cell suspension (taking into account the recommended seeding density for the respective cell type) is transferred into the pre-equilibrated culture container. Further incubation takes place in the incubator at 37° C and 5% CO.sub.2.