Preservation of stem cells

Abstract

The invention relates to a field of stem cell preservation and in particular to use of an aqueous solution comprising polyethylene glycol (PEG) having a molecular weight about 35000 Da as an extracellular agent for preserving stem cells.

Claims

1. A method of preserving stem cells and preventing their degradation, comprising: applying to the stem cells an extracellular agent comprising an aqueous solution comprising polyethylene glycol (PEG) having molecular weight of about 35000 Da, wherein the solution comprises: about 20 to about 40 mmol/l of raffinose, about 70 to about 140 mmol/l of lactobionic acid, about 1 to about 10 mmol/l of MgSO4, about 10 to about 40 mmol/l of KH2PO4, about 1 to about 6 mmol/l of glutathione, about 1 to about 10 mmol/l of adenosine, about 1 to about 5 mmol/l of allopurinol, about 30 to about 150 mmol/l of NaOH, and additionally pH of the solution is in the range of about 6.5 to about 8, and osmolality of the solution is in the range of about 290 to about 320 mOsm/kg, wherein the aqueous solution of the extracellular agent results in a concentration of sodium ions Na+ that is greater than a concentration of potassium ions K+, thereby preventing cell death during storage by reducing ice formation inside a cell.

2. The method according to claim 1, wherein PEG is present in the solution at a concentration of about 0.01 to about 5 mmol/l.

3. The method according to claim 1, wherein PEG is present in the solution at a concentration lower than about 1 mmol/l.

4. The method according to claim 1, wherein PEG is present in the solution at a concentration of about 0.03 mmol/l.

5. The method according to claim 1, wherein the solution comprises Na+ ions at a concentration of at least 30 mmol/l and K+ ions at a concentration of at least about 10 mmol/l.

6. The method according to claim 1, wherein the solution comprises Na+ ions at a concentration of about 125 mmol/l and K+ ions at a concentration of about 25 mmol/l.

7. The method according to claim 1, wherein the solution comprises 30 mmol/l of raffinose.Math.5H2O, 100 mmol/l of lactobionic acid, 5 mmol/l of MgSO4.Math.7H2O, 25 mmol/l of KH2PO4, 3 mmol/l of glutathione, 5 mmol/l of adenosine, 1 mmol/l of allopurinol, 0.03 mmol/l of polyethylene glycol having molecular weight of about 35000 Da and NaOH in an amount sufficient to obtain pH of about 7.4, and water for injections, wherein the solution has osmolality of 300 mOsm/kg and comprises about 125 mmol/l of Na+ ions.

8. The method according to claim 1, wherein the extracellular agent for preserving stem cells is used for cryopreservation of stem cells, and wherein the solution further comprises dimethyl sulfoxide (DMSO).

9. The method according to claim 8, wherein the solution comprises dimethyl sulfoxide (DMSO) at a concentration of about 5 to about 20 vol. % in relation to the total volume of the solution.

10. The method according to claim 9, wherein the solution comprises DMSO at a concentration of about 10 vol. % in relation to the total volume of the solution.

11. The method according to claim 8, wherein the stem cells are stored in the extracellular agent in a temperature range between 196 C. and the freezing temperature of the stem cells, and wherein the viability of the stem cells after storing is of at least 95% relative to the initial viability of the stem cells.

12. The method according to claim 11, wherein the viability of the stem cells after storing is of at least 95.9% relative to the initial viability of the stem cells.

13. The method according to claim 8, wherein the stem cells are stored in a temperature of 196 C.

14. The method according to claim 1, wherein the stem cells are stored in the extracellular agent in a temperature range from 4 C. to room temperature, and wherein a viability of the stem cells after storing is of at least 70% relative to the initial viability of the stem cells.

15. The method according to claim 1, wherein the stem cells are stored in the extracellular agent in a temperature range from 0 to 5 C., and wherein a viability of the stem cells after storing is of at least 90% relative to the initial viability of the stem cells, and wherein the solution further comprises dimethyl sulfoxide (DMSO).

Description

DESCRIPTION OF FIGURES

(1) FIG. 1 shows the results of ADSC cells viability measurements after 12, 24, 36, 48, 72, 96 and 120 hours of storage in various tested formulations.

(2) FIG. 2 shows the number of the viable ADSC cells after 12, 24, 36, 48, 72, 96 and 120 hours of storage in HTS formulation and the solution used according to the present invention.

(3) FIG. 3 shows the results of ADSC cells viability measurements before and after cryopreservation.

(4) FIG. 4 shows the results of ADSC cells viability measurements after thawing and after 12, 24, 36, 48 and 72 of storage in HTS formulation and the solution used according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

(5) The subject invention has been developed based on the observation made by its inventors that the aqueous solution comprising polyethylene glycol (PEG) having a defined molecular weight allows for effective preservation of stem cells while maintaining their viability.

(6) In the use according to the subject invention PEG having molecular weight of about 35000 Da (35 kDA) also called PEG 35, is employed. PEG used according to the present invention can be obtained by any known method, wherein preferably it can be synthetized from PEG molecules having lower molecular weight. In other preferred embodiment of the invention, PEG may be purified by any known technique known in the prior art. In especially preferred embodiment of the invention commercially available PEG can be used.

(7) According to the invention, PEG is preferably used at a concentration from about 0.01 to about 5 mmol/l, for example at a concentration of 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4,5 and 5 mmol/l. In a particularly preferred embodiment of the invention, the PEG concentration is lower than 1 mmol/l and most preferably it is about 0.03 mmol/l.

(8) According to the subject invention, the employed aqueous solution is used as extracellular agent, i.e. agent, in which the sodium ions (Na.sup.+) is greater than the concentration of potassium ions (K.sup.+). Agents of this type prevent the cell death during storage by reducing ice formation inside a cell.

(9) In a preferred embodiment of the invention, the solution contains Na.sup.+ ions at a concentration of at least about 30 mmol/l and K.sup.+ ions at a concentration of at least about 10 mmol/l. In the most preferred embodiment of the invention the solution contains Na.sup.+ ions at a concentration of about 125 mmol/l and K.sup.+ ions at a concentration of about 25 mmol/l.

(10) Preferably, sodium ions are introduced in sodium hydroxide (NaOH), while potassium ions are introduced in potassium dihydrogen phosphate (KH.sub.2PO.sub.4).

(11) In a particularly preferred embodiment of the invention, besides the components mentioned above, i.e. PEG, sodium ions and potassium ions, the solution may contain other components used in the field of cell storageformulations, i.e. an agent comprising an impermeant anion, a compound from the group of sugars, a membrane stabilizing agent, a buffer solution and an energy source.

(12) In an especially preferred embodiment of the invention, the mentioned solution comprises:

(13) TABLE-US-00001 about 20 to about 40 mmol/l of raffinose, about 70 to about 140 mmol/l of lactobionic acid, about 1 to about 10 mmol/l of MgSO.sub.4 about 10 to about 40 mmol/l of KH.sub.2PO.sub.4, about 1 to about 6 mmol/l of glutathione about 1 to about 10 mmol/l of adenosine, about 1 to about 5 mmol/l of allopurinol, about 30 to about 150 mmol/l of NaOH wherein pH of the solution is in the range of about 6, 5 to about 8, and osmolality of the solution is in the range of about 290 to about 320 mOsm/kg.

(14) The anion impermeable to cell membranes, whose role is to prevent the cell swelling caused by hypothermia, is lactobionic acid or its salts.

(15) The function of raffinose is additional osmotic support. In a preferred embodiment of the invention raffinose pentahydrate (raffinose.Math.5H.sub.2O) is used.

(16) The membrane stabilizing agent is magnesium sulphate, preferably magnesium sulphate heptahydrate (MgSO.sub.4.Math.7H.sub.2O), which acts in order to stabilize the electrochemical equilibrium of the cell membrane, which determines the proper transport of Na.sup.+ ions, K.sup.+ ions, phosphorous ions and Ca.sup.2+ ions.

(17) KH.sub.2PO.sub.4 is a buffer system, whose role is to maintain pH of the solution, which provides the acid/base equilibrium. Additionally, it provides the potassium ions to the solution.

(18) Glutathione and allopurinol are agents counteracting the formation and action of free radicals.

(19) Adenosine is a source of ATP precursors, being the energy source.

(20) Water used to prepare the solution used according to the invention is a pharmaceutically acceptable water for injections.

(21) In the most preferred embodiment of the invention, mentioned solution comprises:

(22) TABLE-US-00002 30 mmol/l of raffinose5H.sub.2O, 100 mmol/l of lactobionic acid, 5 mmol/l of MgSO.sub.47H.sub.2O, 25 mmol/l of KH.sub.2PO.sub.4, 3 mmol/l of glutathione 5 mmol/l of adenosine, 1 mmol/l of allopurinol, 0.03 mmol/l of polyethylene glycol having molecular weight of about 35000 Da and NaOH in an amount sufficient to obtain pH of about 7.4, and water for injections, wherein the solution has osmolality of 300 mOsm/kg and comprises about 125 mmol/l of Na.sup.+ ions.

(23) When the use according to the subject invention is intended for cryopreservation, an addition of cryoprotectant is necessary. The cryoprotectant may be any component of this type used in the cryopreservation formulations, wherein preferably it is dimethyl sulfoxide (DMSO). Preferably dimethyl sulfoxide (DMSO) is used at a concentration of about 5 to about 20 vol. %, most preferably at a concentration of about 10 vol. % based on total volume of the solution.

(24) The solution used according to the invention does not contain calcium ions added separately or as components of the compounds included in the composition of the mentioned solution. The water for injections used for the preparation of the solution used according to the invention also does not contains said ions.

(25) According to the invention, the term about used hereinabove and hereinbelow is to be understood as +/5% deviation from the given value, reflecting the inaccuracies which may appear during the process of manufacturing of the composition of the invention, e.g. during measurements of the components of the solution.

EXAMPLES

Example 1

(26) Solution for the Preservation of Stem Cells

(27) In experiments relating to the preservation of stem cells, the formulation StoreProtect Plus (Manufacturer: Carnamedica) having following composition, was used:

(28) TABLE-US-00003 30 mmol/l (17.84 g/l) of raffinose5H.sub.2O, 100 mmol/l (35.8 g/l) of lactobionic acid, 5 mmol/l (1.232 g/l) of MgSO.sub.47H.sub.2O, 25 mmol/l (3.402 g/l) of KH.sub.2PO.sub.4, 3 mmol/l (0.922 g/l) of glutathione 5 mmol/l (1.336 g/l) of adenosine, 1 mmol/l (0.136 g/l) of allopurinol, 0.03 mmol/l (1 g/l) of polyethylene glycol having molecular weight of about 35000 Da and NaOH in an amount sufficient to obtain pH of about 7.4, water for injections,

(29) The solution of Example 1 exhibits osmolality of about 300 mOsm/kg and comprises sodium ions (Na.sup.+) at concentration of about 120 mmol/l and potassium ions (K+) at concentration of about 25 mmol/l.

Example 2

(30) Solution for Cryopreservation of Stem Cells

(31) In experiments related to cryopreservation of stem cells a formulation from Example 1 additionally comprising 10 vol. % of dimethyl sulfoxide (DMSO) in relation to the total volume of the solution, was used.

Example 3

(32) Short-Term Preservation of Stem Cells

(33) Mesenchymal, adipose-derived Stem Cells (ADSCs) isolated from 9 donors were divided into test groups and stored for up to 120 h. Following experimental groups were used: (a) NaCl solution (0.9%), (b) NaCl solution (0.9%) with glucose, (c) Ringer's solution (Fresenius Kabi), (d) Plasmalyte formulation (Baxter) (comprising, per 1000 ml of the solution: 5.26 g of sodium chloride, 0.37 g of potassium chloride, 0.3 g of magnesium chloride (6H.sub.2O), 3.68 g of sodium acetate (3H.sub.2O), 5.02 g of sodium gluconate, and comprising: 140 mmol/l of sodium ions, 5 mmol/l of potassium ions, 1.5 mmol/l of magnesium ions, 98 mmol/l of chloride ions, 27 mmol/l of acetate ions (CH.sub.3COO.sup.), 23 mmol/l of gluconate ions (C.sub.6H.sub.11O.sub.7.sup.), wherein the theoretical osmolarity of the solution is 294 mOsm/l, pH is about 7.4 (from 6.5-8.0), (e) HypoThermosol FRS formulation (HTS, BioLife Solutions) (comprising/: Trolox, Na.sup.+, K.sup.+, Ca.sup.2+, Mg.sup.2+, Cl.sup., H.sub.2PO.sub.4.sup. HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine, glutathione) and (e) the solution as defined in Example 1. The short term preservation (maximum 120 hours) was conducted in temperature of 4 C., wherein in case of groups (d) and (e) also in room temperature. During the experiment the viability of the cells and number of viable cells were observed in selected time points.

(34) For the purposes of counting, the cells were labeled with propidium iodide and counted with an automatic cell counter (NanoEntec).

(35) FIG. 1 shows the results of viability measurements obtained for experimental groups (a), (b), (d) and (e), while FIG. 2 shows the number of viable cells as a function of the preservation time for test groups (d) and (e).

(36) The results shown on FIG. 1 confirm that the highest stem cells viability was obtained in case of solution of the solution of Example 1 (above 90% after 120 hours). The number of viable cells after 120 hours from the beginning of the experiment was also the highest in case of solution of Example 1 (FIG. 2).

Example 4

(37) Cryopreservation of Stem Cells

(38) In the second experiment the usability of the formulation as defined in Example 2 in stem cell cryopreservation was tested. In this experiment the same type of stem cells was used as in Example 1. Certified formulation Stem-Cell Banker (Zenoaq) was used as the reference formulation. The experiment has been conducted in temperature of 196 C. for 7 days

(39) After thawing the cells, the viability measurement was conducted (in the same manner as in Example 1) and the cells were transferred to the storage solutions (HypoThermosol FRS (HTS, BioLife Solutions) (reference formulation) and the solution of Example 1) in order to assess whether the change of storage conditions negatively influences the cells viability.

(40) During said preservation, cells viability was observed in selected time points (12 hours, 24 hours, 48 hours, 72 hours).

(41) FIG. 3 shows the results of stem cells viability measurements after cryopreservation. The measurements confirmed that the formulation of Example 2 provides efficient preservation of stem cells in the frozen state.

(42) FIG. 4 shows the results of stem cells viability measurements after thawing in selected time points for HypoThermosol FRS formulation and the solution of Example 1. The results presented on FIG. 4 clearly show that the solution of Example 1 exhibits better properties than the reference formulation used.