阅读说明：本技术 干细胞的保存 (Preservation of Stem cells ) 是由 P·圣祖德洛 I·卡拉什琴斯卡 于 2020-01-23 设计创作，主要内容包括：本发明涉及干细胞保存领域,特别地涉及包含分子量为约35000Da的聚乙二醇(PEG)的水溶液作为用于保存干细胞的细胞外试剂的用途。(The present invention relates to the field of stem cell preservation, in particular to the use of an aqueous solution comprising polyethylene glycol (PEG) having a molecular weight of about 35000Da as an extracellular agent for the preservation of stem cells.)

1. Use of an aqueous solution comprising polyethylene glycol (PEG) having a molecular weight of about 35000Da as an extracellular agent for the preservation of stem cells.

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

3. The use of any one of the preceding claims, wherein the PEG is present in the solution at a concentration of less than about 1 mmol/l.

4. The use of any one of the preceding claims, wherein the PEG is present in the solution at a concentration of about 0.03 mmol/l.

5. Use according to any one of the preceding claims, wherein the solution comprises Na in a concentration of at least about 30mmol/l⁺Ions and a K concentration of at least about 10mmol/l⁺Ions.

6. Use according to any one of the preceding claims, wherein the solution isContaining Na in a concentration of about 125mmol/l⁺Ions and a K concentration of about 25mmol/l⁺Ions.

7. Use according to any one of the preceding claims, wherein the solution comprises:

and additionally

The pH of the solution is in the range of about 6,5 to about 8, and

the solution has an osmolality in the range of about 290mOsm/kg to about 320 mOsm/kg.

8. Use according to any one of the preceding claims, wherein the solution comprises

And

in an amount sufficient to obtain NaOH having a pH of about 7.4, and

the water for injection is used as the water,

wherein the solution has an osmotic pressure of 300mOsm/kg and comprises about 125mmol/l Na⁺Ions.

9. The use according to any one of the preceding claims, wherein the extracellular agent for preserving stem cells is used for cryopreservation of stem cells.

10. The use of claim 9, wherein the solution further comprises dimethyl sulfoxide (DMSO) at a concentration of about 5% to about 20% by volume relative to the total volume of the solution.

11. The use according to claim 10, wherein the solution comprises DMSO at a concentration of about 10% by volume relative to the total volume of the solution.

Technical Field

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

Prior Art

Stem cells have found widespread use in scientific research and clinical therapy due to their potential for self-renewal and multipotential differentiation. Stem cells have many functions and applications-for example, they can be used in research related to tissue and organ in vitro growth, development of disease models, screening, and therapeutic methods. For example, mesenchymal stem cells may be used to treat diabetes, cirrhosis, skin damage, and myocardial infarction. In addition, they can enhance hematopoiesis and regulate immune response.

Stem cell-based therapies are continually advancing through preclinical and clinical trials. An improved carrier solution for preservation and delivery of cells would allow for expansion of the worldwide application possibilities of advanced stem cell-based therapies. Furthermore, an increase in cell stability, understood as maintenance of viability and functionality, will provide flexibility and expandability to the range of applications of stem cells. The quality of the cells changes over time and due to the influence of various external factors, such as the composition of the carrier solution or temperature conditions during transportation. However, the use of stem cells in all the indicated applications requires the development of efficient methods for their preservation, ensuring not only the maintenance of high cell viability, but also protection from contamination by bacteria and pathogens, and a reasonable cost of culturing the cells.

Preserving organs, tissues and cells under cold storage (i.e. at cryogenic conditions) means using a temperature in the range of 0 ℃ to 5 ℃, e.g. a temperature of about 4 ℃. So-called cryogenic solutions are used for this purpose. At a temperature of 4 ℃, the cells immersed in this solution are maintained in a state called sustained hypothermia. Many solutions for this purpose are known in the prior art, for example(also known as University of Wisconsin (University of Wisconsin) solution or UW solution), or EURO-COLLINS.

However, under such conditions, only short-term storage is possible. In the case of long-term storage, the preserved materials must be frozen in a temperature range between the freezing temperature of the materials and the temperature of liquid nitrogen (-196 ℃). This process is also known as cryopreservation.

There are many technical solutions in the prior art relating to the optimization of preservation methods for organs, tissues and cells, all involving cold storage (coldstorage) and cryopreservation (cryopreservation).

To ensure efficient transplantation, stem cells must be preserved in a manner that maintains their viability. For example, in the case of cryopreservation, great efforts have been made to eliminate cell death caused by freezing, mainly due to intracellular ice formation and chemoosmotic stress. Cryoprotectants, such as dimethyl sulfoxide (DMSO), are used for this purpose.

Document US 6,045,990 describes cell-free solutions for the cryogenic storage of human or animal organs, tissues and cells. The solution comprises at least one electrolyte selected from the group comprising potassium ions, sodium ions, magnesium ions and calcium ions; a macromolecular swelling agent; a biological buffer; a nutritionally effective amount of at least one monosaccharide; mannitol, an impermeable anion of an impermeable cell membrane; a substrate effective for ATP regeneration; and glutathione.

Matsumoto et al published as ` Successful liquid storage of peripheral blood stem cells at a non-freezing temperature below zero `]' Bone Marrow Transpl [ Bone Marrow transplantation](2002),30,777- & 784, describe experiments with several solutions during dry cell preservation at sub-zero but non-freezing temperatures. Mixtures of (i) plasma, ACD-a solution and heparin were tested; (ii) belzer UW solution-containing heparin, potassium lactobionate, KH₂PO4, raffinose, adenosine, glutathione, allopurinol, hydroxyethyl starch, and NaOH, and mixtures of the two with albumin.

Application CN 105145547 describes a fluid for cryopreservation of stem cells, comprising DMSO, umbilical cord mesenchymal stem cell conditioned medium and fetal bovine serum.

CN 104770363 discloses a plasma-free solution for cryopreservation of stem cells, said plasma-free solution comprising DMSO and dextran-40.

Document CN 105961374 discloses a cell cryopreservation fluid, in particular for stem cell cryopreservation, comprising: PBS or saline; a basal medium as a main component, and one or more components selected from the group consisting of: polyethylene glycol, propylene glycol, Ectoin (Ectoin), albumin, trehalose, proline, and poloxamer 188. The fluids mentioned for cryopreservation of cells do not contain serum and DMSO. According to the content of the patent specification, polyethylene glycol is added in order to lower the freezing temperature and increase the dehydration of the cells. This document neither discloses the molecular weight of the polyethylene glycol used, nor defines whether the fluid is of the intracellular or extracellular type.

Application CN 107912419 describes a liquid for cryopreservation of human peripheral blood mononuclear cells (including stem cells), said liquid comprising: dimethyl sulfoxide, polyethylene glycol, human serum albumin, brigmal-A (Plasmalyte-A), trehalose, hydroxyethyl starch, beta-glucan, glucose and vitamin C. The said document does not disclose the molecular weight of the polyethylene glycol. Polyethylene glycol is used especially because polyethylene glycol has good solubility in water and a low melting temperature so that the extracellular solution does not form ice crystals during cooling.

CN 102578077 discloses a serum-free cryoprotectant comprising an intracellular osmoprotectant (DMSO) and an extracellular protectant comprising NaCl, KCl, Na₂HPO₄、KH₂PO₄PEG with molecular weight in the range of 400Da to 2000Da, D-trehalose and type IV collagen. The disclosed reagents can be used for stem cell storage.

As a preferred embodiment of the subject invention, the solution described in example 1 herein is considered a formulation for organ perfusion and cold storage for transplantation. The applicant of the present invention introduced this formulation into the market under the trade name StoreProtect Plus.

Despite the great advances made in this field, there are still a number of problems associated with cryopreservation, i.e. storage of cells in a frozen state. The main drawback of the known solutions and media is that the cell viability decreases with time (under cryogenic conditions and during cryopreservation) and also during transport between the laboratory and the recipient site.

In view of the above-mentioned prior art, there is also an unmet need to provide a reagent that can be effectively used for preserving stem cells, which can also be used in typical cryopreservation conditions.

Unexpectedly, an aqueous solution comprising polyethylene glycol (PEG) having a molecular weight of about 35000Da can be successfully used as an extracellular agent for the preservation of stem cells, and after addition of dimethyl sulfoxide (DMSO), can also be used as an agent for the cryopreservation of stem cells.

Subject matter of the invention

The subject of the invention is the use as defined in claim 1. Preferred embodiments of the invention are defined in the dependent claims 2-11.

Advantages of the invention

According to tests conducted by the inventors, the use of the subject invention allows for the efficient preservation of collected stem cells. The viability of stem cells preserved according to the invention exceeds that obtained with typical, commercially available storage formulations.

Additionally, the mentioned experiments demonstrate that the use according to the invention also allows for efficient cryopreservation of stem cells after addition of dimethyl sulfoxide (DMSO). Also in this case, the results obtained are at least comparable to those of typical, commercially available formulations, and even exceed them.

Thus, the use according to the invention allows a wider temperature range to be used during cell transport, making the cells more tolerant to prolonged transport times and varying temperature conditions. Thus, the quality of the cells preserved according to the present invention is also improved.

Drawings

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

Fig. 2 shows the number of viable ADSC cells after 12, 24, 36, 48, 72, 96 and 120 hours of storage in HTS formulations and solutions used according to the present invention.

Fig. 3 shows the results of the ADSC cell viability measurement before and after the cryopreservation.

Fig. 4 shows the results of ADSC cell viability measurements after thawing and after storage 12, 24, 36, 48 and 72 in HTS formulations and solutions used according to the present invention.

Detailed Description

The subject invention was developed based on the observation of its inventors that an aqueous solution comprising polyethylene glycol (PEG) having a defined molecular weight allows for efficient preservation of stem cells while maintaining their viability.

In the use according to the subject invention, PEG (also known as PEG 35) having a molecular weight of about 35000Da (35kDA) is used. The PEG used according to the present invention may be obtained by any known method, wherein preferably said PEG may be synthesized from PEG molecules having a lower molecular weight. In other preferred embodiments of the invention, the PEG may be purified by any known technique known in the art. In a particularly preferred embodiment of the invention, commercially available PEG may be used.

According to the invention, PEG is preferably used in a concentration of about 0.01mmol/l to about 5mmol/l, for example in a concentration of 0.02mmol/l, 0.03mmol/l, 0.04mmol/l, 0.05mmol/l, 0.06mmol/l, 0.07mmol/l, 0.08mmol/l, 0.09mmol/l, 0.1mmol/l, 0.2mmol/l, 0.3mmol/l, 0.4mmol/l, 0.5mmol/l, 0.6mmol/l, 0.7mmol/l, 0.8mmol/l, 0.9mmol/l, 1.0mmol/l, 1.5mmol/l, 2.0mmol/l, 2.5mmol/l, 3.0mmol/l, 3.5mmol/l, 4.0mmol/l, 4.5mmol/l and 5 mmol/l. In a particularly preferred embodiment of the invention, the PEG concentration is less than 1mmol/l and most preferably about 0.03 mmol/l.

According to the subject invention, the aqueous solution used is used as an extracellular agent, i.e., wherein sodium ions (Na) are present⁺) Greater than potassium ion (K)⁺) A concentration of the agent. This type of agent prevents cell death during storage by reducing the formation of intracellular ice.

In a preferred embodiment of the invention, the solution contains Na in a concentration of at least about 30mmol/l⁺Ions and a K concentration of at least about 10mmol/l⁺Ions. In thatIn the most preferred embodiment of the invention, the solution contains Na in a concentration of about 125mmol/l⁺Ions and a K concentration of about 25mmol/l⁺Ions.

Preferably, the sodium ions are introduced in the form of sodium hydroxide (NaOH) and potassium dihydrogen phosphate (KH)₂PO₄) Introducing potassium ions.

In a particularly preferred embodiment of the invention, the solution may contain, in addition to the above-mentioned components (i.e. PEG, sodium and potassium ions), further components used in the field of cell storage formulations, i.e. reagents comprising impermeable anions, compounds from the group of sugars, membrane stabilizing/buffering solutions and energy sources.

In a particularly preferred embodiment of the invention, the solution mentioned comprises:

wherein

The pH of the solution is in the range of about 6,5 to about 8, and

the solution has an osmolality in the range of about 290mOsm/kg to about 320 mOsm/kg.

The anion impermeable to the cell membrane, which functions to prevent cell swelling caused by low temperature, is lactobionic acid or a salt thereof.

Raffinose functions as an additional osmotic support. In a preferred embodiment of the present invention, raffinose pentahydrate (raffinose.5H) is used₂O)。

The membrane stabilizer is magnesium sulfate, preferably magnesium sulfate heptahydrate (MgSO)₄·7H₂O), which acts to stabilize the electrochemical equilibrium of the cell membrane, which determines Na⁺Ion, K⁺Ions, phosphorus ions and Ca²⁺Proper transport of ions.

KH₂PO₄Is a buffer system that acts to maintain the pH of the solution, thereby providing an acid/base equilibrium. Additionally, it also provides potassium ions to the solution.

Glutathione and allopurinol are agents that combat the formation and action of free radicals.

Adenosine is a source of ATP precursor as an energy source.

The water used for the preparation of the solutions used according to the invention is pharmaceutically acceptable water for injection.

In a most preferred embodiment of the invention, the solution referred to comprises:

and

in an amount sufficient to obtain NaOH having a pH of about 7.4, and

the water for injection is used as the water,

wherein the solution has an osmotic pressure of 300mOsm/kg and comprises about 125mmol/l Na⁺Ions.

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

The solutions used according to the invention do not contain calcium ions added separately or as a component of the compounds included in the composition of the solutions mentioned. The water for injection used for the preparation of the solutions used according to the invention also does not contain said ions.

In accordance with the present invention, the term "about" as used above and below should be understood as a deviation of +/-5% from a given value, reflecting inaccuracies that may occur during the manufacture of the composition of the present invention, for example, during measurement of a component of a solution.

Examples

Example 1

Solution for stem cell preservation

In experiments relating to stem cell preservation, the formulation StoreProtect Plus (manufacturer: Carnamedica) with the following composition was used:

and

in an amount sufficient to obtain NaOH having a pH of about 7.4,

The water for injection is used as the water,

the solution of example 1 exhibited an osmotic pressure of about 300mOsm/kg and contained sodium ions (Na) at a concentration of about 120mmol/l⁺) And potassium ion (K +) at a concentration of about 25 mmol/l.

Example 2

Solution for cryopreservation of stem cells

In experiments related to the cryopreservation of stem cells, the formulation from example 1 was used, which additionally contained 10% by volume of Dimethylsulfoxide (DMSO) relative to the total volume of the solution.

Example 3

Short term preservation of stem cells

Mesenchymal adipose-derived stem cells (ADSCs) isolated from 9 donors were divided into test groups and stored for up to 120 h. The following experimental groups were used: (a) NaCl solution (0.9%), (b) NaCl solution containing glucose (0.9%), (c) Ringer's solution (Ringer's solution) (FreseniusKabi), and (d) Bowman's force (plasma) preparation (Baxter) (containing 5.26g of sodium chloride, 0.37g of potassium chloride, 0.3g of magnesium chloride (6H) per 1000ml of solution₂O), 3.68g of sodium acetate (3H)₂O), 5.02g of sodium gluconate, and comprising: 140mmol/l sodium ions, 5mmol/l potassium ions, 1.5mmol/ll magnesium ion, 98mmol/l chloride ion, 27mmol/l acetate ion (CH)₃COO^-) 23mmol/l of gluconate ion (C)₆H₁₁O₇ ^-) Wherein the solution has a theoretical osmolality of 294mOsm/l and a pH of about 7.4 (from 6.5-8.0)), (e) hypo thermolosol FRS formulation (HTS, BioLife Solutions (BioLife Solutions)) (contains/: trolox, Na⁺、K⁺、Ca²⁺、Mg²⁺、Cl^-、H₂PO₄ ^-HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine, glutathione) and (e) a solution as defined in example 1. Short-term storage (up to 120 hours) is carried out at a temperature of 4 ℃ in the case of groups (d) and (e) also at room temperature. During the experiment, cell viability and the number of viable cells were observed at selected time points.

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

Fig. 1 shows the viability measurements obtained for experimental groups (a), (b), (d) and (e), while fig. 2 shows: number of viable cells as a function of storage time for test groups (d) and (e).

The results shown in fig. 1 confirm that the highest stem cell viability (above 90% after 120 hours) was obtained with the solution of example 1. In the case of the solution of example 1, the number of viable cells was also the highest 120 hours after the start of the experiment (fig. 2).

Example 4

Cryopreservation of stem cells

In a second experiment, the formulation defined in example 2 was tested for its usefulness in stem cell cryopreservation. In this experiment, the same type of stem cells as in example 1 was used. Certified preparation Stem-Cell Banker (Zenoaq) was used as reference preparation. The experiment was carried out at a temperature of-196 ℃ for 7 days.

After thawing the cells, viability measurements were performed (in the same way as in example 1) and the cells were transferred into storage solutions (hypo thermolosol FRS (HTS, bio-life solutions company) (reference formulation) and the solution of example 1) to assess whether changes in storage conditions negatively affect cell viability.

During the storage period, cell viability was observed at selected time points (12 hours, 24 hours, 48 hours, 72 hours).

Figure 3 shows the results of stem cell viability measurements after cryopreservation. The measurements demonstrate that the formulation of example 2 provides efficient preservation of dry cells in the frozen state.

Figure 4 shows the results of stem cell viability measurements at selected time points after thawing for the hypo thermosol FRS formulation and the solution of example 1. The results shown in fig. 4 clearly show that: the solution of example 1 showed better properties than the reference formulation used.