CZ309446B6 - Means for cryopreservation of human or animal cells - Google Patents

Abstract

A means of preserving human or animal stem cells at very low temperatures from -18 °C to -196 °C, which consists of an aqueous solution of trehalose, which contains 250 to 400 mmol/l of trehalose. The solution can also contain K+ cations of up to 50 mmol/l, Na+ cations of up to 80 mmol/l, Cl- anions of up to 40 mmol/l, PO43- and/or HPO42- and/or H2PO4- anions of up to 65 mmol/l, Mg2+ and/or Ca2+ cations of up to 2 mmol/l, SO42- and/or HSO4- anions of up to 2 mmol/l, while the pH of the solution is 7.1 to 7.6 , and the total osmolality of the solution is not higher than 900 mosmol/l. Also described is the use of the above composition for long-term storage of stem cells, in particular mesenchymal stem cells, for more than three days at very low temperatures from -18°C to -196°C.

Description

Means for cryopreservation of human or animal cells

Field of technology

The invention relates to a means for preserving human or animal cells at very low temperatures. Furthermore, the invention relates to the use of a buffered or non-buffered aqueous solution of trehalose for long-term storage of stem cells at very low temperatures.

Current state of the art

Stem cells are a highly valued fraction in the biopharmaceutical industry for their multipotent potential. Their limited life ex vivo is currently solved by their cryopreservation, i.e. by deep freezing to a temperature of -18 °C to -196 °C. Deep-frozen stem cells can be used at any time if necessary, and their lifespan is extended by several years in this state. A key step in the entire stem cell freezing process is the solution in which the cells are stored. Its composition is crucial for the resulting properties of the cells after they are thawed. From the point of view of the biopharmaceutical industry and the current rules for the use of cells for therapeutic purposes, it is desirable that the thawed cells can be directly applied to the patient without further steps of washing or centrifuging the cells. This fact places great demands on the composition of the solution with regard to the safety of the individual components contained in the solution. Dimethyl sulfoxide (DMSO), ethylene glycol or glycerol are commonly used for cryopreservation of cells.

The disadvantage of using these cryoprotective agents is their significant cytotoxicity and significant adverse side effects when administered to patients. Therefore, cryoprotective solutions are developed, which are primarily based on the use of non-toxic components while maintaining cryopreservation properties.

Trehalose appears to be a very potent component of cryoprotective solutions. Trehalose, a disaccharide of glucose, is synthesized by lower organisms in cases where they are exposed to stress, cold, high temperatures or desiccation, it is known as an anti-stress factor. Trehalose, unlike DMSO, does not pass through the cell membrane and during freezing protects the cells extracellularly due to slight dehydration of the cells.

Dash and colleagues tested trehalose (Curr Stem Cell Res Ther., 2008) for freezing fish embryonic stem cells. Trehalose added to the cryoprotectant solution containing DMSO increased the viability of thawed cells.

A very positive effect of 1M trehalose solution on freezing stem CD34 + cells isolated from peripheral blood is described by Martinetti (Biomed Rep., 2017).

The use of extracellular as well as intracellular trehalose in cryosolution favorably affects the vitality of thawed stem cells isolated from umbilical cord blood (Motta et al., Cryobiology, 2014).

Campbell et al describe (Cryobiology, 2012) the preferred use of a 0.2 to 0.4M trehalose solution in combination with sodium carbonate and EMEM (Eagle's Minimum Essential Medium) culture medium for freezing bovine endothelial cells.

A 24-hour pre-incubation of cells with trehalose before the actual freezing process proves to be very advantageous. The advantage of preincubation of cells with 200mM trehalose solution is also described by Petrenko et al. (Cryo Letters. 2014).

Patent application CN 105961374 describes a cryoprotective solution for preservation

-1 CZ 309446 B6 mesenchymal stem cells. This solution mainly contains polyethylene glycol and albumin, while it also contains trehalose as one of the components.

Application CN 105494317 describes the freezing of stem cells isolated from adipose tissue in a solution containing mainly lipoprotein, trehalose and glycerin.

Patent application CN 104012519 describes an advantageous combination of trehalose and erythropoietin in a cryoprotective solution.

Patent application US 2011008300 describes the advantageous use of a solution containing trehalose and glycerin for cryopreservation of adipose tissue.

Patent application KR 20140046690 describes the freezing of porcine spermatogonial cells in a solution containing 200 mM trehalose, fetal bovine serum, DMSO and culture medium Alpha MEM (Alpha Modification of Eagle's Minimum Essential Media).

Similarly, application CN 102578077 is focused on the issue of freezing mesenchymal stem cells in a cryosolution containing DMSO, trehalose and other components. The cryosolution is used without serum.

Korean patent KR 20120078094 describes an advantageous combination of DMSO, trehalose, catalase and the caspase inhibitor zVAD-fmk for freezing stem cells isolated from amniotic fluid.

A solution containing trehalose and other pharmaceutical ingredients is described in Czech patent application PV 2016-284. The application describes the hypothermic storage of cells in this solution at a temperature reduced to -4 °C.

The solutions known so far do not allow deep freezing using pharmaceutically incompatible components, which significantly prevents the immediate use of such solutions after thawing in clinical practice.

The essence of the invention

The above-mentioned shortcomings are eliminated by a means for preserving stem cells at very low temperatures, which consists of an aqueous solution of trehalose, which contains trehalose disaccharide in an amount of 250 to 400 mmol/1.

According to the invention, the solution consists of a buffered aqueous solution of 250 to 400 mmol/1 trehalose, which contains simple salts to buffer pH fluctuations, the pH of the solution being 7.1 to 7.6, and the total osmolarity of the solution not being higher than 900 mosmol/1. In this application, osmolarity is understood as osmolarity calculated in a manner customary in this field of technology, or, in case it would be higher than calculated, osmolarity measured.

This solution preferably contains as necessary components K ⁺ cations in the amount of 20 to 50 mmol/1, Na ⁺ cations in the amount of 20 to 80 mmol/1, Cl anions in the amount of 0.5 to 40 mmol/1, and PO4 ³ anions and /or HPO4 ² and/or H2PO4 in a total amount of 20 to 65 mmol/1. The upper limit of the trehalose content is determined by the fact that the total osmolarity of the solution should not be higher than 900 mosmol/1. The upper limit of the trehalose content in the buffered solution is therefore lower than 900 mosmol/1 by the amount of dissociated salts. The concentrations of the mentioned components are chosen within the mentioned range so that the pH value of the solution is 7.1 to 7.6. In the simplest embodiment of the invention, the composition according to the invention does not need to contain any other intentionally added components. In any case, the optimal total osmolarity of the solution is not higher than 900 mosmol/1.

In other embodiments of the invention, the solution may also contain Mg ²⁺ and/or Ca ²⁺ cations in

-2 CZ 309446 B6 in a total amount of 0 to 2 mmol/1 and SO4 ² and/or HSO4 anions in a total amount of 0 to 2 mmol/1. If Mg ²⁺ and/or Ca ²⁺ cations are present as an intentionally added component, they are contained in the solution in a total amount of 0.1 to 2 mmol/1. If SO4 ² · and/or HSO4 · anions are present as an intentionally added component, they are contained in the solution in a total amount of 0.5 to 2 mmol/1.

In a preferred embodiment of the invention, the solution contains K ⁺ cations in an amount of 28 to 32 mmol/1, Na ⁺ cations in an amount of 20 to 50 mmol/1, Mg ²⁺ and/or Ca ²⁺ cations in a total amount of 0.4 to 1.2 mmol/1, Cl anions in the amount of 0.5 to 20 mmol/1, PO4 ³ and/or HPO4 ² and/or H2PO4 anions in the total amount of 40 to 50 mmol/1, SO4 ² and/or HSO4 anions in the total amount of 0 .9 to 1.2 mmol/1.

It is particularly advantageous if the aqueous solution does not contain, in addition to K ⁺ cations, Na ⁺ cations, Mg ²⁺ and/or Ca ²⁺ cations, Cl' anions, PCX ^3- and/or HPO4 ² · and/or H2PO4 ·, SO4 anions ² · and/or HSO4· and trehalose no other intentionally added components. Of course, it cannot be ruled out that there are trace amounts of substances in the solution, the presence of which is conditioned by production or otherwise random.

More specifically, the device according to the invention is designed to store cells at low and very low temperatures from -5°C to -273°C. The composition according to the invention is particularly suitable for storing stem cells at temperatures from -18°C to -196°C, preferably -60°C to -196°C, most often 80°C to -196°C. The mode of freezing the product according to the invention can have a different course. Programmable freezing units (e.g. IceCube; SY-LAB) or other temperature-optimized devices can be used: Mr. Frosty™ (Thermo Fisher Scientific) or CoolCell® (BioCision). Advantageously, the suspension of cells in the composition according to the invention can also be frozen by directly placing the sample in liquid nitrogen vapor. Liquid nitrogen or its vapors are the most suitable from the point of view of freezing and storing the sample.

The composition according to the invention consists of pharmaceutically acceptable compounds and can be used directly to apply the stem cells that are frozen in it. After thawing the stem cells, their sufficient viability is ensured.

The product according to the invention is intended in particular for the cryopreservation of mesenchymal stem cells obtained from bone marrow, adipose tissue, peripheral blood or from extraembryonic tissues (placenta, umbilical cord, umbilical cord blood) or embryonic, neural, induced or limbal stem cells of mammals, including humans. It can also be used to preserve stem cells attached to a biological carrier. From the essence of the invention itself, i.e. the osmotic effect of the solution on the cells before freezing incl. stabilization of the cell membrane, it is possible to expect a positive effect of the solution on other cell types as well.

Stem cells preserved in the composition according to the invention can be applied after thawing without the need to wash off this composition directly to the patient, especially intravenously, intraarterially, intramuscularly, subcutaneously, subconjunctivally or intrathecally, either alone or with a carrier.

The carrier of stem cells can be biodegradable or non-degradable material. The carrier can be functional biomaterials. These can be synthetic biocompatible polymers (polylactic acid PLA, polyglycolic acid PGA, polyethylene glycol PEG, polycaprolactone PCL, polyhydroxybutyrate PHB and others), natural polymers and polysaccharides (collagen type I, hyaluronic acid, fibrin, chitosan, cellulose, starch and others) or extracellular matrix (ECM) in the form of hydrogel, nanofibers or microfibers. Alternatively, for applications requiring a carrier, the stem cell cultures can be performed in the presence of such carriers and stored and administered together in the composition of the invention.

The trehalose solution according to the invention can be used for long-term storage of deep-frozen mesenchymal stem cells. Deep freezing means freezing to

-3 CZ 309446 B6 very low temperatures from -18 °C to -196 °C. Lower temperatures are also possible, although less practical. Long-term storage means storage for more than three days, but also several weeks or months, for example up to five years.

Buffered aqueous solution of trehalose, containing trehalose in the amount of 250 to 400 mmol/1, K ⁺ cations in the amount of 28 to 32 mmol/1, Na ⁺ cations in the amount of 20 to 50 mmol/1, Mg ²⁺ and/or Ca ²⁺ cations in a total amount of 0.4 to 1.2 mmol/1, Cl anions in an amount of 0.5 to 20 mmol/1, anions PO4 ³ · and/or HPO4 ² · and/or H2PO4· in a total amount of 40 to 50 mmol/ 1, SO4 ² · and/or HSO4 · anions in a total amount of 0.9 to 1.2 mmol/s, can be used especially for long-term storage of deep-frozen mesenchymal stem cells.

Examples of implementation of the invention

Example 1: Isolation and Culture of Bone Marrow MSCs (BM-MSCs)

Bone marrow aspiration was performed with a trepanobioptic needle, which is part of a sampling set containing saline solution with heparin and gentamicin. After transporting the bone marrow to the room for sterile processing of biological material, the sample was mixed with Gelofůsin infusion solution. The amount of Gelofusin added corresponded to 25% of the bone marrow volume. The mixture was allowed to sediment for 10 to 30 minutes, and then the ring layer of mononuclear cells was collected. In the removed layer of cells, the cellularity of the mixture was analyzed with a hematology analyzer. 10 ml of complete culture medium (Alpha MEM Eagle medium containing 5% human platelet lysate and gentamicin) and a defined amount of isolated cell suspension (5 to 10 million nucleated cells) were added to each of the prepared culture flasks with an area of 75 cm ² for starting the primoculture. Cell cultivation took place at a temperature of 37 ± 0.5 °C and 5% CO2. During the cultivation of the cells, the complete culture medium was continuously replaced with possible rinsing of the cells with 10 ml of PBS buffer per culture bottle. According to the number of growing cells, their passage took place, i.e. the controlled enzymatic separation of adherent cells and their transfer to a larger cultivation area. This process always began with an optical inspection of the culture under a microscope; reaching 80 to 90% coverage of the culture bottom cells was optimal for culture growth. For passaging, it was necessary to aspirate the culture medium, rinse the culture with 10 ml of PBS buffer and add 1 ml of TrypLE Select CTS enzyme solution. The culture bottles were incubated for about 4 minutes at 37±0.5 °C. The correct course of the enzymatic reaction was checked under a microscope. If the cells moved with the flowing liquid after tapping the culture bottle, 1 ml of 20% HSA (human serum albumin) infusion solution and 6 ml of PBS buffer were added to each bottle. This mixture was mixed by gentle pipetting and was transferred to a centrifuge tube and centrifuged (230 g, 5 min). The cell pellet was resuspended in complete culture medium and the cell suspension divided into the required number of culture bottles. A final cell suspension was achieved by the third passaging of the cells. It was additionally washed once more with PBS buffer and centrifuged (230 g, 5 minutes). To create the preparation according to the embodiment of the invention, the pellet obtained by the above-mentioned procedure was resuspended in the solution of the preparation shown in example 2A (with 250 mM trehalose, which is hereinafter referred to as TR-250), 2B (with 300 mM trehalose, referred to as TR-300), 2C ( with 350 mM trehalose, designated TR-350), 2D (with 400 mM trehalose, designated TR-400), and 2E (only aqueous solution with 400 mM trehalose designated TRH-400). The label mM or mmol/1 indicates the number of millimoles in a liter of solution.

The content of frozen stem cells was usually 5 x 10 ^s to 1 x 10 ⁷ MSCs/ml. The cell suspension thus prepared was continuously mixed by gentle shaking for at least ten minutes at room temperature. At the same time, the viability of the input cell suspension was determined. Subsequently, the suspension (TR-250 and TR-300) was stored in a -70 °C freezer in a CoolCell® freezer (BioCision). In parallel, the samples were stored directly at a temperature of -70 °C or -196 °C for 24 hours (table 1). Table 2 shows the viability values of cells frozen by simply storing them at -196 °C, however, the cells are in suspension in different solutions (TR

-4 CZ 309446 B6

350, TR-400 and TRH-400). Subsequently, all samples were placed in a Dewar container containing liquid nitrogen. After thawing them in a water bath at 37 °C, the samples were tested according to Example 3.

Example 2: Possible preparations of 100 ml of the solution according to the invention

Example 2A. Preparation of the solution according to the invention with 250 mM trehalose (TR-250)

Weigh out 0.408 g of KH2PO4 and 0.213 g of Na2HPO4, dissolve in 20 ml of distilled water; weigh 0.007 g of CaCb x 2H2O and dissolve in 10 ml of distilled water; weigh 0.012 g of MgSO4 and dissolve in 10 ml of distilled water. Mix the solutions prepared in this way and adjust the pH to 7.4. Then dissolve 9.45 g of trehalose in the mixture and adjust the final volume to 100 ml with distilled water. The resulting osmolarity of the solution is 358 mosmol/1.

Example 2B. Preparation of the solution according to the invention with 300 mM trehalose (TR-300)

Weigh out 0.408 g of KH2PO4 and 0.213 g of Na2HPO4, dissolve in 20 ml of distilled water; weigh 0.007 g of CaCb x 2H2O and dissolve in 10 ml of distilled water; weigh 0.012 g of MgSO4 and dissolve in 10 ml of distilled water. Mix the solutions prepared in this way and adjust the pH to 7.4. Then dissolve 11.34 g of trehalose in the mixture and adjust the final volume to 100 ml with distilled water. The resulting osmolarity of the solution is 408 mosmol/1.

Example 2C. Preparation of the solution according to the invention with 350 mM trehalose (TR-350)

Weigh out 0.408 g of KH2PO4 and 0.213 g of Na2HPO4, dissolve in 20 ml of distilled water; weigh 0.007 g of CaCb x 2H2O and dissolve in 10 ml of distilled water; weigh 0.012 g of MgSO4 and dissolve in 10 ml of distilled water. Mix the solutions prepared in this way and adjust the pH to 7.4. Then dissolve 13.23 g of trehalose in the mixture and adjust the final volume to 100 ml with distilled water. The resulting osmolarity of the solution is 458 mosmol/1.

Example 2D. Preparation of the solution according to the invention with 400 mM trehalose (TR-400)

Weigh out 0.408 g of KH2PO4 and 0.213 g of Na2HPO4, dissolve in 20 ml of distilled water; weigh 0.007 g of CaCb x 2H2O and dissolve in 10 ml of distilled water; weigh 0.012 g of MgSO4 and dissolve in 10 ml of distilled water. Mix the solutions prepared in this way and adjust the pH to 7.4. Then dissolve 15.12 g of trehalose in the mixture and adjust the final volume to 100 ml with distilled water. The resulting osmolarity of the solution is 508 mosmol/1.

Example 2E. Preparation of an aqueous unbuffered solution according to the invention with 400 mM trehalose (TRH-400)

Weigh and dissolve 15.12 g of trehalose and adjust the final volume to 100 ml with distilled water. The resulting osmolarity of the solution is 400 mosmol/1.

Example 3: Viability of BM-MSCs after their freezing and subsequent thawing (storage in liquid nitrogen)

The viability of MSCs isolated from human bone marrow preserved in TR-250, TR-300, TR-350, TR-400 and TRH-400 solution immediately after thawing was evaluated. First, the suspension was stained with propidium iodide and cell viability was determined by flow cytometry. Viability determined by propidium iodide staining reflects the viability of individual cells in suspension. Propidium iodide is a substance that does not penetrate the membrane of living cells. In damaged and non-viable cells, propidium iodide binds to DNA. The viability of BM-MSCs monitored by propidium iodide staining immediately after thawing reached more than 50% in samples frozen by shock storage to -70 °C or to -196 °C (Table 1). The most suitable se

-5 CZ 309446 B6 shows the direct storage of the suspension of cells in the composition according to the invention to -70 °C or -196 °C in TR-250 and TR-300 solution (table 1). However, the best results were achieved using the TR-400 solution with the highest concentration of trehalose. The latter protects cells from frost damage the most, cell viability after thawing is over 60% (table 2).

Table 1 - MSCs from bone marrow, cell viability in TR-250 and TR-300 solution in different freezing modes

TR-250 % live (propidium iodide negative) cells sample before freezing 90.8 COOLCELL 14.2 direct storage down to -70 °C 54.7 direct storage to -196 °C 52.9

TR-300 % live (propidium iodide negative) cells sample before freezing 90, 8 COOLCELL 12.1 direct storage down to -70 °C 51.0 direct storage to -196 °C 59.7

Table 2 - MSCs from bone marrow, viability of cells stored directly to -196 °C in various solutions according to the invention

Save to -196°C % live (propidium iodide negative) cells sample before freezing 91.1 TR-350 54, 5 TR-400 63.9 TRH-400 39.1

The thawed cells were also tested in parallel using subsequent cultivation and their ability to adhere to the culture plastic and their morphological properties were monitored. The basic property of mesenchymal stem cells is their ability to adhere to culture plastic. Morphologically, viable mesenchymal stem cells have an elongated fibroblastic shape. An equal volume of cells corresponding to the number of 100,000 fresh cells was plated in a 24-well culture plate. After 48 hours, the cells were evaluated under a microscope. The highest degree of adhesion was observed in the suspension of cells frozen by direct storage to -70 °C or -196 °C in the solution according to the invention (TR-250, TR-300, TR-350 and TR-400). All adhered cells corresponded to the morphology of mesenchymal stem cells. These observations are consistent with those observed in viability detection using propidium iodide.

Orientation experiments have shown that the trehalose content can be higher than 400 mmol/l, but its effect does not increase significantly, while with a total osmolarity higher than 900, the solution is already pharmaceutically unsuitable, and on the contrary, the trehalose content is lower than 60 mmol/l 1 no longer has an observable effect.

Claims (5)

1. Means for cryopreservation of human or animal stem cells, characterized in that it consists of a buffered aqueous solution of trehalose, which contains trehalose in an amount of 250 to 400 mmol/1, while the solution contains K ⁺ cations in an amount of up to 50 mmol/1, cations Na ⁺ in an amount of up to 80 mmol/1, Cl anions in an amount of up to 40 mmol/1, anions PO4 ³ and/or HPO4 ² and/or H2PO4 in a total amount of up to 65 mmol/1, cations Mg ²⁺ and/or Ca ^{2 +} in a total amount of up to 2 mmol/1, SCE ^2- and/or HSO4· anions in a total amount of up to 2 mmol/1, while the pH of the solution is 7.1 to 7.6 and the total osmolarity of the solution is not higher than 900 mosmol/ 1. 2. The composition according to claim 1, characterized in that the solution contains K ⁺ cations in an amount of 20 to 50 mmol/1, Na ⁺ cations in an amount of 20 to 80 mmol/1, Cl anions in an amount of 0.5 to 40 mmol/1 , PO4 ³ and/or HPO4 ² and/or H2PO4 anions in a total amount of 20 to 65 mmol/1. 3. The composition according to claim 2, characterized in that the solution contains Mg ²⁺ and/or Ca ²⁺ cations in a total amount of 0.1 to 2 mmol/1, SO4 ² and/or HSO4 anions in a total amount of 0.5 to 2 mmol/l. 4. The composition according to claim 3, characterized in that the solution contains K ⁺ cations in an amount of 28 to 32 mmol/1, Na ⁺ cations in an amount of 20 to 50 mmol/1, Mg ²⁺ and/or Ca ²⁺ cations in total an amount of 0.4 to 1.2 mmol/1, Cl anions in an amount of 0.5 to 20 mmol/1, anions of PO4 ³ and/or HPO4 ² and/or H2PO4· in a total amount of 40 to 50 mmol/1, anions of SO4 ² · and/or HSO4· in a total amount of 0.9 to 1.2 mmol/1. 5. Use of the composition according to any one of claims 1 to 4, to store stem cells for at least three days at very low temperatures from -18°C to -196°C.