A preservation method that uses trehalose and does not include other cryoprotective agents in the cryopreservation protocol.

Abstract

This invention provides a cell cryopreservation method that avoids or improves upon the use of DMSO as a cryoprotective agent. [Solution] Cell material containing living cells is preserved during a cryopreservation protocol by combining the cell material with a cryoprotective formulation / medium / solution containing an effective amount of trehalose (in the absence of DMSO and / or any other added cryoprotective agents). That is, the cryopreservation protocol does not contain any cryoprotective agents other than trehalose, and the cryopreservation protocol includes exposing the cell material to a cryoprotective formulation containing an effective amount of trehalose to act as a cryoprotective agent, cooling the cell material to a predetermined temperature below -20°C at a cooling rate in the range of -3°C / min to -50°C / min, and obtaining warmed cryopreserved cell material.

Description

Technical Field

[0001] (Statement Regarding Federal Support of Research) This invention was made with government support under Grant HL142371 from the National Heart, Lung, and Blood Institute of the National Institutes of Health. The United States government has certain rights in this invention.

[0002] (Cross - Reference to Related Applications) This non - provisional patent application claims the benefit of U.S. Provisional Application No. 63 / 183,678. The disclosure of the prior application is incorporated herein by reference in its entirety.

[0003] (Technical Field) This disclosure relates to the field of cell and tissue preservation. In particular, the present invention uses trehalose but does not contain other added cryoprotective agents such as dimethyl sulfoxide (DMSO), glycerin / glycerol, ethylene glycol, propylene glycol, etc. For example, it relates to a method for cryopreserving cell materials such as stem cells, hematopoietic stem cells, lymphocytes, white blood cells, T cells (and T cell subsets and CAR T cells), and pancreatic islets.

Background Art

[0004] (Background) Most cells used in research are added with 5 - 10% DMSO to the cells in suspension in cryovials, then slowly cooled at about - 1°C / min to induce nucleation at a high sub - zero temperature (usually exceeding - 10°C), or without inducing nucleation, and stored at less than - 80°C or - 135°C, and then cryopreserved.

[0005] However, cell types and tissues that are difficult to preserve, as well as cell harvests for cell therapy applications, etc. ​There are situations where rate is important. Improving cell viability and yield and preserving them is a traditional approach. Alternative protocols and solutions are needed to enable the preservation of cell types, which is currently difficult to achieve. New cell therapies have been developed, and cell and tissue-based screening for drug development has been advanced. As SEI becomes more widespread, effective preservation solutions and protocols are needed, As a result, the potential use and benefits of emerging therapies such as stem cell transplantation and assays can be realized. Cut.

[0006] With the increasing demand for bringing pharmaceuticals to market more quickly and at a lower cost, Furthermore, there is a need for more cost-effective high-throughput screening technologies and services. Shortening the lead time for discovery and validation is a critical area of ​​development, and pharmaceutical companies are potentially The focus is shifting to defining how specific drugs are toxic. Cells and tissues Assays based on this are a trend in such screening. (More than 20 years since 1997) Previously, pharmaceutical and biotechnology companies had already spent $42 billion on research and development. The screening costs were approximately $5.9 billion. Environmental companies monitor cleanup and remediation activities. They are shifting to visual and quality control. Furthermore, the company is screening products and services. In order to do so, external sources are increasingly being used. A cost-effective, reliable, and quantitative cell and tissue assay system. Therefore, it is desirable to increase the availability of cell products and the use of such products. A preservation methodology that increases utilization efficiency is highly desirable.

[0007] DMSO is the most effective cryoprotective agent discovered and widely used. Cryopreservation is usually done by freezing in culture medium using DMSO at a slow cooling rate, and then... Includes storing at temperatures below -135°C for use. Cell yield and viability are extremely important. A possible example is starting a culture for a bioreactor protein production run. Minimizing the costly delays involved, and administering cells to patients for the treatment of various diseases such as cancer. Cell therapy involving several cells, such as fibroblasts and keratinocytes, is one example. Hepatocytes and other cell types such as cardiomyocytes can be easily cryopreserved, but if not frozen sufficiently, Cell yields are often well below 50%.

[0008] The current opinion is that DMSO should be removed before injecting cells into the patient. (Caselli et al., Respiratory depression and somnolence in children receiving di methylsulfoxide and morphine during hema topoietic stem cell transplantation. Matologica, 94:152-3, 2009; Junior et al ., Neurotoxicity associated with dimethy l sulfoxide-preserved hematopoietic prog enitor cell infusion. Bone Marrow Transp lant, 41:95-6, 2008; Mueller et al., Neu rotoxicity upon infusion of dimethylsulfate oxide-cryopreserved peripheral blood ste m cells in patients with and without pre -existing cerebral disease. Eur J Haemat ol, 78:527-31, 2007; Otrock et al., Tran sient global amnesia associated with the infusion of DMSO-cryopreserved autologo us blood stem cells. Haematologica, 93:3 6-7, 2008; and Schlegel et al., Transien t loss of consciousness in pediatric rec ipients of dimethylsulfoxide (DMSO)-cryo preserved peripheral blood stem cells in dependent of morphine co-medication. Hae matologica, 94:1473-5, 2009). Therefore, the time required to effectively use such cells increases.

[0009] The mechanism of DMSO cytotoxicity has not been clearly elucidated, but it is thought to modify membrane fluidity, induce cell differentiation, and cause cytoplasmic microtubule changes and metal complexes (Barne tt, The effects of dimethylsulfoxide and glycerol on Na+, K+-ATPase and membrane structure. Cryobiology. 1978;15(2):227-​​ 9; Katsuda et al., The influence of dime thyl sulfoxide on cell growth and ultras tructural features of cultured smooth mu scle cells. J Electron Microsc (Tokyo). 1984;33(3):239-41; Katsuda et al., Dimet hyl sulfoxide induces microtubule format ion in cultured arterial smooth muscle c Cell Biol Int Rep. 1987;11(2):103- 10; Miranda et al., Alteration of myobla st phenotype by dimethyl sulfoxide. Proc Natl Acad Sci US A. 1978;75(8):3826-30 ). DMSO also dose-dependently reduces collagen mRNA expression (Zeng et al., Dimethyl Sulfoxide Decrease Type -I and -III Collagen Synthesis in Human Hepatic Stellate Cells and Human Foreski n Fibroblasts. Advanced Science Letters, 3:496-499, 2010). More recently, DMSO has been shown to be involved in cell cycle progression and meiotic spindles. It affects pyramidal tissue (Li et al., Dimethyl Sulfoxid e Perturbs Cell Cycle Progression and Sp indle Organization in Porcine Meiotic Oo cytes. PLoS One. 2016 Jun 27;11(6):e0158 074), protein aggregation (Giugliarelli et al., Evidence of DMSO-Induced Protein Aggregation in Cells. J Phys Chem A. 2016 Jul 14;120(27 ):5065-70), and macroscopic molecular changes that have the potential to interfere with various cellular processes. (Tuncer et al., Low dose dimethyl sulfo xide driven gross molecular changes have the potential to interfere with various cellular processes. Sci Rep. 2018;8(1): 14828) has been reported.

[0010] Therefore, using DMSO as a cryoprotective agent avoids or improves the outcome of cell cryopreservation. A method of preservation is needed. In this regard, disaccharides such as trehalose are used as cryoprotectants. It has been widely studied. The main hypothesis that trehalose is an effective cryoprotective agent is that This means that trehalose should be present on both sides of the cell membrane. Trehalose is a mammalian substance. Not metabolized by animal cells, and active mammalian trans trehalose for uptake There is no port mechanism. Therefore, prior to the invention of the methodology disclosed herein, trehalose (single The use of (German) results in very low survival rates and metabolic function values ​​(especially in the absence of DMSO). This was expected. [Overview of the project] [Problems that the invention aims to solve]

[0011] The methodology described herein addresses the above-mentioned need for cellular materials for a wide variety of potential applications. By providing more efficient, cost-effective, and safer storage and transportation methods This provides an improvement over existing cell and tissue therapies. See Figure 1.

[0012] The methodology of this disclosure also applies to, for example, stem cells, hematopoietic stem cells, mesenchymal stem cells (e.g., human-derived stem cells). Leaf stem cells, lymphocytes, leukocytes, T cells (and T cell subsets and CAR T cells) and The aim is to increase the usability of cell materials such as pancreatic islets and to change their lifespan. This enables the increased use of cellular materials (in some cases, cells can be used without any intermediary processes). (It can be used and / or injected directly into the patient after thawing.) The application of the methodology of this disclosure is also detailed. Cell and tissue research, cell and tissue-based engineered regenerative medicine products, and transplantation and toxicology studies Includes cell and tissue banks for leaning. [Means for solving the problem]

[0013] (Summary of the invention) This disclosure relates to cryopreservation protocols that do not use other conventional cryoprotective agents (e.g., D MSO, glycerin / glycerol, ethylene glycol, propylene glycol, etc. This provides an improved preservation method for using trehalose in the absence of DMSO.

[0014] In some embodiments, the present disclosure describes a conventional cryoprotective substance (e.g., DMSO, which is toxic) It is known that certain cells or tissues are cryopreserved at temperatures below -80°C. By replacing (which is designed to be removed after being refurbished), The objective is to provide a cryopreservation methodology that achieves protective effects on cells or tissues and low toxicity. The methodology of this disclosure is for use with highly toxic cryoprotective agents (for example, when cells are immersed in a cryopreservation solution). Then, DMSO or other conventional freezing agents are used when the product is to be frozen and stored at temperatures below -80°C. Provides an inexpensive and safe method for cryopreservation without the use of preservatives. (DMSO) Since conventional cryoprotective agents such as these are not used, the toxicity experienced by cells (during the preservation process, preservation) The temperature is kept low (during, during, and after rewarming), and the cells are left undisturbed after thawing without any intervention. It can be used and / or injected directly into the patient. In some embodiments, it is thawed The cells or tissues are suspended in the culture medium and the culture process is immediately (i.e., rewarming process) It may be started immediately after thawing (for example, without washing after thawing cells or tissues). In some embodiments, the methodology of this disclosure applies to all cellular functions (e.g., stem cells, pancreatic islets, intercellular matrix). This refers to the cryopreservation of cultured cells in a manner that maintains the cells (including leaf stem cells, etc.). Therefore, the efficiency of using and / or transplanting these cells is improved. For example, several In that embodiment, the methodology of the present disclosure does not require further processing / washing after rewarming from storage. Instead, ready-to-use, on-demand, ready-to-order human mesenchymal stem cells (hM) derived from bone marrow are available for therapeutic use. The aim is to provide SC). In some embodiments, the methodology of this disclosure is used for trehalose This methodology targets the cryopreservation of Pan T cells using a cryopreservation solution, where the cells are placed in the cryopreservation solution. DMSO or other conventional materials used when immersed and then frozen and stored at -80°C or below. Freezing preservative (glycerin / glycerol, ethylene glycol, propylene glycol) It does not contain other added cryoprotective agents such as (etc.). [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 illustrates the patient population that may ultimately benefit from cell and tissue therapy (the total U.S. patient population is 122 million). [Figure 2] Figure 2 is an explanatory diagram of the data obtained from the experiment, showing the effect of the cooling rate on the viability of hMSCs after cryopreservation in the absence of trehalose and at the DMSO vs. DMSO-only control concentrations. The data are shown as mean ± 1 standard error. [Figure 3] Figures 3A and 3B show data obtained regarding cell viability after cryopreservation at -15°C / min using combinations of DMSO and trehalose (cells were cryopreserved and re-prepared with various concentrations of DMSO and trehalose. Cryostor-5 containing 5% DMSO was used as a control. Cell number, viability (Figure 3A), and metabolic activity (Figure 3B) were measured. The values ​​are the average (±SEM) of 9 replicates from 3 experiments with 0.2–0.6 M trehalose and Cryostor-5 controlled with 3 replicates from 1 experiment with 0.8 M trehalose). [Modes for carrying out the invention]

[0016] (Detailed explanation) (Terms and Definitions) The following description contains numerous details to help you understand this disclosure. However, Furthermore, the methods of this disclosure may be carried out without these details, and many of the embodiments described may be derived from the described embodiments. Those skilled in the art will understand that the numbers may be transformed or modified.

[0017] Firstly, in the development of any such actual embodiment, system-related constraints and To achieve specific goals for the developer, such as complying with business-related constraints, various implementations are required. Note that implementation-specific decisions regarding the number may be made. Furthermore, such development efforts may be complex. This may be rough and time-consuming, but nevertheless, for those skilled in the art who have an interest in this disclosure It will be understood that this is a routine task for them. In addition, the terms used / disclosed herein The composition being considered includes several components other than those cited (i.e., other cryoprotective substances excluded). It can also include (). In the summary and this detailed explanation, each number is expressed by the term "approximately". It should be read once as modified (unless already explicitly modified). Unless otherwise indicated in the context, it should be read again as unmodified. be.

[0018] Where used herein, the term "about" in relation to quantity refers to the stated value. It includes and has a meaning indicated by the context. For example, it is related to the measurement of a specific quantity. It includes at least the degree of error. When used in the context of a range, the modifier "about" means Furthermore, it is considered to disclose the range defined by the absolute values ​​of the two endpoints. It should be done. For example, if the range is "approximately 2 to approximately 4", the range "2 to 4" should also be disclosed.

[0019] Unless otherwise specified herein, the modifier "approximately" relating to temperature (°C) refers to the stated temperature. Or the temperature range, and the stated temperature or temperature range + / 1~4% (the stated temperature or (This refers to the end point of the temperature range.) Regarding cell viability and cell retention (%), see separately specified herein. Unless explicitly stated otherwise, the modifier "approximately" in relation to cell viability and cell retention (%) is used. This refers to the stated value or range of values, as well as the stated value or range of values ​​+ / 1 to 3%. If the expression has either parts per million (ppm) or parts per billion (ppb) units, Regarding the quantity, unless otherwise explicitly stated in this specification, the adjusting agent "about" is cell-derived. Regarding survival rate and cell retention rate (%), the stated value or range of value, and the stated value or This refers to a range of ±1 to 3%. Regarding the expression of content in units of μg / mL, this specification specifically refers to the range of ±1 to 3%. Unless otherwise specified, the modifier "approximately" in relation to values ​​in μg / mL refers to the stated value or range of the value. This refers to the range and the stated value or value range + / 1 to 4%. Regarding molar concentration (M), Unless otherwise specified in the specification, the modifier "approximately" with respect to molar concentration (M) refers to the stated value or This refers to the range of values, as well as the stated value or the range of values ​​+ / 1 to 2%. Cooling rate (°C / min) Regarding this, unless otherwise explicitly stated in this specification, the cooling rate (°C / min) is as follows: The modifier "approximately" refers to the stated value or range of values, as well as the stated value or range of values ​​+ / 1 This refers to approximately 3%.

[0020] Furthermore, in the summary and this detailed description, the categories listed or described as useful, appropriate, etc. The enclosure is considered to include at least all points within the range that include the endpoint, It is intended to include support for any conceivable sub-ranges within that range. For example, "the range of 1 to 10" means each possible number along the continuum from approximately 1 to approximately 10. It should be interpreted as indicating something. In addition, for example, + / 1~4% is a sequence of 1~4 This should be interpreted as indicating each possible number along the continuation. Furthermore, in this embodiment One or more data points may be joined together or used to create a range. It may be combined with one of the data points in the specification, and therefore within this range Each includes possible values ​​or numbers. Therefore, (1) a number of specific data points within a range are clearly defined. (2) Even if explicitly identified, even if a small number of specific data points within the range are referenced. , or (3) even if data points within the range are not explicitly identified, (i) the inventors It is important to recognize and understand that any possible data point within the range is considered to have been identified. (ii) The inventors have identified the entire range, each conceivable subrange within the range, and within the range It should be understood that you have knowledge of each possible point. Furthermore, this specification The subject matter of this application disclosed exemplary may include any elements not specifically disclosed herein. It can be properly implemented even if it does not exist.

[0021] Unless explicitly stated otherwise, "or" refers to an inclusive "or" and not an exclusive one. It does not mean "or". For example, condition A or B is satisfied by either of the following: : A is true (or exists) AND B is false (or does not exist), A is false (or does not exist) AND B is true (or exists), AND both A and B are true. (Or it exists.)

[0022] In addition, the use of "a" or "an" is used to describe the elements and components of the embodiments described herein. This is used for the purpose of [doing something]. This is merely for convenience and to give a general meaning to the concepts described herein. This is done for the purpose of [something]. This explanation should be read as including one or at least one of the following: Unless otherwise specified, the singular form includes the plural form.

[0023] The terms and expressions used in this specification are for illustrative purposes only and are not intended to limit their scope. It should not be interpreted as "including" or "com "prising", "having", "containing" The words "to be involved" or "to participate," and their variations, are used in a broad sense. The intention is that the following subjects, equivalents, and enumerated items are listed.

[0024] Furthermore, any reference to "one embodiment" or "embodiment" as used herein is prohibited. , at least one of the specific elements, features, structures, or properties described in relation to the embodiments This means that it is included in the embodiments. In various parts of this specification, "in one embodiment" means The appearance of such a phrase does not necessarily refer to the same embodiment.

[0025] As used herein, the term "room temperature" refers to a temperature of approximately 18°C ​​to approximately 25°C (at standard pressure). It refers to. In various examples, room temperature is approximately 18°C, 19°C, 20°C, 21°C, and 22°C. It could be approximately 23°C, 24°C, or 25°C.

[0026] As used herein, "cell material" or "cell sample" means that the material is natural or human-derived. Regardless of whether it is manufactured or not, biomaterials containing cellular components, whether natural or artificial This term still includes cells, tissues, and organs. Such terms also include cells, tissues, and organs, etc. This refers to any type of viable material that is cryopreserved. In some embodiments, this includes cells, tissues, and Mammalian organs (human organs, etc.), mammalian cells (human cells, etc.) and mammalian tissues ( It could be human tissue, etc.

[0027] As used herein, the term “cell” can mean, for example, stem cells, hematopoietic stem cells, phosphorus Pacocytes, leukocytes, T cells (and T cell subsets and CAR T cells), pancreatic islets, somatic cells (groups) (including all types of cells in tissue or organ), fibroblasts, keratinocytes, hepatocytes, heart Any type of cell, such as muscle cells, chondrocytes, smooth muscle cells, progenitor cells, oocytes, and germ cells. Includes cells. Such cells may form tissues or organs. Some embodiments Therefore, the cells are derived from mammalian tissues or organs, such as the human tissues or organs mentioned above.

[0028] Where used herein, “storage protocol” or “freezing protocol” means “living This refers to a process for preserving the storage life of cells containing biomaterials. Col can be preserved by freezing, vitrification, and / or freeze-drying or drying. This may include the preservation of anhydrous organisms.

[0029] As used herein, the term "freezing" refers to a preservation method that promotes ice formation. As the temperature decreases, not only physiological changes, such as those on water-forming ice, but also chemical changes occur. Freezing occurs, which subsequently affects the viability and survival of cells and tissues upon thawing. As the temperature drops, heat is removed, molecular processes slow down, and even before freezing, the cells This leads to various structural and functional changes. As a result, the cell becomes more resistant to further damage. Cascades of biochemical and biophysical changes that can make the body susceptible and cause irreversible damage. Experience the road.

[0030] When cells are cryopreserved by freezing, ice first forms in the extracellular space. It separates as ice crystals, and the residual solute is concentrated in the residual liquid phase. As a result, water is released into the extracellular space. To re-establish osmotic equilibrium within the cell, cells move across the plasma membrane to the extracellular space. If cooled too much, the time it takes for water to move out of the cell decreases, and intracellular ice forms, causing the cell to freeze. This causes irreparable damage. If cells cool too slowly, more Water is released from the cell, increasing the solute concentration inside the cell. This increase in solute concentration can lead to an increase in salt concentration, which can denature proteins and membranes, and precipitation of buffer solutions. pH changes, increased protein concentration that allows for cross-sectional possibilities, or structurally important water It is called "solution-effect" damage because it encompasses many changes, including simple removal. Furthermore, as it is pushed together by the forming ice, it concentrates at a slower cooling rate. The cells are then isolated in ice-free vitrification channels and stored at cryogenic temperatures. It can be stored at a certain temperature. Maximum cell viability is usually due to the balance between the risks of osmotic dehydration and intracellular ice formation. This is achieved with a moderate intermediate cooling rate. Rapid cooling allows for intracellular ice formation.

[0031] During rewarming, the process reverses, ice is replaced by water, and the cryoprotectant (CPA) is applied to the system. It is removed from the cell. However, physical and chemical changes are required to return the cell to physiological temperature. This can still cause damage. Recrystallization can occur as the sample is heated. Recrystallization is the process in which metastable ice crystals formed during freezing reform into larger crystals during rewarming. This is when the opportunity is given. These ice crystals are similar to crystals formed during freezing. It can potentially damage cells. Another concern during rewarming is the removal of the cryoprotectant. A was added to the sample before freezing, and for compounds like DMSO, water was removed from the cells. It replaces it. DMSO does not move across the cell membrane as easily as water, so it is uneven. A balance can occur, and as a result, the cells absorb water faster than the DMSO is removed, leading to swelling. There is a tendency for this to occur. If the swelling is too great, it can cause irreversible damage to the cells. Therefore, even if the freezing protocol works, if the unwinding is not properly controlled, the cells Survival is still not very good. All of these factors affect the overall survival of cells during cryopreservation. This affects existence. Therefore, optimization for a given cell type may be necessary (Ba ust JM, Campbell LH, Harbell JW. (2017) Best practices for cryopreserving, thawi In V itro Cell Dev Biol Anim. 53(10): 855-871 ).

[0032] As used herein, the term "vitrification" means without or substantially without ice crystal formation. This refers to solidification without the formation of ice crystals, but in cryopreservation by freezing, cells must be frozen. If frozen, it is stored in the vitrified channel within the sample. In some embodiments, it is stored The sample (e.g., tissue or cell material) is vitrified and / or frozen in glass form ( Overall, the process (from initial cooling to the end of rewarming) can be achieved without the formation of ice crystals. It can be vitrified. In some embodiments, the sample to be preserved (e.g., tissue or cell material) The samples (e.g., tissue or cell material) to be preserved will not solidify with substantial ice crystal formation. If possible, vitrification and / or glassy cryopreservation can be achieved. It is possible (i.e., vitrification and / or glassy cryopreservation (in its entirety, initial cold (From re-warming to the end of re-warming) a small or limited amount, less than the amount that would cause tissue damage. (This can even be achieved in the presence of a large amount of ice.)

[0033] When used herein, the sample to be preserved (e.g., organ, tissue, or cell material) When it reaches the glass transition temperature (Tg), it vitrifies. The vitrification process is carried out at nuclei on ice. As the temperature decreases, the viscosity of the antifreeze solution decreases, inhibiting formation and growth. This is accompanied by a significant increase. Generally, the lowest temperature at which a solution can be supercooled without freezing is A uniform nucleation temperature T at which ice crystals nucleate and grow. h Thus, crystalline solids are formed from the solution. The vitrification solution has a glass transition temperature T. g It has the property that at this temperature the solute vitrifies, Alternatively, it becomes an amorphous solid.

[0034] When used herein, "glass transition temperature" refers to the point at which the sample shifts more from the liquid phase to the solid phase. All molecular motion ceased, and a glass transition was observed in both the vitrified and frozen samples. This refers to the glass transition temperature of a solution or formulation under certain conditions. Generally, the methodology of this disclosure is used It is performed under physical pressure. However, the sample being preserved (e.g., tissue or cell material) Higher pressure can be used as long as it does not cause significant damage.

[0035] As used herein, “physiological pressure” refers to the pressure that tissue experiences during normal functioning. This refers to the following. Therefore, the term "physiological pressure" refers to normal atmospheric conditions, as well as angiogenic tissue, etc. This includes the higher pressures that various tissues experience under diastolic and systolic conditions.

[0036] As used herein, the term "sugar" may refer to any sugar. In application, sugars are polysaccharides. As used herein, the term "polysaccharide" means This refers to sugars that contain two or more monosaccharide units. In other words, the term polysaccharides includes disaccharides and trisaccharides. It contains oligosaccharides such as [list of oligosaccharides], but does not contain monosaccharides. Sugars also contain at least one polysaccharide. In some cases, it may be a mixture of sugars such as trehalose. In some embodiments, sugar (trehalose) (excluding) may be at least one member selected from the group consisting of disaccharides and trisaccharides. In some embodiments, the sugar (excluding trehalose) is a disaccharide such as sucrose. In some embodiments, the sugar (excluding trehalose) is a trisaccharide such as raffinose. (Apart from trehalose) Sugars include sucrose and / or raffinose and / or other disaccharides. Alternatively, a combination of trisaccharides may be used.

[0037] As used herein, the term “functional after cryopreservation” with respect to cryopreserved materials refers to the same term as used herein. , the cryopreserved material such as organs, tissues, or cells after cryopreservation is acceptable and / Or it means retaining the desired function. In some embodiments, cells after cryopreservation. The material retains all of its intended functions. In some embodiments, by the method of this disclosure The cryopreserved cell material preserved retains at least 50% of its intended function, for example, the intended function At least 60%, for example, at least 70% of the intended function, for example, less than the intended function At least 80%, for example, at least 90% of the intended function, for example, at least the intended function It retains 95%, for example, 100% of the intended function. For example, it preserves the viability of cells. In addition to this, the physiological function of cells and / or tissues / cells (for example, those to be transplanted) It may also be important for them to maintain / preserve their ability to integrate with the surrounding organization.

[0038] As used herein, the term "sterile" refers to living bacteria, microorganisms, and organisms that do not grow. This means that it does not include other organisms that can do the same thing.

[0039] As used herein, "substantially free of cryoprotective substances other than trehalose" The term refers to a quantity of cryoprotective substances (other than trehalose) less than 0.01 w / w%. In some embodiments, the method of this disclosure substantially does not contain DMSO (i.e., Cryoprotective agent (trehalose) for cell materials, etc. (DMSO is present in amounts less than 0.01 w / w%) Using and / or achieving a medium / solution and / or cell material that substantially does not contain (other than) This can be done. In some embodiments, the method of the present disclosure can be used with any additive other than trehalose. Using and / or achieving a medium / solution and / or cell material that is substantially free of cryoprotective agents Obtain. Other cryoprotective substances that can be excluded in this regard are those that allow cells to be immersed in a cryopreservation solution. Then, when frozen and stored at -80°C or below, one or more conventionally used cryoprotective materials Ingredients (usually added for their function): DMSO, glycerin, acetamide, agar - Alginate, alanine, albumin, ammonium acetate, antifreeze protein, pork Diols (2,3-butanediol, etc.), chondroitin sulfate, chloroform, choline cyclohexanediol, cyclohexanedione, cyclohexanetriol, dex Transyl benzoate, diethylene glycol, dimethylacetamide, dimethylformamide (n-di Methylformamide, dimethyl sulfoxide, erythritol, ethanol, ethyl Ethylene glycol monomethyl ether, formamide, glucose, Lycerol, glycerophosphate, glyceryl monoacetate, glycine, glycoprotein, hyaluronic acid Droxyethyl starch, inositol, lactose, magnesium chloride, magnesium sulfate, Maltose, mannitol, mannose, methanol, methoxypropanediol, methyl Cetoamide, methylformamide, methylurea, methyl glucose, methylglycerol phenol, pluronic polyol, polyethylene glycol, polyvinyl pyrrolid Proline, propanediol (etc.), 1,2-propanediol and 1,3-propanediol (Pyridiolic acid), pyridine N-oxide, raffinose, ribose, serine, sodium nitrate Sodium nitrite, sodium sulfate, sorbitol, triethylene glycol, acetic acid These may be trimethylamine, urea, valine, and xylose.

[0040] (Embodiment) This disclosure relates to a cryopreservation protocol using DMSO, glycerin / glycerol, and ethyl acetate. In the absence of any other conventional cryoprotectant such as cellulose glycol or propylene glycol, Methodologies containing rehalose (for example, at a rate of 1°C / min used in nucleated mammalian cells) (including a rapid cooling rate which is faster than conventional slow cooling) and composition, or Methodologies and compositions that do not contain and / or substantially contain cryoprotective agents other than halos. To include.

[0041] The cryopreservation method described herein uses trehalose. The sample to be preserved is DM In the absence of conventional cryoprotectants such as SO, immersion in a cryoprotectant mixture containing trehalose. It may be used in or perfused with it, or may not contain any cryoprotectant other than trehalose. The methodology and composition may be less than or substantially less than trehalose. Freeze-protecting formulations that do not contain, or substantially contain, externally added freeze-protecting agents. It may be immersed or perfused with it. The use of trehalose is for rapid cooling rate and Related to this, the rapid cooling rate is over 1°C / min to approximately 80.0°C / min (for example, approximately 37°C to 0°C). Temperatures in the range of 0°C to approximately -80°C or below, or in the range of approximately 37°C to 0.0°C to approximately -135°C or below. During cooling from a certain temperature, or approximately 3°C / min to approximately 50.0°C / min (for example, approximately 37°C to 0.0°C) Temperatures in the range of ℃ to approximately -80℃ or below, or in the range of approximately 37℃ to 0.0℃ to approximately -135℃ or below. During cooling from a certain temperature, or at a temperature in the range of approximately 10°C / min to approximately 30°C (for example, approximately 3 From temperatures in the range of 7°C to 0.0°C to approximately -80°C or below, or from approximately 37°C to 0.0°C to approximately -13°C From temperatures in the range of 5°C or below, or in the range of approximately 15°C / min to approximately 25.0°C / min (for example) Cooling from approximately 37°C to below -80°C, or between approximately 37°C and below -135°C. middle).

[0042] In some embodiments, rapid / rapid cooling is performed when the cells are transferred to a freezer at their final storage temperature. Before that, it may be carried out by plunge freezing into liquid nitrogen.

[0043] In the method disclosed herein, the metabolic activity of the stored cell material is maintained for 6 hours after preparation. Within 24 hours of being ready, or within 48 hours of being ready, or ready Within 96 hours, the control value (i.e., without an intermediate washing step after thawing) was obtained; however This allows for complete recovery (reducing processing time and variability). The control value is stored. Cell material exposed to trehalose preparations in growth media suitable for a specific tissue, and the same cell type. The fresh cell material is evaluated / set. The recovered metabolic activity is preserved by the method disclosed herein. If cryopreserved cell material is intended for research or therapeutic use (e.g., transplantation), Until it is put into use (for example, several hours, several days, or at least three days, or less) It will be maintained for a period of at least 5 days, or at least 7 days, etc.

[0044] In embodiments, the present disclosure describes how trehalose is used in the absence of conventional cryoprotectants (such as DMSO). Freeze-protection compositions that do not contain cryoprotective agents other than trehalose, or are substantially free of such additives. A cryoprotective composition that does not contain any of the above and has minimal damage to tissue / cell material. This document describes a cryoprotection composition effective for thawing cryopreserved samples containing textile / cellular materials. Protective agents / formulations are any other materials suitable for cryopreservation of biological materials (other than additional cryoprotective agents). It may contain (other than additional sugars).

[0045] The method disclosed herein involves cell materials (e.g., stem cells, hematopoietic stem cells, lymphocytes, leukocytes, T cells). (and T cell subsets and CAR T cells) and pancreatic islets, etc.) are protected by conventional cryoprotective agents (DMS) Contact with a cryoprotectant solution containing an effective amount of trehalose in the absence of (e.g., O). Includes. In some specific embodiments, during cooling and rewarming, cells of the cell material (e.g., Stem cells, hematopoietic stem cells, lymphocytes, white blood cells, T cells (and T cell subsets and CAR T A disaccharide (for example, At least one other sugar, such as sucrose, may also be present in the cryoprotectant formulation / solution.

[0046] In some embodiments, the cell cryopreservation material preserved by the method of the present disclosure (e.g.) , stem cells, hematopoietic stem cells, lymphocytes, leukocytes, T cells (and T cell subsets and CAR T cells and pancreatic islets, etc., perform at least 50% of their intended function, for example, at least the intended function 60%, for example, at least 70% of the intended function, for example, at least 8% of the intended function 0%, for example, at least 90% of the intended function, for example, at least 95% of the intended function For example, it retains 100% of the intended function.

[0047] In the embodiment, the formulation / solution / culture medium containing trehalose is used in the desired dose (effective dose, etc.). The presence of trehalose on / in cells or tissues improves survival rates (after cryopreservation). Store for any desired period, such as until heated and / or until tissue damage during heating is prevented / protected. It can be brought into contact with the sample.

[0048] In some embodiments, the cells to be cryopreserved are, for example, in at least a basic salt solution. An energy source (e.g., glucose) and a slow-release system that can maintain a neutral pH at a cooling temperature. It may be in contact with a freeze-compatible pH buffer consisting of a buffer. Known materials of this type include For example, Dulbecco's modified Eagle medium (DMEM) is one such example. This material can also be frozen. It may be included as part of a preservative composition. For example, Campbell et al. ”Cryopreservation of Adherent Smooth Mu scle and Endothelial Cells with Disaccha rides,” In: Katkov I. (ed.) Current Fron tiers in Cryopreservation. Croatia: In T ech (2012); and Campbell et al., "Developme nt of Pancreas Storage Solutions: Initia l Screening of Cytoprotective Supplement s for β-cell Survival and Metabolic Stat us after Hypothermic Storage,” Biopreser vation and Biobanking 11(1): 12-18 (2013 See ( ). Each of these disclosures is incorporated herein by reference in its entirety.

[0049] In some embodiments, trehalose and / or any other sugars (if present) The total amount of trehalose and other sugars may be added to the cryopreserved composition in any effective amount (i.e., (In the absence of conventional cryoprotectants (such as DMSO), for example, from approximately 100 mM to approximately 900 mM M, about 150mM to about 800mM, about 200mM to about 700mM, about 250mM to about 60 It may exist at 0 mM, approximately 275 mM to approximately 500 mM, and approximately 300 mM to approximately 450 mM.

[0050] The cryopreservation composition may also contain a solution well suitable for the storage of cells, tissues, and organs. (or may be based on it). The solution may contain well-known pH buffers. In several embodiments, the solution may be, for example, dextrose, monobasic and dibasic phosphorus. Eurocholine solution is composed of potassium acid, sodium bicarbonate, and potassium chloride. However, Taylor et al., "Comparison of Unis ol with Euro-Collins Solution as a Vehic le Solution for Cryoprotectants,” Transp Lantation Proceedings 33: 677-679 (2001) It is described in [the relevant document]. That disclosure is incorporated herein by reference in its entirety. The cryoprotectant solution is Unisol, Hypothermosol (BioLife S Formulations in alternative solutions such as olutions and Lifor (Detraxi, Inc.) It may be converted.

[0051] The cells in the cell material that may be used in the method of this disclosure are any suitable cell composition. In some embodiments, cells may be stem cells, hematopoietic stem cells, lymphocytes, leukocytes, T cells. Cells (and T cell subsets and CAR T cells), skin cells, keratinocytes, skeletal muscle cells Cells, cardiomyocytes, lung cells, mesenteric cells, adipocytes, stem cells, hepatocytes, epithelial cells, Kupffer cells Cells, fibroblasts, neurons, cardiomyocytes, muscle cells, chondrocytes, pancreatic acinar cells, Langerhans Islets of Bruns, osteocytes, myoblasts, satellite cells, endothelial cells, adipocytes, preadipocytes, bile ducts It may be a combination of cutaneous cells and progenitor cells, or any combination of these cell types. In embodiments, such cells / tissues used in the methods of the present disclosure may be any appropriate movements Species, for example, humans, dogs (for example, dogs), cats (for example, cats), horses (for example, horses) It may be derived from mammals such as pigs, sheep, goats, or cattle.

[0052] Once the cryopreservation composition is prepared (and any other conventional cryoprotective agents added (e.g.) For example, DMSO, glycerin / glycerol, ethylene glycol, propylene glycol When trehalose associates with the cellular material being preserved in the absence of (etc.), for cryopreservation The cooling can be carried out at the above rapid cooling rate (for example, faster than conventionally used with DMSO). When a fast cooling rate is used, trehalose alone does not need to be placed inside the cell. That is, when used in the culture medium surrounding the cell material being stored (extracellular trehalose). ), any additional materials may be used in addition to the above. Cell materials listed below Additional materials such as those discussed in the protocol for preserving the following patents and publications :Fahy et al., U.S. Patent No. 6,395,467; Wowk et al., U.S. Patent No. 6,194, Patent No. 137; Fahy et al.'s U.S. Patent No. 6,187,529; Toner et al.'s U.S. Patent No. 6 ,127,177; Fahy et al. U.S. Patent No. 5,962,214; Calaco et al. U.S. Patent No. 5,955,448; Beattie et al.'s U.S. Patent No. 5,827,741 ; U.S. Patent No. 5,648,206 by Goodrich et al., U.S. Patent No. 5,648,206 by Khirabadi et al. 225A2, corresponding to application No. 09 / 691,197. Its disclosure is by reference in its entirety. This is incorporated herein.

[0053] The cryopreservation portion of the preservation protocol typically keeps cells / tissues well below their freezing point in water. For example, this includes cooling to a temperature of approximately -80°C or lower, more typically to approximately -135°C or lower. A freezer known to those skilled in the art (i.e., capable of achieving the desired rapid / rapid cooling rate) Any method of preservation can be used. For example, a cooling protocol for cryopreservation. The freezing temperature is lower than approximately -20°C (i.e., low temperature), for example, approximately -80°C or is lower than that (i.e., low temperature), or about -135°C or lower (i.e., It can be any suitable type, which may be low temperature.

[0054] In some embodiments, the storage protocol is set from a temperature control start point (+4 to -30°C) to -8 This may include continuous controlled-rate cooling to 0°C or any of the cooling temperatures disclosed above. The rapid cooling rate is set according to the characteristics of the cells / tissues being cryopreserved. For example, cryopreservation The cooling protocol for this is greater than approximately -1.0°C / min, greater than approximately -4.0°C / min. Or greater than approximately -6.0°C / min, or greater than approximately -8.0°C / min, or approximately -10.0°C / min Greater than °C / min, or greater than approximately -14.0 °C / min, or greater than approximately -25.0 °C / min Speeds greater than -30°C / min, for example, greater than -35°C / min (and / or flat It may be by a uniform cooling rate, or by rapid freezing in liquid nitrogen. good.

[0055] Cooling rate (and / or average cooling rate), e.g., continuous rate cooling (or other types of cooling) For example, temperatures of approximately -1 to -80°C per minute, approximately -3 to -50°C per minute, and approximately -5 to -35°C per minute. , approximately -7 to -30°C per minute, or approximately -10 to -25°C per minute, or approximately -4 to -10 °C, approximately -4°C to approximately -8°C per minute, approximately -4 to approximately -6°C per minute, approximately -6 to approximately -10°C per minute, approximately -6 to approximately -9°C, approximately -6 to approximately -8°C per minute, approximately -6 to approximately -7°C per minute, or approximately -7 to approximately - It may be 10℃, approximately - per minute. 7 to approximately -9℃ per minute, approximately -7 to approximately -8℃ per minute, approximately - It can be 8 to approximately -9°C, or approximately -9 to approximately -10°C per minute.

[0056] Samples to be preserved (e.g., cellular material and / or tissues) will be approximately - When cooled to 40°C to below -80°C, they typically do not reach the glass transition temperature of the frozen liquid. To further cool it to its full freezing temperature, it is transferred to liquid nitrogen or the gas phase of liquid nitrogen. Obtain. The sample to be stored (e.g., cell material and / or tissue) is further cooled to the freezing temperature. Before discarding, check the temperature ranges: approximately -40°C to approximately -75°C, approximately -45°C to approximately -70°C, and approximately -50°C to approximately -60°C. °C, approximately -55°C to approximately -60°C, approximately -70°C to approximately -80°C, approximately -75°C to approximately -80°C, approximately - 40℃ to approximately -45℃, approximately -40℃ to approximately -50℃, approximately -40℃ to approximately -60℃, approximately -50℃ to The sample may be cooled to approximately -70°C, or to approximately -50°C to approximately -80°C. Alternatively, if the sample is desired It may be cooled to -120°C before further cooling to the freezing temperature.

[0057] In embodiments, the heating method may be used to heat the sample. This may include, for example, convection, electromagnetic, and microwave heating.

[0058] In embodiments, cryopreserved cell materials preserved by the method of the present disclosure are, for example, research. Alternatively, on-demand use as a therapeutic measure and / or medical measure and / or regenerative medicine. This includes creating a large supply of cryopreserved cell material (hMSCs, etc.) for use in various applications. It can be used for appropriate purposes. With regard to therapeutic use, cryopreserved cell material can be used for disease or condition It may be administered to human or animal patients to treat or prevent the condition. For example, it may be cryopreserved. If the cell material is hMSC, the cryopreserved cell material is suitable for allogeneic hMSC transplantation. This will improve severe health disparities, especially in children (Donnenberg AD, Gorantla VS, Schneeberger S, Moore LR, Brandacher G, Stanczak HM, Koch EK, Lee WA. Clinical implementation of a procedure re to prepare bone marrow cells from cad averic vertebral bodies. Regen Med. 2011 ;6(6):701-6; Gragert L, Eapen M, William s E, Freeman J, Spellman S, Baitty R, Ha rtzman R, Rizzo JD, Horowitz M, Confer D , Maiers M. HLA match likelihoods for he matopoietic stem-cell grafts in the US registry. N Engl J Med. 2014;371(4):339 -48; Ustun C, Bachanova V, Shanley R, Ma cMillan ML, Majhail NS, Arora M, Brunste in C, Wagner JE, Weisdorf DJ. Importance of donor ethnicity / race matching in unr elated adult and cord blood allogeneic h ematopoietic cell transplant. Leuk Lymph oma. 55(2):358-64, 2014) - Racial and ethnic minorities Therefore, the likelihood of finding a match in the bone marrow registry for hMSCs is extremely low (Grage (See rt, 2014).

[0059] Cryopreserved cell material can be administered to patients in any appropriate manner. In one embodiment, cryopreserved cell material is applied locally to a patient (for example, to burns, wounds, or It can be delivered (in the treatment of skin disorders). In some embodiments, cryopreserved filtrates The cellular material can be delivered to the local implant site within the patient, or by intravenous injection. Any of these administration methods, or any combination thereof, in the treatment of the patient It can be used for this purpose. [Examples]

[0060] Using bone marrow-derived human mesenchymal stem cells (hMSCs) from various commercially available sources, hBM-MSCs were isolated from normal, healthy adult donors. (Purchased from a supplier (Rooster-Bio, etc.) along with appropriate growth medium.) They were propagated according to the manufacturer's instructions.

[0061] Many studies have been conducted using trehalose as a cryoprotective agent (CPA), but In most cases, trehalose is not used as a primary CPA, but is typically used as a cryoprotectant. It is used as part of a cocktail. Efforts to use trehalose as a primary CPA are Trehalose is introduced into cells in various ways so that it is present on both sides of the membrane. This mainly includes (Stewart et al., Intracellular Delivery of Trehalose for Cell Banking, Langmuir, 2019, 35(23): 7414-7422)(Table e 1) Our previous research has opened up a method for introducing trehalose into cells before preservation. It included the act of emitting (Brockbank et al., Lessons fr om nature for preservation of mammalian cells, tissues, and organs, In Vitro Cel l. Dev. Biol., 2011; U.S. Patent No. 8,017,311; Cam pbell et al., Cryopreservation of Adhere nt Smooth Muscle and Endothelial Cells w ith Disaccharides, Current Frontiers in Cryopreservation, 2012; Campbell et al., Comparison of electroporation and Chari ot(trademark) for delivery of β-galactosidase i nto mammalian cells: strategies to use t rehalose in cell preservation, In Vitro Cell. Dev. Biol., 2010; Campbell et al., Culturing with trehalose produces viabl e endothelial cells after cryopreservati on, Cryobiology 64 (2012) 240-244). Disaccharide cells. For various other methods identified in the literature that may lead to internal delivery, see the table. Please refer to II (below).

[0062] [Table 1]

[0063] Prior to the development of the methodology disclosed herein, the need to have trehalose on both sides of the cell membrane was deemed necessary for cryopreservation. It has long been considered the best strategy for maximizing protection with the remaining trehalose. Stewart et al., Intracellular Delivery o f Trehalose for Cell Banking, Langmuir, 2019, 35(23): 7414-7422). Introducing trehalose into cells Despite the development of several methods, each protocol has its drawbacks, and these methods The results regarding whether it can consistently protect cells are mixed. Research is also being conducted using trehalose, which has a cooling rate (>50°C / min) (Heo et al., “Universal” vitrification of ce lls by ultra-fast cooling, Technology (S ingap World Sci), 2015, 3(1), 64-71; Lie bermann et al., Potential Importance of Vitrification in Reproductive Medicine, Biology of Reproduction, Volume 67, Issue e 6, 2002, pages 1671-1680). However, faster cooling The rejection rate was used in conjunction with small amounts (≤300 μL), and as a result, these samples were glassed. It is condensed (without ice formation). These types of protocols are mainly for reproductive tissue processes. Used, typically with the use of a cryoprotectant cocktail containing trehalose. Slightly slow cooling. The decongestion rate (-5 to -60°C / min) is also used, but these studies are based on the interaction with DMSO. This includes the use of trehalose as a supplementary cryoprotectant in the formula (Barbas et al., Cryopreservation of domestic an imal sperm cells, Cell and Tissue Bankin g, 2008, 10(1), 49-62), on the other hand, the approach used in this disclosure Extracellular trehalose is used as a cryoprotective agent for sole.

[0064] Prior to the development of the methodology of the present invention, the inventors of this application were Xiaoming He Merley Professor at RAND University (Zhang et al., Cold-Responsive Na noparticle Enables Intracellular Deliver y and Rapid Release of Trehalose for Org anic-Solvent-Free Cryopreservation, Nano Lett. 2019, 19, 9051-9061; Rao et al., Nanoparticle-Mediated Intracellular Deli very Enables Cryopreservation of Human A dipose-Derived Stem Cells Using Trehalos e as the Sole Cryoprotectant, ACS Appl M (ater Interfaces, 2015, 7(8), 5017-5028) Therefore, we previously evaluated the nanotechnology developed for intracellular trehalose delivery, and bone Marrow-derived MSCs. Unfortunately, trehalose nanotechnology is likely from the University of Maryland. Instability of trehalose nanoparticles during storage and transport from Charleston, SC. These studies failed for this reason. During these studies, MSCs were found to be trehalose in cells. If it is present only externally and used as a negative control, it is fairly well preserved by freezing. Survival was observed. Due to these unexpected results, the exogenous trehalose used The range was expanded to 0.2~0.8M, compared with cooling rates of -1, -5 and -15°C / min. Ta.

[0065] At cooling rates of -5 and -15°C / min, the 0.2M trehalose group reacts with DMS at each cooling rate. The group had the same or higher survival rate as the control group. Figure 2 shows the pooled results of the two experiments. The results shown are in the absence of trehalose and at DMSO versus DMSO-only control concentrations. This shows the effect of cooling rate on the survival rate of hMSCs after cryopreservation, and the data are mean ±1 standard error. It is shown as a difference.

[0066] In preliminary experiments, DMSO control was clearly the best at a cooling rate of 1°C / min (untreated) With a tolerance of 65.3 ± 2.7%, the best overall result was 0.2M at a cooling rate of -15℃ / min. The rehalose group was the largest group (78.9 ± 3.5% of the untreated control). The DMSO group was the largest group at -1°C / min. A statistically significant difference was observed between the trehalose group (-15°C / min) and the T-test group. (p<0.05, Figure 2). These results suggest that the unexpected extracellular trehalose benefit is greater than that of DMSO. It offers better rapid cooling rates than the ±1°C / min typically used for cell cryopreservation by freezing. This demonstrated that.

[0067] Similar results were obtained for exogenous training of pan-T cells at a cooling rate of -15°C / min. Obtained using halos, and this methodology is discussed for other cellular materials (e.g.) This is expandable to include, but is not limited to, T cell subsets and CAR T cells. This reflects what is expected to happen.

[0068] Extracellular trehalose in combination with several concentrations of 0-5% DMSO (0.2-0.0%). Experiments were conducted to evaluate the 8M concentration, and a positive control containing 5% DMSO was used. It was compared with stor-5.

[0069] Pan-T cells were grown and then harvested and counted. Approximately 10 × 10 6 Individual cells were used for each sample. The cells were mixed with DMSO and trehalose in 1 mL of water. The samples were resuspended in various combinations of -15°C and equilibrated on ice for 20 minutes. It was cooled at ℃ / min ~ -80℃, and then transferred to liquid nitrogen vapor phase storage for approximately 10 days. After storage, The sample was rapidly thawed in a 37°C water bath, transferred to a 15 mL centrifuge tube, and then mixed with 10 mL of culture medium. Diluted with . Aliquots were taken for cell counting. Then the cells were pelleted and 3 Resuscitation was resuspended in mL of culture medium and placed in a 12-well plate, 1 sample per well. Resazurin Before measuring viability using the dye (300 μl), the cells were placed in a 37°C incubator. The cells were allowed to recover for 60 minutes. After leaving the cells at 37°C for 3 hours, the excitation wavelength was 544 nm, and the emission wave was... The plate was read using a 590 nm fluorescence microplate reader. Cell counting This was done by mixing a fixed amount of 20 μl of cells with 20 μl of trypan blue. We obtained counts of both living and dead cells. We repeated this experiment 2-3 times and combined the results. The results are shown in Figures 3A and 3B.

[0070] In Figures 3A and 3B, the combination of DMSO and trehalose was used at -15°C / Cell survival after cryopreservation in minutes. Cells were cryopreserved and subjected to various concentrations of DMSO and trehalose. The samples were re-prepared using the solution. Cryostore-5 containing 5% DMSO was used as a control. The following parameters were used: cell count, viability (A), and metabolic activity (B). The values ​​ranged from 0.2 to 0. Mean (±SEM) of 9 replicates from 3 experiments with 6M trehalose and 0.8M trehalose This is a Cryostor-5 control study involving three replicates of a single experiment using a halo.

[0071]

number

[0072] These experiments demonstrated that the presence of DMSO is not required.

[0073] In other words, trehalose alone provides adequate protection, similar to that of the control Cryostor-5. The following was provided. Statistical analysis of cell count showed no significant difference between the control group and the experimental group, except in two cases. This was not shown. Regarding the number of viable cells (Figure 3A), control and 5% containing 0.2M trehalose were used. Between DMSO (p<0.05), the number of dead cells (Figure 3A) was controlled and 0.2M trehalose. A significant difference was observed between 0% DMSO (p<0.05) containing rose. The analysis excluded 0.2M trehalose (p>0.05), which includes 0.2M trehalose. No statistically significant difference was observed in any of the groups containing DMSO (Figure 3B). Comparisons between groups containing different concentrations of DMSO were made using 0% DMSO containing 0.2M trehalose. , 1% DMSO containing 0.2M and 0.6M trehalose, and 0.2M trehalose Excluding the group containing 5% DMSO (p>0.05), no significant differences were observed. This was the most interesting and unexpected finding. The group that did not exhibit the desired results was the group that was preserved using only trehalose without DMSO. No attempts were made to introduce trehalose into cells deemed necessary in the literature. Rehalose, except for the lowest exogenous concentration, showed similar cell count, viability, and metabolism to the positive control. It showed activity.

[0074] These experiments showed that exogenous trehalose alone can protect cells during cryopreservation. We demonstrated the ability to create a suitable alternative cryoprotective agent to DMSO. Trehalose is Compared to DMSO, it has fewer side effects on patients, and the cells are thawed without any intermediary steps. It is anticipated that it may later be injected directly into the patient.

[0075] All documents and patent references cited throughout this disclosure are incorporated by reference in their entirety. The above description is included. The above description is provided herein with reference to specific means, materials, and embodiments. However, this specification is not intended to be limited to the details disclosed herein, but rather to include additional information. This patent covers all functionally equivalent structures, methods, and uses as described in the attached claims. Furthermore, although only a few exemplary embodiments are described in detail above, those skilled in the art will understand that... Regardless of the storage protocol, this substantially deviates from the disclosure of storage methods using trehalose. Without needing to do so, it is easy to understand that many modifications are possible in the exemplary embodiments. Therefore, all such modifications are defined in the following claims. In the claims, means plus fun The action clause is a structure described herein as performing the listed functions, and It is intended to cover not only structural equivalents but also equivalent structures. Therefore Nails and screws are not structurally equivalent in that they both employ a cylindrical surface to fasten wooden parts together. However, while screws use a spiral surface, in environments where wooden parts are secured, nails and screws have similar structures. It is permissible to explicitly use the word "means" along with the function to which the patent claim relates. Except for the above, any limitation of the claims herein is 35 US The applicant's explicit intention is not to invoke Section C §112(f).

Claims

1. A method for preserving cellular materials, as follows: A step of subjecting cell material containing living cells to a cryopreservation protocol, The aforementioned cryopreservation protocol does not include any cryoprotective agents other than trehalose, Tsu The aforementioned cryopreservation protocol is as follows: The cell material contains an effective amount of trehalose to act as a cryoprotective agent. Exposure to cryoprotective agent preparations, The cell material is cooled to below -20°C at a cooling rate in the range of -3°C / min to -50°C / min. To cool to a constant temperature, and Obtaining warmed cryopreserved cell material including, Process; Includes, Percentage of cell viability of the cryopreserved cell material after the cryopreserved cell material has been warmed. is greater than 50%; method.

2. The method according to claim 1, wherein the effective amount of trehalose is in the range of 200 to 800 mM. Law.

3. The method according to claim 1, wherein the cooling rate is in the range of 5°C / min to -30°C / min.

4. The cryopreservation protocol involves preserving the cell material at a temperature of -80°C or below for a predetermined period of time longer than one hour. The method according to claim 3, comprising storing for a specified period of time.

5. The cryopreservation protocol involves preserving the cell material at -135°C or below for a predetermined time longer than one hour. The method according to claim 3, comprising storing for the duration of the period.

6. The method according to claim 1, wherein the cell material includes T cells.

7. The method according to claim 1, wherein the cell material includes mesenchymal stem cells.

8. The method according to claim 7, wherein the mesenchymal stem cells are human mesenchymal stem cells.

9. Cell survival of the cryopreserved cell material after the cryopreserved cell material has been heated. The method according to claim 1, wherein the percentage is at least 60%.

10. Cell survival of the cryopreserved cell material after the cryopreserved cell material has been heated. The method according to claim 1, wherein the percentage is at least 70%.

11. A method for preserving cellular materials, as follows: A step of subjecting cell material containing living cells to a cryopreservation protocol, wherein the cryopreservation The protocol is as follows: The cell material is exposed to a cryoprotective agent formulation containing an effective amount of trehalose and then frozen. To act as a protective agent, The cell material is cooled to a predetermined temperature of less than -20°C at a cooling rate in the range of -3°C / min to -50°C / min. To cool to a certain temperature, and Obtaining warmed cryopreserved cell material including, Process; Includes, Cell survival of the cryopreserved cell material after the cryopreserved cell material has been heated. The rate (cell viability (%)) is greater than 50%, The cryopreservation protocol involves DMSO, glycerin, acetamide, agarose, and algin. Salts, alanine, albumin, ammonium acetate, antifreeze protein, butanediol, 2 ,3-butanediol, chondroitin sulfate, chloroform, choline, cyclohexanediol ol, cyclohexanedione, cyclohexanetriol, dextran, diethylene Glycol, dimethylacetamide, dimethylformamide, n-dimethylformamide Dimethyl sulfoxide, erythritol, ethanol, ethylene glycol, ethylene Glycol monomethyl ether, formamide, glycerin, glycerophosphate, monoacetate Lyceryl, glycine, glycoprotein, hydroxyethyl starch, inositol, lactose, Magnesium chloride, magnesium sulfate, maltose, mannitol, mannose, methano methylacetamide, methylformamide, methylurine Phenol, methyl glucose, methylglycerol, phenol, pluronic polyol, Polyethylene glycol, polyvinylpyrrolidone, proline, 1,2-propanediol and 1,3-propanediol, pyridine N-oxide, raffinose, ribose, serine Sodium nitrate, sodium nitrite, sodium sulfate, sorbitol, triethylene sulfate It does not contain any additives of lycoprazole, trimethylamine acetate, urea, valine, or xylose. stomach, method.

12. The effective amount of trehalose is in the range of 200 to 800 mM, and the cryopreservation protocol Kor cools the cell material at a cooling rate in the range of -5°C / min to -30°C / min. The method according to claim 11, including the method according to claim 11.

13. The cryopreservation protocol involves preserving the cell material at -135°C or below for a predetermined time longer than one hour. The method according to claim 12, comprising storing for the duration of the period.

14. The method according to claim 13, wherein the cell material includes T cells.

15. The cell material includes mesenchymal stem cells, and the mesenchymal stem cells are human mesenchymal stem cells. The method according to claim 11.

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