Capillary vitrification methods and materials for preserving biological samples

Abstract

To provide an improved vitrification method for vitrifying a biological substance by high-speed drying while preserving proteins and nucleic acids.SOLUTION: A method of preserving a biological sample comprises providing a biological sample comprising at least one cell, contacting the biological sample with a vitrification medium comprising a vitrification agent and a lysis agent to form a vitrification mixture, and vitrifying the vitrification mixture to produce a storage stable sample.SELECTED DRAWING: Figure 2

Description

Related Applications

[0001] This application is derived from and claims priority to U.S. Provisional Application No. 62 / 982,856, filed February 28, 2020, the entire contents of which are incorporated herein by reference. [Technical Field]

[0002] The present disclosure relates to the preservation of biological samples, and in particular to the vitrification of biological material for the preservation of blood, saliva or tissue samples or portions thereof. [Background technology]

[0003] Vitrification, the process of directly transitioning from a liquid to an amorphous glassy state, is commonly used for the preservation of biological materials by cooling them to cryogenic temperatures at rapid rates. By using cryogenic temperatures, vitrification avoids the damaging effects of ice crystals known to form during conventional cryopreservation. However, to prevent ice nucleation during cooling, extremely high concentrations (6–8 M) of cryoprotectants (CPAs) are required, which can be toxic. The most commonly used CPAs include dimethyl sulfoxide (DMSO), glycerol, ethylene glycol (EG), and 1,2-propanediol (PROH). Consequently, the uptake and removal of CPAs from cells requires numerous steps and complex, precise procedures. Summary of the Invention [Problem to be solved by the invention]

[0004] Anhydrous vitrification at ambient temperature offers an alternative strategy for preserving biological materials. In nature, various organisms can survive extreme dehydration, often associated with the accumulation of large amounts (up to 20% of the dry weight) of glass-forming sugars, such as trehalose and sucrose, in the intracellular space. However, long-term preservation by dry storage has a major limitation: biological materials degrade due to cumulative chemical stress caused by the high concentration of the vitrification solution in the extracellular space. This means that irreversible cellular damage, including damage to proteins and nucleic acids, occurs before the cells and vitrification solution reach a low enough water content for vitrification. Therefore, improved vitrification methods that rapidly dry biological materials while preserving proteins and nucleic acids are desirable. [Means for solving the problem]

[0005] The following summary is intended to facilitate understanding of the various aspects described herein, but is not intended to be a complete description, which will be best understood when taken together with the specification, claims, drawings, and abstract.

[0006] Various aspects of the present disclosure provide methods for preserving proteins (particularly intracellular nucleic acids) or other biological material from cells, blood, saliva, tissue, or other samples of an organism or from the biological sample. The methods include providing a biological sample containing at least one cell containing nucleic acids, contacting the biological sample with a vitrification medium containing a vitrification agent and a lysis agent to form a vitrification mixture, and vitrifying the vitrification mixture to produce a storage-stable sample. In various aspects, the storage-stable sample can be stored at temperatures higher than cryogenic temperatures, such as at or above room temperature, and optionally for periods of 20 days or more.

[0007] Also provided is a method for preserving tissue cells that provides structural support while preventing the infiltration of materials that could damage the cells, thereby more effectively preserving cellular structure for future histological or other tissue studies. The method comprises contacting a target biological tissue with a polymer and a vitrification agent, and optionally also contacting the target biological tissue with a crosslinking agent and / or energy suitable for linking the polymer to at least one component of the extracellular structure of cells within the tissue, and vitrifying the tissue to form a vitrified tissue sample, optionally also cutting the tissue into at least one tissue slice (either before or after vitrification). Optionally, the method further comprises rehydrating the vitrified tissue using a releasing agent and / or energy that releases the polymer from the tissue, thereby rendering the highly preserved, viable tissue available for analysis.

[0008] The drawings are not necessarily to scale, and some structures may be exaggerated or reduced in size for the purpose of illustrating the details of particular components. Therefore, specific structural and functional details disclosed herein should not be construed as limiting the invention, but merely as representative examples for teaching those skilled in the art various ways of utilizing the invention. The illustrated embodiments will be better understood by considering the detailed description and the accompanying drawings. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram illustrating an example of a vitrification method that may be employed in one or more embodiments illustrated and described herein. [Figure 2] FIG. 1 is a schematic diagram illustrating another example of a vitrification method according to one or more embodiments illustrated herein, depicting a biological sample comprising blood cells and a vitrification medium for vitrification within a membrane. [Figure 3]FIG. 1 is a schematic diagram illustrating an example method for extracting RNA from a storage-stable sample produced according to one or more embodiments illustrated and described herein. [Figure 4] FIG. 1 illustrates an example system for isolating a desired substance, optionally separating it from contaminants such as bacteria or other unwanted organisms. [Figure 5] Electrophoresis gel images showing the degradation of RNA extracted from Comparative Examples A-C, which used conventional nucleic acid preservation techniques, and Example 1, which used one or more embodiments of the nucleic acid preservation method illustrated and described herein. [Figure 6A] 1 is a graph showing the GAPDH cycle threshold values ​​of Example B and Comparative Examples D and E according to one or more embodiments illustrated herein. [Figure 6B] 1 is a graph showing GAPDH cycle threshold fold change for Example B and Comparative Examples D and E according to one or more embodiments illustrated herein. [Figure 6C] 1 is a graph showing VEGF mRNA fold change for Example B and Comparative Examples D and E according to one or more embodiments illustrated herein. DETAILED DESCRIPTION OF THE INVENTION

[0010] Various embodiments described herein provide methods for preparing and preserving cells, tissues, or cellular materials, such as proteins (e.g., antibodies or other proteinaceous materials) and nucleic acids, using a vitrification medium containing a vitrification agent and, optionally, a lysis agent. In various embodiments, a storage-stable sample can be produced by contacting a biological sample containing at least one cell containing nucleic acid with the vitrification medium and vitrifying it. Specifically, in various embodiments, preservation or storage of nucleic acids at temperatures above cryogenic temperatures, particularly at or above room temperature, can maintain biological materials in a robust and highly preserved state. Such embodiments not only significantly reduce preservation costs by, for example, eliminating the need for refrigeration or freezing, but also simplify sample preparation for storage without adversely affecting the quality of nucleic acids (e.g., DNA and / or RNA). This allows for future reconstitution of high-quality tissues, cells, proteins, and nucleic acids, such as RNA and DNA.

[0011] Additionally, various embodiments described herein may allow for the processing and storage of saliva, blood, tissue, or other biological samples with minimal pre-processing. After storage, the sample may be used for any of a wide variety of purposes. For example, tumor tissue samples may be stored long-term and later processed according to appropriate procedures, such as for RNA mapping analysis. As another example, whole blood samples may be stored without separating plasma from red and white blood cells, allowing for future use of any or at least one component of the stored sample.

[0012] As used herein, the following terms or phrases, in one or more aspects, are intended to have the exemplary meanings set forth below.

[0013] The term "amorphous" or "glass" refers to a non-crystalline material in which there is no long-range order of atomic positions (order parameter: 0.3 or less). The change from liquid to glassy solid occurs at the glass transition temperature T gIn some embodiments, the vitrification medium can be or form an amorphous material. In other embodiments, the biological material can be an amorphous material.

[0014] "Glass transition temperature" refers to the temperature above which a substance behaves like a liquid but below which it behaves like a solid, i.e., it becomes amorphous / glassy. This temperature is not fixed but varies depending on the timescale of the measurement method used. In some embodiments, the glassy state can refer to the state a biological composition enters when its temperature drops below its glass transition temperature. In other embodiments, the glassy state can refer to the state a vitrification mixture and / or vitrification agent enters when its temperature drops below its glass transition temperature. In still other embodiments, the glassy state can refer to a state that possesses the mechanical rigidity of a crystal or gel while retaining the characteristics of a liquid due to the random, disordered arrangement of molecules.

[0015] "Crystal" means a three-dimensional structure of atoms, ions, or molecules consisting of a periodically repeating, ordered geometric arrangement, also called a lattice or unit cell.

[0016] "Crystalline" refers to a form of matter that is composed of components arranged in a regular structure at the atomic level, unlike glassy or amorphous solids. A crystalline solid is formed at a temperature above the crystallization temperature. T c and solidify.

[0017] As used herein, "vitrification" refers to the process of converting a material into an amorphous material. An amorphous solid can be one that does not possess any crystalline structure.

[0018] As used herein, "vitrification mixture" means a heterogeneous mixture of at least one biological material and a vitrification medium, which includes at least one vitrification agent, optionally a dissolving agent, and optionally other materials.

[0019] As used herein, the terms "biological material" and "biological sample" refer to material that can be isolated or derived from at least one living organism. Examples of biological material include, but are not limited to, proteins, cells, tissues, organs, cell-based constructs, blood or fractions thereof, nucleic acids, or combinations thereof. In some embodiments, biological material can refer to mammalian cells. In other embodiments, biological material can refer to human mesenchymal stem cells, mouse fibroblasts, white blood cells, red blood cells, platelets, bacteria, viruses, mammalian cells, liposomes, enzymes, tissues (e.g., intestinal, liver, neural cells, etc.), or combinations thereof. In other embodiments, biological material can refer to reproductive cells, such as sperm cells, spermatocytes, oocytes, eggs, embryos, germinal vesicles, combinations thereof, etc. In other embodiments, biological material can refer to whole blood, red blood cells, white blood cells, platelets, blood plasma, blood serum, algae, fungi, or combinations thereof.

[0020] As used herein, a "vitrification agent" refers to a substance that causes the formation of an amorphous structure or inhibits the crystallization of other materials when a mixture of the vitrification agent and at least one other material is cooled or dried. The vitrification agent(s) may also provide protection against osmotic pressure or allow cell survival during dehydration. In some embodiments, the vitrification agent(s) may be any aqueous solution that creates an amorphous structure suitable for preserving biological materials. In other embodiments, the vitrification agent may be incorporated into cells, tissues, or organs.

[0021] As used herein, the terms "storable," "storage," and "storage-stable" refer to the ability to preserve biological material in a viable state so that it can be used in the future.

[0022] As used herein, "above cryogenic temperatures" refers to temperatures above -80°C. As used herein, "room temperature" refers to a temperature range of 18°C ​​or higher and 37°C or lower.

[0023] As used herein, "hydrophilic" means preferentially attracting or associating with water molecules. A hydrophilic material has a particular affinity for water, resulting in maximum contact with water and a small contact angle with water.

[0024] As used herein, "hydrophobic" means having a low affinity for water. Hydrophobic substances naturally repel water, forming droplets, and have a small contact angle with water.

[0025] As used herein, "room temperature" refers to a temperature of about 16°C or higher and about 30°C or lower.

[0026] Various embodiments described herein provide methods for preserving biological material from a biological sample. In various embodiments, a vitrification mixture is formed by contacting a biological sample containing at least one cell with a vitrification medium. As described in more detail below, the vitrification medium includes at least a vitrification agent and an optional dissolution agent. The vitrification mixture is vitrified to produce a storage-stable sample that can be stored for future use. The storage-stable sample can then be rehydrated and processed to extract or characterize the biological material or a portion thereof, which can be used for quantitative and / or qualitative and / or clinical analysis.

[0027] In various embodiments, the vitrification medium comprises at least a vitrification agent and a dissolution agent. Examples of vitrification agents include, but are not limited to, dimethyl sulfoxide, glycerol, sugars (e.g., trehalose), polyhydric alcohols, methylamines, betaines, antifreeze proteins, synthetic antinucleating agents, polyvinyl alcohol, cyclohexanetriols, cyclohexanediols, inorganic salts, organic salts, ionic liquids, or combinations thereof. In some embodiments, the vitrification medium comprises one, two, three, four, or five or more vitrification agents.

[0028] The vitrification agent is included in the vitrification medium at a concentration that depends on the identity of the vitrification agent. In some embodiments, the concentration of the vitrification agent is below a concentration that is toxic to the biological sample being vitrified. As used herein, "toxicity" refers to the lack of function or viability in future use of the sample, or the unsuitability of the biological sample for future analysis. In various embodiments, the concentration of the vitrification agent is from 500 micromolar (μM) to 6 molar (M), or any value or range therebetween. In one example, in various embodiments, trehalose is included at a concentration of from 1 millimolar (mM) to 6M, optionally from 150 mM to 6M. In some embodiments, the total concentration of all vitrification agents combined is from 1 mM to 6M, optionally from 1 mM to 6M.

[0029] Optionally, the vitrification media provided herein contain a lysing agent. The lysing agent is included in the vitrification medium to partially or completely perforate or render porous the cell membrane (and optionally the cell nucleus) to facilitate the transport of the vitrification agent into the cell and the evacuation of water from the cell, thereby enabling rapid vitrification. By way of example, the lysing agent may be a detergent. Suitable detergents include, but are not limited to, sodium dodecyl sulfate (SDS), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (e.g., Triton X-100), (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) (CHAPS), guanidine hydrochloride, other similar agents, and combinations thereof. Other lysing agents are also possible, provided they do not interfere with the vitrification process or are not toxic to the biological material of interest. In various embodiments, the lysis agent is present in the vitrification medium in an amount of 0.01 weight percent (wt%) to 5 wt% or more. The specific amount of lysis agent can vary depending on the particular embodiment, and more specifically, the desired amount of cell membrane penetration. For example, in some embodiments, the lysis agent may completely lyse the cells, while in other embodiments, the lysis agent may only puncture or perforate the cell membrane to facilitate enhanced transport of the vitrification agent through the cell membrane.

[0030] In some embodiments, the vitrification medium may further comprise other components, such as, but not limited to, water, other solvents, buffers, at least one salt, an RNase inhibitor, a DNAse inhibitor, or combinations thereof. A buffer is any agent with a pKa of 6-8.5 at 25°C. Examples of buffers include choline, betaine, HEPES, TRIS, PIPES, MOPS, and the like, among others. In some embodiments, the buffer is a buffer containing a large (greater than 120 kDa) organic ion, such as choline, betaine, or HEPES. In embodiments where a buffer is included, the buffer is provided at a concentration appropriate to stabilize the pH of the vitrification medium at a desired level.

[0031] Examples of the salt include, but are not limited to, sodium salts, potassium salts, chloride salts, and combinations thereof. When the salt is contained in the vitrification medium, the salt may be provided at a concentration of 1 millimolar (mM) to 500 mM. For example, the salt may be provided at a concentration of 1 mM to 500 mM, 1 mM to 400 mM, 1 mM to 300 mM, 1 mM to 250 mM, 1 mM to 200 mM, 1 mM to 150 mM, 1 mM to 100 mM, 1 mM to 75 mM, 1 mM to 50 mM, 1 mM to 25 mM, 25 mM to 500 mM, 25 mM to 400 mM, 25 mM to 300 mM, 25 mM to 250 mM, or 25 mM to 200 mM. In some embodiments, the ATP may be present at a concentration of from 25 mM to 150 mM, from 25 mM to 100 mM, from 25 mM to 75 mM, from 25 mM to 50 mM, from 50 mM to 500 mM, from 50 mM to 400 mM, from 50 mM to 300 mM, from 50 mM to 250 mM, from 50 mM to 200 mM, from 50 mM to 150 mM, from 50 mM to 100 mM, from 50 mM to 75 mM, or any range or sub-range therein.

[0032] In some embodiments, RNase inhibitors and / or DNase inhibitors can be included in the vitrification medium to prevent nucleic acid degradation. Any known RNase inhibitors and / or DNase inhibitors used in the art can be used, provided that they do not interfere with vitrification. However, it should be understood that in various embodiments, RNase inhibitors and / or DNase inhibitors may not be necessary to preserve nucleic acids.

[0033] In some embodiments, such as when the vitrification medium is used with a blood sample, the vitrification medium may further include at least one anticoagulant. Alternatively, a blood sample may be collected in one or more anticoagulants and then contacted with the vitrification medium. Suitable anticoagulants include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), dioxalate, heparin, sodium citrate, sodium fluoride, and combinations thereof. When an anticoagulant is included, the anticoagulant is present in the vitrification medium in an amount of 0.1 mg / mL to 5 mg / mL, or the same amount is used in other methods. The amount of anticoagulant in an embodiment will vary depending on the specific anticoagulant selected. For example, EDTA may be present in an amount of 1-2 mg / mL of blood, heparin may be present in an amount of 0.2 mg / mL of blood, oxalate may be present in an amount of 1-2 mg / mL of blood, and sodium fluoride may be present in an amount of 2 mg / mL of blood. As another example, sodium citrate may be present in a 1:9 ratio, where 9 parts blood and 1 part sodium citrate. Those skilled in the art will appreciate that other amounts of anticoagulant may be present as long as they prevent clotting of the blood sample.

[0034] In various embodiments, a biological sample containing at least one biological material is contacted with the vitrification medium to form a vitrification mixture. In various embodiments, the vitrification mixture is incubated prior to vitrification. For example, the vitrification mixture can be incubated for 5 to 60 minutes, 5 to 45 minutes, 5 to 30 minutes, or 5 to 20 minutes. Incubation can be performed at any suitable temperature. In various embodiments, incubation is performed at room temperature (i.e., 18°C ​​to 37°C, optionally about 25°C). After incubation, the vitrification mixture containing the biological sample and the vitrification medium is vitrified to produce a storage-stable sample. Vitrification can be performed according to any known vitrification method.

[0035] Vitrification is a method for rapidly cooling liquid materials or transferring small amounts of biological material directly into liquid nitrogen. They are often prepared by immersion or soaking. Cooling reduces the molecular mobility of the material before it can be compressed into a thermodynamically favorable crystalline state. Additives can be used to prevent the crystallization of the main components, resulting in amorphous / vitrified materials. By incorporating appropriate glass-forming agents, biological materials can be encased within a vitrified matrix even at temperatures higher than cryogenic temperatures. Vitrification is achieved by dehydration.

[0036] Some animals and many plants can survive complete dehydration. This ability to survive in dry conditions (anhydrobiosis) is due to several complex physiochemical and genetic mechanisms within the cell. Among these mechanisms is the intracellular accumulation of sugars (e.g., monosaccharides, disaccharides, and oligosaccharides) that act as protectants during desiccation. Trehalose is an example of a disaccharide that is naturally produced by desiccation-tolerant organisms.

[0037] Sugars such as trehalose can protect desiccation-tolerant organisms in several different ways. Due to the special arrangement of hydroxyl groups on the trehalose molecule, trehalose molecules can effectively replace hydrogen-bonded water molecules on the surface of folded proteins without altering their conformational geometry or folding. Furthermore, sugar molecules can bind to the phospholipid head groups of lipid bilayers, preventing cytoplasmic leakage during rehydration. Furthermore, many sugars have high glass transition temperatures, allowing them to form glasses at room temperature or even at temperatures higher than cryogenic temperatures, even with low water content. The highly viscous "glassy" state reduces molecular mobility, thereby preventing degradative biochemical reactions that can lead to cell function loss, cell death, and even protein and nucleic acid degradation.

[0038] In some embodiments, vitrification of biological samples involves dehydration in the presence of the glass-forming sugar trehalose, as disclosed in N Chakraborty, et al., Biopreservation and Biobanking, 2010, 8 (2), 107-114. Referring to FIG. 1, system 10 is the most common approach to dehydrating biological materials. Sessile droplets 11 are placed on a substrate 12 and evaporatively dried within an enclosure 16 containing a low-humidity environment 13. The humidity, pressure, and temperature within the enclosure can be operably controlled via a regulator 17. However, evaporative drying of sessile droplets in system 10 is inherently slow and non-uniform. When biological materials are dried in a glass-forming solvent, a glassy skin forms at the liquid / vapor interface 14 of the sample. This glassy skin slows (and ultimately prevents) the sample from drying beyond a predetermined dryness point. Consequently, the water content throughout the sample is significantly non-uniform spatially. As a result, cells trapped within the partially dried sample below the glassy membrane may not vitrify but may decompose due to their high molecular mobility.

[0039] In another embodiment, vitrifying the biological sample comprises dehydrating by capillary drying in the presence of the glass-forming sugar trehalose, as disclosed in U.S. Patent No. 10,433,540. Figure 2 shows an example of an apparatus for carrying out such a method.

[0040] The vitrification process can be performed on or within a membrane, which may have one or more capillary channels (optionally, a continuous capillary channel) therein. The capillary can provide an interface through which rapid evaporation occurs. The membrane can be composed of multiple capillary channels (optionally, multiple adjacent capillary channels). The capillary network of porous materials, such as membranes, can be formed from materials that are not toxic or reactive toward biological materials or biological samples and do not chemically or physically react with the vitrification medium. The membrane can be made of a hydrophilic or hydrophilically modified material. In some embodiments, the membrane can be partially soluble or time- or stimulus-dependently soluble in the vitrification mixture or reconstitution solution, optionally with the aid of a supporting structure, as described below. The membrane material can be a suitable polymer, metal, ceramic, glass, or a combination thereof. In some embodiments, the continuous capillary network is formed from materials such as polydimethylsiloxane (PDMS), polycarbonate, polyurethane, polyethersulfone (PES), and polyester (e.g., polyethylene terephthalate), among others. Examples of capillary channels with membranes as suitable surfaces for use in the devices and methods provided herein include hydrophilic filtration membranes, such as those available from EMD Millipore (Billerica, Massachusetts, USA). In certain embodiments, the porous material does not substantially bind to, alter, or chemically or physically associate with components of the biological sample and / or vitrification medium. Optionally, the porous material is underivatized. Optionally, capillary channels can be formed in a substrate (e.g., a wall of a dry space) of a desired material and thickness by PDMS forming techniques, laser drilling, or other pore-forming techniques known in the art.

[0041] In some embodiments, the capillary network provided by the porous material has pores with cross-sectional dimensions of about 100 μm or less, optionally 20 μm or less, which provide internal capillaries that support vitrification. Optionally, larger pore sizes, such as cross-sectional dimensions of about 100 μm or less, can be used when the capillary network is used for tissue vitrification. In some embodiments, cross-sectional dimensions of about 20 μm or less can be used when targeting cell samples (other than tissue). In some embodiments, the pores can have an average opening of from about 100 μm to about 0.1 μm, such as about 90 μm, about 80 μm, about 70 μm, about 60 μm, about 50 μm, about 40 μm, about 30 μm, about 20 μm, about 19 μm, about 18 μm, about 17 μm, about 16 μm, about 15 μm, about 14 μm, about 13 μm, about 12 μm, about 11 μm, about 10 μm, about 9 μm, about 8 μm, about 7 μm, about 6 μm, about 5 μm, about 4 μm, about 3 μm, about 2 μm, about 1.0 μm, about 0.9 μm, about 0.8 μm, about 0.7 μm, about 0.6 μm, about 0.5 μm, about 0.4 μm, about 0.3 μm, about 0.2 μm, etc. Optionally, the length of the capillary channel can be determined by the thickness of the substrate forming the channel or by one or more individual channels. Optionally, the length of the capillary channel is about 1 millimeter or less (although it should not be construed as limited to such dimensions). Optionally, the length of the capillary channel is about 0.1 microns to about 1000 microns, or any value or range therebetween. Optionally, the length of the capillary channel is about 5 to about 100 microns, optionally about 1 to about 200 microns, and / or optionally about 1 to about 100 microns. Optionally, the length of the capillary channel is about 5 microns, about 10 microns, about 15 microns, about 20 microns, about 25 microns, about 30 microns, about 35 microns, about 40 microns, about 45 microns, about 50 microns, about 55 microns, about 60 microns, about 65 microns, about 70 microns, about 75 microns, about 80 microns, about 85 microns, about 90 microns, about 95 microns or about 100 microns.In some embodiments, the length of the capillary channels varies among the plurality of capillary channels, optionally with non-uniform variability.

[0042] The cross-sectional area of ​​one or more of the capillary channels is about 8000 μm 2 The following options are 2000 μm 2 Optionally, the cross-sectional area may be less than or equal to about 0.01 μm 2 ~about 8000μm 2 , approximately 100 μm as an option 2 ~about 2000μm 2 Optionally, the cross-sectional area of ​​one or more of the capillary channels is less than about 100 μm 2 Below, approximately 200μm 2 Below, approximately 300μm 2 Below, approximately 400μm 2 Below, approximately 500μm 2 Below, approximately 600μm 2 Below, approximately 700μm 2 Below, approximately 800μm 2 Below, approximately 900μm 2 Below, approximately 1000μm 2 Below, approximately 1100μm 2 Below, approximately 1200μm 2 Below, approximately 1300μm 2 Below, approximately 1400μm 2 Below, approximately 1500μm 2 Below, approximately 1600μm 2 Below, approximately 1700μm 2 Below, approximately 1800μm 2 Below, approximately 1900μm 2 Below, approximately 2000μm 2 Below, approximately 3000μm 2 Below, approximately 4000μm 2 Below, approximately 5000μm 2 Below, approximately 6000μm 2 Below, approximately 7000μm 2 Less than or equal to 8000 μm 2 The following is said to be true.

[0043] As shown in FIG. 2, the capillary-assisted vitrification device 20 includes a capillary plate / membrane 22 disposed within an enclosure 28. The capillary membrane is depicted as containing a biological sample within the pores of the membrane. Optionally, the capillary membrane in some embodiments may be replaced with multiple filtration layers or a separate membrane without multiple distinct channels. In other embodiments, multiple membranes may be used, stacked together to form a vitrification membrane suitable for vitrification of the biological sample, with the biological sample sandwiched between or contained within the membranes. Optionally, the plate / membrane 22 includes multiple, optionally substantially parallel, capillary channels, each including a first opening 23 and a second opening 25. When a vitrification mixture 24 is placed in the first opening 23, the vitrification mixture 24 flows through the capillary channels while the surface of the vitrification mixture 24 is exposed to ambient atmosphere 29 through the second opening 25. In various embodiments, the ambient atmosphere 29 has a humidity lower than that of the vitrification mixture. Vitrification is achieved by drying the vitrification mixture until it reaches a glassy state by capillary action. The chemistry, humidity, pressure, and temperature within the enclosure 28 are regulated by one or more control mechanisms 21.

[0044] The adjustment mechanism 21 is shown simplified, but is illustrative only and may consist of multiple systems and mechanisms to achieve the most desirable drying and vitrification conditions. Optionally, in some embodiments, the vitrification mixture 24 is sandwiched between two plates / membranes similar to component 22, thereby providing the benefits of capillary drying on both the top and bottom surfaces of the vitrification mixture 24.

[0045] In some embodiments, a low-humidity gas flow (relative humidity less than 30%) is supplied through the second opening 25 of the capillary plate / membrane or from the opposite side of the membrane, which is the vitrification medium, to enhance the capillary effect. The low-humidity gas can be an inert or relatively inert gas, such as nitrogen, argon, or xenon. In some embodiments, a reduced pressure or vacuum is maintained within the enclosure 28. In some embodiments, suction / pressure is supplied through the second opening 25 to speed up the drying process. Note that maintaining a low ambient humidity (optionally 5% relative humidity or less) is essential to prevent rehydration after drying. For further details on capillary drying, see U.S. Pat. No. 10,433,540.

[0046] The vitrification method may be carried out at a temperature between -80°C and +60°C. In some cases, this temperature range allows for increased mobility of water molecules in the sample without being detrimental to the integrity or viability of the biological material. This will vary from material to material and depending on the composition of the vitrification medium. In some embodiments, the vitrification temperature is between 0.1°C and 40°C. Optionally, the vitrification temperature is between 4°C and 26°C. Optionally, the vitrification temperature is about 25°C.

[0047] The vitrification method can be carried out in a dry atmosphere or environment. A dry environment is an environment with a humidity level below saturation humidity. In some embodiments, the humidity level of the environment (e.g., the environment on the second side of the capillary tube) is 30% or less, optionally 20% or less, optionally 10% or less, or optionally 5% or less relative humidity. Optionally, the humidity of the dry environment is 1%-30%, or any value or range therebetween, optionally 1%-5%.

[0048] The vitrification method can be carried out in a low-pressure environment [less than 1 atm (760 mmHg)]. A low-pressure environment has a favorable effect on the vitrification rate. Optionally, the pressure of the environment is 100 mmHg or 0.1 atm. Optionally, the pressure of the environment is 10 mmHg to 760 mmHg, or any value or range therebetween. Optionally, the pressure of the environment is 10 mmHg to 200 mmHg.

[0049] The vitrification method may be carried out for a drying time, which is a time sufficient to promote adequate drying for vitrification of the vitrification medium. Optionally, the drying time is between 1 second and 1 hour. Optionally, the drying time is between 1 second and 50 minutes, and further optionally, between 5 seconds and 60 minutes. Drying times may vary depending on the type or properties of the sample and the details of the capillary channel.

[0050] In still other embodiments, vitrification may be performed on or between membranes, or on or between one or more filter papers. Depending on the particular embodiment, other vitrification methods may be used.

[0051] After vitrification, the sample is shelf-stable and can be stored viably and substantially undegraded at temperatures above cryogenic temperatures for future use. In some embodiments, the vitrified mixture after vitrification can be sealed and stored in a glassy state in a water- and air-impermeable protective enclosure. In some embodiments, the shelf-stable sample can be stored at temperatures between -196°C and +60°C or above, between 16°C and 60°C or above, or between 18°C ​​and 60°C or above, while unused. In some embodiments, the storage period is 1 day or more, 5 days or more, 10 days or more, 20 days or more, 30 days or more, 45 days or more, 60 days or more, or more than that.

[0052] In various embodiments, when the storage-stable sample is desired for use, it is rehydrated (or reconstituted) and processed according to a specific procedure depending on the intended use of the sample. In some embodiments, the storage-stable sample is rehydrated with a rehydration solution, which may precipitate proteins and one or more nucleic acids. In some embodiments, the storage-stable sample is reconstituted by treating the storage-stable sample with a lysis buffer, such as may be included in an extraction and / or purification kit, to completely lyse cellular material.

[0053] The sample may be subjected to a variety of protocols. However, in some embodiments, the sample is processed to extract nucleic acids, such as DNA and / or RNA. For example, the DNA and / or RNA may be pelleted and / or bound and / or washed and / or eluted and / or dried and / or dissolved after rehydration, depending on the particular extraction method used.

[0054] In some embodiments, RNA is extracted using a GITC-based method: the storage-stable sample is rehydrated and phase-separated, and propanol is added to the supernatant. The mixture is centrifuged to form a pellet containing the RNA. The RNA pellet is then washed, dried, and reconstituted for analysis.

[0055] In some embodiments, RNA is extracted using the TRIspin method, where the storage-stable sample is rehydrated and phase separated, and ethanol is added to the supernatant. The RNA is then bound, washed, and eluted before being available for analysis.

[0056] In some embodiments, RNA is extracted using a column-based method, in which the storage-stable sample is rehydrated prior to the addition of ethanol, and the RNA is then bound, washed, and eluted before being available for analysis.

[0057] Other extraction methods are contemplated. That is, in some embodiments, regardless of the specific extraction method used, the cell lysis step, which is typically the first step of any such extraction method, can be performed during the vitrification process described above, with the remaining steps being performed after rehydration. As described above, the embodiments described herein can provide samples that are capable of rapid vitrification and are storage-stable, allowing them to be used for a variety of optional processes after storage.

[0058] In some embodiments, DNA or RNA can be extracted from vitrified cells. For example, any of a number of commercially available DNA or RNA extraction kits or similar methods can be used to extract DNA or RNA from the storage-stable sample. In various embodiments, the sample can be reconstituted using a lysis buffer used in the extraction kit.

[0059] Additionally, in various embodiments described herein, whole blood or portions thereof can be stored at temperatures higher than cryogenic temperatures. For example, whole blood and / or serum and / or plasma and / or red blood cells and / or platelets and / or lymphocytes may be collected in a tube (optionally with an anticoagulant) or on a membrane, contacted with the vitrification medium, and stored at room temperature for 5 to 20 minutes. Vitrification can be achieved by incubation. The sample can be stored at room temperature until use. DNA and / or RNA and / or protein extraction can be performed by treating the sample with Trizol. This not only reduces storage costs but also allows stored blood samples to be used for various post-storage procedures, improving sample versatility. For example, the storage-stable sample can be rehydrated, allowing extraction of leukocyte lysate with a lysis buffer. The lysate can be transferred to a spin column with ethanol and washed with a wash buffer. Total RNA can then be eluted with RNase-free water, allowing for quantitative, qualitative, and clinical analysis of RNA.

[0060] In some embodiments, whole blood can be collected and centrifuged, and one or more layers (e.g., plasma layer, buffy coat, red blood cell layer, etc.) can be transferred to the vitrification medium matrix and vitrified. After storage, the sample can be reconstituted with a diluent (e.g., PBS with or without protease inhibitors) and subjected to qualitative and / or quantitative analysis.

[0061] Additionally, while various embodiments have been described herein using a biological sample in the form of whole blood, it will be appreciated that other types of biological samples may be used, as generally described herein. In some embodiments, the biological sample may be in the form of tissue. In some such embodiments, the tissue may be homogenized using a lysis solution, pulverized, or enzymatically digested. In alternative embodiments, the tissue may be subjected to a cryosection method, which may be used in various embodiments.

[0062] In some embodiments, the biological sample is heterogeneous. A heterogeneous sample is one that contains at least one contaminating organism (not of interest) that also has nucleic acids. Such nucleic acids may be stored with the biological sample and potentially contaminate downstream analysis of the sample. As such, additional preparation steps may be required to selectively isolate the desired biological sample components from the undesired contaminants. As a non-limiting example, the biological sample may be saliva. Saliva is known to contain cells of both the host organism that produces the saliva and bacterial or viral contaminants.

[0063] Heterogeneous samples can be subjected to a pretreatment process to remove or reduce the amount of contaminating organisms (optionally bacteria, viruses, yeast, etc.) in the sample. The pretreatment process can be performed simultaneously with the vitrification process. However, in some embodiments, the pretreatment process is performed prior to the actual vitrification. The pretreatment process can be contacting the biological sample with an isolation medium (optionally in particulate or membrane form) containing or binding to a molecule selective for at least one contaminating organism (optionally bacteria and / or viruses and / or yeast and / or other non-target organisms). In some embodiments, the sample is contacted with the surface of the isolation medium containing a separation agent specific for at least one non-target organism, optionally mannose-binding lectin (MBL), optionally mannose-binding lectin having the NCBI reference sequence (sequence: NP_000233). MBL is a C-type lectin that binds to N-acetylglucosamine and mannose residues in bacteria, yeast, some parasites, and viruses. By contacting a biological sample with a surface that contains MBL, non-target organisms can be selectively isolated from target cells. This improves the method's ability to selectively isolate target cells from a biological sample and, optionally, to vitrify those cells.

[0064] Referring to Figure 4, in some embodiments, a sample is subjected to a membrane system that removes contaminating organisms using at least one separation membrane 32. Figure 4 depicts a plurality of particles 36 having a separation agent bound to their surface. The particles may be incubated with the vitrification mixture and / or biological sample. The particles selectively bind contaminating organisms via at least one separation agent, optionally via at least one separation agent bound to mannose-binding lectin (MBL). The sample containing the particles is placed on separation membrane 32. Separation membrane 32 has a pore size that allows the biological sample to pass through but leaves the particles on the surface.

[0065] Alternatively or additionally, at least one separation agent may be bound to the separation membrane itself, such that upon contact with a biological sample or vitrification mixture, the separation membrane binds contaminating organisms to the separation membrane while allowing desired substances in the biological sample to pass through and contact the vitrification membrane, thereby vitrifying the biological sample. The separation membrane may be any substantially porous membrane system that allows the passage of cellular material not bound to the separation agent within or on the separation membrane.

[0066] The separation medium can be a suitable polymer (e.g., polyvinylidene fluoride (PVDF)), metal, ceramic, glass, or a combination thereof. In some embodiments, the separation medium can be made of PVDF, cellulose esters, nitrocellulose, or other desired materials. At least one separation agent can be bound to or otherwise associated with the suitable separation medium. When a biological sample is contacted with the separation medium, contaminating cells / organisms can selectively bind to the separation agent. This allows the cells of interest to pass through the system and can be collected or harvested, or vitrified, as provided elsewhere herein.

[0067] The separation membrane (optionally comprising a separation agent) may be laminated to a vitrification membrane, as described elsewhere herein, suitable for vitrification of biological samples. The vitrification membrane may be any membrane that comprises or forms a capillary network. Optionally, such porous membranes may be formed from materials that are non-toxic or non-reactive with biological materials or biological samples, and do not react chemically or physically with the vitrification medium. The materials may be suitable polymers, metals, ceramics, glass, or combinations thereof. In some embodiments, continuous capillary networks are formed from materials such as polydimethylsiloxane (PDMS), polycarbonate, polyurethane, polyethersulfone (PES), and polyesters (e.g., polyethylene terephthalate), among others. Examples of capillary channels with membranes as suitable surfaces for use in the devices and methods described herein include hydrophilic filtration membranes, such as those available from EMD Millipore (Billerica, Massachusetts, USA). In certain embodiments, the porous material does not substantially bind to, alter, or chemically or physically associate with components of the biological sample and / or vitrification medium. Optionally, the porous material is underivatized. Optionally, capillary channels can be formed in a substrate (e.g., a wall of a dry space) of a desired material and thickness by PDMS molding techniques, laser drilling, or other pore-forming techniques known in the art.

[0068] After the biological sample passes through a separation membrane containing a separating agent to separate or remove cellular material from non-target organisms, the remaining cellular material is collected within or on the membrane, vitrified, and optionally preserved. The separation membrane may be laminated onto a vitrification membrane. After contact with the biological sample, the separation membrane may be removed and subjected to analysis or discarded, and the remaining biological sample may be vitrified on or within the vitrification membrane by the methods described herein. Alternatively, the separation membrane may remain attached to the vitrification membrane, and the entire membrane system may be subjected to vitrification as described herein. The separation membrane may then be peeled or removed from the vitrification membrane before resolubilization of the biological material. The pretreatment process described above results in the removal of most cellular material from non-target organisms, thereby improving the isolation and preservation of the target biological sample material.

[0069] Optionally, the separation agent may be bound to beads or particles. In some embodiments, beads bearing a separation agent may be mixed with the biological sample and vitrification mixture, which may then be poured into the membrane system. It will be appreciated that this process allows sufficient contact time between the beads and the vitrification mixture to capture target pathogens. Referring to FIG. 4, when the vitrification mixture is poured into the membrane system, the beads with trapped pathogens are separated from the vitrification mixture and remain on the upper surface of the membrane system. To achieve this, the pore size of the membrane system must be smaller than the size of the beads but larger than the size of the cells that must pass through the vitrification membrane. The size of the beads may be between 6 microns and 500 microns, and optionally between 10 microns and 500 microns. The beads may be made of a polymer, such as polystyrene, or an iron oxide, such as magnetite (Fe3O4), functionalized to bind the separation agent.

[0070] As provided herein, the biological sample in various embodiments can be tissue or other parts of an organism. Preserving tissue samples under non-freezing conditions is typically difficult. Simply vitrifying a tissue sample can result in insufficient stability of the tissue material due to uneven drying or insufficient preservation of tissue structure. Therefore, a tissue vitrification method is provided that not only preserves the molecular material of the tissue, but also maintains the overall structure and other properties of the tissue, dramatically improving the effectiveness and feasibility of future tissue analysis. Tissues that can be used as biological samples in the methods provided herein include, but are not limited to, tissues from neurons, liver, heart, kidney, blood vessels, kidney, lung, larynx, stomach, esophagus, pancreas, thyroid, muscle, epithelium, hair, or any other tissue type recognized as biological tissue.

[0071] In the vitrification methods provided herein, a vitrification agent (optionally a sugar) may be introduced to a tissue sample, and the tissue sample may be contacted with a polymer or polymer-forming agent (e.g., a PEG hydrogel). For example, the tissue may be injected with a trehalose-PEG hydrogel precursor and an attachment agent suitable for covalently or ionically bonding or associating the tissue or a portion thereof with the polymer.

[0072] The polymer may be any molecule that can be used as, or can be used to generate, a polymer suitable for preserving tissue at temperatures higher than cryogenic temperatures. Such polymers include, but are not limited to, polyalkyl alcohols and glycols, such as polyoxyethylene and polyoxyethylene derivatives; neophenyl glycol diacrylate (NPGDA); polyethylene oxide (PEO); polyacrylamide (PAAm); polyhydroxyethyl methacrylate (PHEMA); polyacrylic acid (PAA); polyvinyl alcohol (PVA); poly(N-isopropylacrylamide) (PNIPAM); polyvinylpyrrolidone (PVP); polylactic acid (PLA); polyglycolic acid (PGA); polycaprolactone (PCL); gelatin; alginate; carrageenan; chitosan; hydroxyalkyl cellulose; alkyl cellulose; silicone; rubber; agar; carboxyvinyl copolymer; polydioxolane; polyacrylic acid; polyvinyl chloride; maleic anhydride; styrene-styrene polymers; dextrans; heparin and polymers of heparin; glutamic acid polypeptides; aspartates; or combinations thereof.

[0073] The polymer is optionally linear, branched, liable, or a combination thereof. Optionally, the polymer is homomeric or heteromeric. Examples of polymeric moieties include at least one carbohydrate molecule or polyoxyethylene (also known as polyethylene glycol or "PEG") molecule.

[0074] Optionally, the polymer is polyethylene glycol. Optionally, the polyethylene glycol comprises 2 to 20,000 ethylene glycol units. Optionally, the number of ethylene glycol units is 2 to 10,000, optionally 2 to 5,000, or optionally 2 to 2,000. In various embodiments, the polyethylene glycol (PEG) is a polyethylene glycol derivative, such as, but not limited to, polyethylene glycol-vinyl sulfone. The PEG can be a linear or branched PEG molecule. Optionally, the branched PEG can be a PEG molecule having 2, 4, 6, 8, or other number of arms.

[0075] In some embodiments, the method further comprises adding a crosslinker along with the polymer-forming agent. The crosslinker may be any agent suitable for joining two or more monomers / polymers. Optionally, the crosslinker has one or more acrylate or methacrylate functional groups. Examples of crosslinkers include 2-hydroxyethyl methacrylate (HEMA), acrylic acid, methacrylic acid, adipic acid hydrazide diamide acrylate, acrylamide, methacrylamide, alkyl-(meth)acrylamide, N-mono(meth)acrylamide, N,N-di-C1-C4 alkyl-(meth)acrylamide (N,N-di-C1-C4 alkyl-(meth)acrylamide), N-butyl (meth)acrylate, N-butyl (meth)acrylate, methyl (meth)acrylate, ethyl (meth)acrylate, dimethicone, isobornyl (meth)acrylate, cyclohexyl (meth)acrylate, hydroxyethyl ... methacrylate, hydroxypropyl acrylate, hydroxypropyl methacrylate, hydroxybutyl acrylate, N-(2-hydroxyethyl)acrylamide [N-(2-hydroxyethyl)acrylamide], N-methylacrylamide, N-butoxymethylacrylamide, N-methoxymethylacrylamide, N-methoxymethylmethacrylamide, 2-acrylamidoglycolic acid, 2-carboxyethyl 2-carboxyethyl acrylate, 2-hydroxy-5-methoxyacetophenone, 2-hydroxyethyl cellulose, 2-hydroxyethyl disulfide, and the like, or combinations thereof.

[0076] Optionally, the method further includes at least one attachment agent present in the vitrification mixture. Optionally, the attachment agent is a boronic acid. Without being bound by any particular theory, it is believed that the boronic acid may covalently attach macromolecules to membrane proteins of tissue cells. This results in the formation of a hydrogel between the tissue cells, which provides support to the cells and tissue against shrinkage and collapse during vitrification and sectioning. After the hydrogel is formed, the tissue can be vitrified. The vitrified specimen can then be sectioned, if desired. After sectioning the tissue, the boronic acid can be released by adding a mixture of glucose and water, removing the hydrogel from the membrane proteins. The boronic acid and hydrogel can then be washed away, yielding a tissue specimen with its native structure preserved by the vitrification method.

[0077] This embodiment may enable histological analysis of the sample. Traditional tissue processing for histological analysis involves injecting low-melting-point paraffin or agarose into the tissue prior to freezing or vitrification before sectioning the tissue sample. While paraffin provides the necessary rigidity to maintain cellular structure, it can also lead to tissue contamination. Agarose can affect RNA extraction and adhere to the tissue wall. Therefore, the use of trehalose-PEG hydrogel (as an example presented herein) allows the hydrogel to be debonded and washed away during a glucose-water wash, thereby supporting cellular structure and enabling tissue vitrification without causing contamination or adversely affecting downstream processing. Other advantages will be apparent to those skilled in the art.

[0078] Optionally, the vitrification medium includes a switchable support material that can be switched between a relatively high-viscosity state and a relatively low-viscosity state in response to two different stimuli. Before application of the stimuli, the switchable support material behaves as a highly flowable, low-viscosity liquid, making it suitable for in situ perfusion of tissue matrices using the methods presented herein. Upon application of a first stimulus, the material switches from a low-viscosity liquid (sol state) to a stress-relieving fluid (gel state), providing sufficient rigidity and support to substantially maintain the structural shape of the tissue and / or membrane during vitrification. Upon application of a second stimulus, the support material reverts from a rigid material to a low-viscosity liquid, allowing for easy removal.

[0079] In some embodiments, various wavelengths of light may be used as stimuli. Ultraviolet (UV) or visible light can be used as the conversion stimulus. In some embodiments, photoconvertible hydrogel support materials exhibit rigidity under visible light but can completely disassemble into a solution state under UV light. Examples of photoconvertible materials include supramolecular hydrogels composed of host-guest complexes of azobenzene (azo) and cyclodextrin (CD), optionally as described in Vapaavuori, et al., J. Mat. Chem. C., 2018; 6:2168-2188 or Rosales, et al., Bioconjugate Chem., 2018; 29: 905-913. There are three main types of cyclodextrins: α-CD, β-CD, and γ-CD, which differ in cavity size. They form a cone structure with six, seven, and eight linked glucopyranose subunits, respectively, and are hydrophilic due to the hydroxyl groups located on the exterior of the cone. However, the hydrophobic interior of the cone allows it to accept hydrophobic molecules such as azobenzene (azo) to form host-guest complexes in aqueous solution. Azobenzene is a well-known photoresponsive molecule. Under visible light (<520 nm), it transforms into a thermodynamically stable trans-state. Upon UV irradiation (<375 nm), it photoisomerizes to its cis-isomer. Trans-azo possesses a shape that allows it to enter the cavity of CDs through hydrophobic interactions in aqueous solution and form host-guest complexes. Upon photoisomerization to cis-azo, its shape no longer fits into the CD cone, resulting in disassembly. By using this photoreversible azo-CD complex as a crosslinker, we can prepare photoconvertible hydrogels that gel under visible light and easily disassemble into liquids (sols) under UV light. This sol-gel transformation is reversible and can be completed within 2 minutes. For other methods and materials for forming transformable support materials, see Koopmans and Ritter, Macromolcules, 2008; 41:7418-7422.

[0080] Optionally, the methods provided herein further comprise the step of including a state-changeable support material within the vitrification medium and applying a stimulus to convert the state-changeable support material to a viscous state, optionally followed by the step of subjecting the vitrification medium to a vacuum vitrification process provided herein to preserve tissue or other cellular material in a vitrified state while providing sufficient support to maintain other physical and / or chemical properties of the biological sample. [Example]

[0081] The following examples are presented for the purpose of illustrating various aspects, but are not intended to limit the scope of the claims. The properties, characteristics, parameters, etc. of the various examples and comparative examples below, and of the materials used in the examples and comparative examples, are approximate.

[0082] Example 1 Cell culture: LINTERNA Jurkat T cells (stably expressing tGFP with a G418 resistance gene) were obtained from Innoprot (Spain). Cells were cultured in RPMI 1640 (Gibco), 10% heat-inactivated fetal bovine serum (Hyclone), 1X Glutamax (Gibco), and G418 (Gibco) at 37°C and 5% CO2. Cultures were performed in 25cm plates. 2 The cells were maintained in T-flasks (Corning Incorporated, New York, USA) at 37°C, equilibrated with 10% CO2 and 90% air. To maintain the cells, fresh culture medium was replaced every 3 days.

[0083] In Example A, 5 x 10 6A sample of Jurkat T cells was incubated for 10-15 minutes in 250 μL of vitrification medium containing 600 mM trehalose, 5% glycerol, and 0.01% Triton X-100. The sample was then sandwiched between two 1.2-micrometer pore-sized PES membrane scaffolds and vitrified for approximately 6 minutes under a vacuum of -29 mmHg to a moisture retention ratio (MRR) of 0.01. The sample was then stored at 25°C or 55°C for 3 days before RNA extraction.

[0084] RNA was extracted using the PureLink RNA Mini Kit (Invitrogen). Vitrified cells were rehydrated with 0.6 mL of lysis buffer containing 1% 2-mercaptoethanol and incubated at room temperature for 15 minutes. The lysate was passed 10 times through a 21-gauge needle to obtain a homogenous lysate. An equal volume of 70% etonal was added to the lysate. The lysate / supernatant was then extracted by centrifugation (12,000 × g, 15 seconds, room temperature) and passed through an RNA-binding spin column by centrifugation (12,000 × g, 15 seconds, room temperature). DNA contamination was then removed by on-column DNase digestion (PureLink DNase Set; Invitrogen), followed by two wash steps with 700 μL Wash Buffer-I and 500 μL Wash Buffer-II containing ethanol (centrifugation: 12,000 × g, 15 seconds). Contaminants and inhibitors were then removed by two wash steps (centrifugation: 12,000 × g, 15 seconds) with ethanol. The spin column containing the bound RNA was dried for 2 minutes, and then 50 μL of RNase-free water was added to the spin column, which was then incubated for 2 minutes. The pure RNA was eluted by centrifugation (12,000 × g; room temperature) in a new tube.

[0085] As a control, RNA was extracted from a sample of the same number of fresh cells using the same method as for the vitrified cells (Comparative Example A).

[0086] In Comparative Example B, samples containing the same number of cells were cryogenically frozen and stored for 2 hours at −80° C. The cells were thawed, and RNA was extracted using the same RNA extraction method as in Example A.

[0087] In Comparative Example C, 5X10 6 Jurkat T cells were incubated for 10–15 minutes in 250 μL of vitrification medium containing 600 mM trehalose and 5% glycerol, but without Triton X-100. The samples were vitrified for approximately 6 minutes to achieve an MRR of 0.01. The samples were then stored at room temperature for 3 days. The cells were then rehydrated in lysis buffer, and RNA was extracted using the same method as for the vitrified cells. The vitrification procedure for Comparative Example C used the same vitrification procedure as for Example A. Cells were stored at either 25°C or 55°C for 3 days before RNA extraction.

[0088] For all samples, 5 μg of RNA per well was loaded onto a 1.2% agarose gel (Native Sybrsafe) and separated by electrophoresis. Figure 5 shows an image of the gel. If the total RNA on the denaturing gel is intact, clear bands of 28S and 18S rRNA should be observed (eukaryotic samples). The 28S rRNA band should be approximately twice as intense as the 18S rRNA band. This 2:1 (28S:18S) ratio is a good indicator of intact RNA. As seen in Comparative Example A (lane 1) and Comparative Example C (lane 3), partially degraded RNA will have a smeared appearance, less clear rRNA bands, or no 2:1 ratio. Completely degraded RNA will have a very low molecular weight smear (Comparative Example B; lane 2). As can be seen in FIG. 5A, Example A (lane 4), which was vitrified and stored (storage at 55°C for 1 week) according to various embodiments described herein, has clear 28S rRNA bands and 18S rRNA bands with a good intensity ratio, suggesting that the RNA is intact even after vitrification and storage at room temperature.

[0089] Similar results were obtained by RNA quantification. Table 1 shows the spectrophotometric quantification results for the RNA prepared for each sample as described above.

[0090] [Table 1]

[0091] Vitrification in the presence of the lysing agent Triton-X100 demonstrated a clear increase in intact RNA compared to cells vitrified in the absence of the lysing agent. Comparative Example B was almost completely degraded. The inclusion of the lysing agent in the vitrification medium improved RNA quality whether stored at 25°C or at a higher temperature of 55°C for 3 days, demonstrating the robust preservation potential of cell samples prepared as described herein.

[0092] Example 2 LINTERNA Jurkat T cells were obtained and cultured as in Example 1.

[0093] 5X10 6 A sample of Jurkat T cells was incubated for 10-15 minutes in 250 μL of vitrification medium containing 600 mM trehalose, 5% glycerol, and 0.01% Triton X-100. The sample was then vitrified for approximately 6 minutes to achieve an MRR of 0.01. The sample was then stored at either 25°C or 55°C for 3 days before RNA extraction. RNA extraction was performed as described in Example 1.

[0094] For comparison, samples of fresh cells stored at 4°C were subjected to RNA extraction and analysis in the same manner as for the vitrified cells.

[0095] RNA extracted from each sample was subjected to RT-PCR using VEGF (a member of the PDGF / VEGF growth factor family) or GAPDH RNA templates. Figure 6A shows the cycle threshold (Ct) values ​​of GADPH mRNA in cells prepared using each preparation and storage technique. Figure 6B shows the fold change in Ct values ​​of GADPH mRNA in cells prepared using each preparation and storage technique. Figure 6C shows the fold change in VEGF mRNA in cells prepared using each preparation and storage technique. As shown in Figures 6A and 6B, there was no significant change in Ct values ​​between fresh and vitrified cells for the GAPDH gene, regardless of storage temperature (25°C or 55°C). Furthermore, robust VEGF mRNA was observed (Figure 6C). Therefore, we concluded that the vitrification method described herein can be used to vitrify and preserve cells without degrading VEGF mRNA.

[0096] Example 3 Various animal tissues were obtained and subjected to vitrification. Mouse intestinal and liver samples were humanely collected using approved animal protocols. The tissues were cut into small sections (approximately 1 mm thick, 15 mg in weight) and incubated for 20 minutes in vitrification medium containing 600 mM trehalose, 5 wt% glycerol, and 0.5 wt% Triton X-100. The samples were vitrified for 5 minutes to an MRR of 0.01, as in Example 1, and stored at either 25°C or 55°C for one week.

[0097] A second group of samples was vitrified in the same vitrification medium, but with the addition of a trehalose polymer, azobisisobutyronitrile (AIBN) (5.28 mg, 3.22 x 10 -2 The polymer was synthesized by dissolving 1000 mg (634 mmol) of trehalose monomer (1.38 mmol) and styrenyl ether trehalose monomer (634 mg, 1.38 mmol) in a mixture of dimethylformamide (DMF) (2.31 mL) and HO (4.61 mL). Oxygen was removed by three freeze-pump-thaw cycles, and polymerization was carried out at 75 °C. The polymerization was stopped after 8.5 h by immersing the vial in liquid nitrogen.

[0098] The third group of samples was vitrified in a vitrification medium containing 600 mM trehalose, 5 wt% glycerol, and 0.5 wt% Triton X-100, containing 8-arm polyethylene glycol conjugated to a boronic acid. This polymer was prepared as an 8-arm PEG amine (400 mg, 10 kDa, 4 × 10 -2 mmol) and 4-formylphenylboronic acid (96 mg, 6.40 × 10 -1 Prepare NaBHCN (37.7 mg, 6.00 × 10 mmol) by dissolving in 2.8 mL MeOH. -1 (mmol) and stir at 25° C. The remaining vitrification and storage protocols remain unchanged.

[0099] After storage, tissue samples were reconstituted in lysis buffer, and mRNA was isolated as described in Example 1. RNA was quantitatively analyzed by spectrophotometric analysis using a Take3 Plate on a Synergy H1 Hybrid MF (BioTek Instrument). Briefly, 2 μL of each sample was added per well to the Take3 plate, and ultrapure RNase-free water was used as a blank. Total RNA concentration was calculated based on the A260 reading using Gen5 software (1.0 corresponds to less than 40 μg / mL ssRNA). The A260 / A280 ratio was used as a measure of RNA quality (A260 / A280 ratios of 1.8 to 2.1+ indicate highly pure RNA). Table 2 shows results confirming the abundant yield of intact mRNA. [Table 2]

[0100] More reliable results are expected by using samples incubated with polyethylene glycol conjugated to boronic acid or samples incubated with trehalose polymers.

[0101] Example 4 When storing biological samples from non-sterile environments, there is always the possibility of contamination with materials from non-target organisms. To address this, procedures have been developed to selectively separate biological samples to eliminate unwanted bacterial contamination. A three-layer system was formed by assembling two layers, either an 8 μm nitrocellulose membrane bound to mannose-binding protein (MBL) or an 8 μm nitrocellulose membrane in its commercially available form, on top of a PES membrane, and vitrification was carried out using this system in the same manner as in Example 1.

[0102] To test the ability to selectively isolate desired nucleic acids over bacterial nucleic acids, three samples of various bacterial strains were constructed to run on the assembly system. The bacteria used in the test were E. coli, Staphylococcus epidermidis, and Pseudomonas aeruginosa. Each bacterium was cultured in a medium at 10 4 100 μL of bacterial material was diluted to 10 colony forming units (CFU). 6 The pellet containing each bacterium was then resuspended. The resuspended solution was then applied to the nitrocellulose membrane surface of the assembled three-layer membrane system and incubated at room temperature for 30 minutes. The three membranes were then separated and washed with PBS. The wash solution was then subjected to cell counting, plated on agar plates, and incubated at 37°C for 2 days. Table 3 shows the results for E. coli as an average of two experiments performed in triplicate. [Table 3]

[0103] Table 4 shows the results as the mean of two experiments performed in triplicate for S. epidermidis. [Table 4]

[0104] These results demonstrate that the MBL-binding filter selectively and reliably binds bacteria in samples, confirming that this system can selectively remove contaminating bacteria from samples containing mRNA.

[0105] The PES membranes were also analyzed for cell counts. The PES membranes were washed with PBS, and the cells were subjected to cell counts. Table 5 shows the results as the average of two experiments performed in triplicate. [Table 5]

[0106] In both cases, the PES membranes recovered >84% of the original cell mass, and the amount of Jurkat cells was independent of whether the nitrocellulose layer contained MBL or not. These results demonstrate a robust filter system that is selective for bacterial cells and can be used for the selective preservation of biological cells. [Application example] (Application example 1) 1. A method for preserving a biological sample, comprising: providing a biological sample containing at least one cell; contacting the biological sample with a vitrification medium comprising a vitrification agent and a dissolution agent to form a vitrification mixture; vitrifying the vitrification mixture, thereby producing a storage-stable sample. (Application example 2) In the method according to Application Example 1, storing the storage-stable sample at a temperature of 16°C or higher and 30°C or lower (optionally above 30°C (optionally above 50°C)). (Application example 3) The method according to Application Example 1 or 2, wherein the biological sample contains nucleic acid (optionally RNA). (Application example 4) A method according to any one of Application Examples 1 to 3, wherein the biological sample comprises whole blood, plasma, or serum. (Application example 5) The method according to Application Example 4, wherein the biological sample comprises whole blood, serum, or plasma, and the vitrification mixture further comprises an anticoagulant. (Application example 6) A method according to any one of Application Examples 1 to 5, wherein the vitrification mixture further comprises a buffer. (Application example 7) The method according to any one of Application Examples 1 to 6, wherein the vitrification agent comprises dimethyl sulfoxide, glycerol, sugars, polyhydric alcohols, methylamines, betaines, antifreeze proteins, antinucleating agents, polyvinyl alcohol, cyclohexanetriols, cyclohexanediols, inorganic salts, organic salts, ionic liquids, or combinations thereof. (Application example 8) The method according to Application Example 7, wherein the vitrification agent comprises trehalose. (Application example 9) A method according to any one of Application Examples 1 to 8, wherein the dissolution agent comprises a detergent. (Application example 10) The method described in Application Example 9, wherein the detergent comprises sodium dodecyl sulfate (SDS), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (e.g., Triton X-100), (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) (CHAPS), guanidine hydrochloride, or a combination thereof. (Application example 11) The method according to any one of Application Examples 1 to 10, further comprising: enclosing the storage-stable sample in a water- and air-impermeable protective enclosure; storing the protective enclosure at a temperature of -196°C to +60°C for a storage period of 20 days or more; A method comprising: (Application example 12) 12. The method according to any one of Application Examples 1 to 11, wherein the step of vitrifying the vitrification mixture comprises drying the vitrification mixture at a temperature higher than cryogenic temperatures until the vitrification mixture is in a glassy state. (Application example 13) The method described in Application Example 12, wherein the vitrification medium is located within one or more capillary channels. (Application example 14) A method according to any one of Application Examples 1 to 13, wherein the vitrification process is carried out for a drying time of 1 second to 1 hour (optionally, 10 minutes or less). (Application example 15) The method according to any one of Application Examples 1 to 14, further comprising: The method comprises the step of incubating the vitrification mixture for at least 5 minutes and not more than 30 minutes prior to vitrification. (Application Example 16) The method according to Application Example 15, wherein the culture is carried out at a temperature of 18°C ​​or higher and 37°C or lower. (Application example 17) A method according to any one of Application Examples 1 to 16, wherein the biological sample is pretreated by contacting it with a separation medium containing at least one separation agent. (Application example 18) The method according to Application Example 17, wherein the separation agent is a mannose-binding lectin. (Application Example 19) The method according to Application Example 17, wherein the separation medium is made of nitrocellulose or polyvinylidene difluoride. (Application Example 20) A method according to any one of Application Examples 1 to 19, wherein the biological sample comprises tissue and the vitrification mixture further comprises a support material (optionally, the support material comprises a polymer support material). (Application Example 21) The method according to Application Example 20, wherein the polymer is suitable for forming a hydrogel. (Application example 22) The method according to Application Example 20, wherein the polymer is polyethylene glycol. (Application Example 23) 23. The method according to any one of Application Examples 20 to 22, wherein the vitrification medium further comprises at least one adhesion agent. (Application Example 24) The method according to Application Example 23, wherein the attachment agent is a boronic acid. (Application Example 25) 25. The method of any one of Applications 20 to 24, wherein the support material is a switchable support material, the method further comprising: applying a stimulus to the vitrification mixture to convert the state-transformable support material into a gel state.

[0107] While various embodiments are disclosed herein, it will be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various alternative forms. Therefore, the specific details disclosed herein should not be construed as limiting, but merely as a representative basis for any embodiment of the invention and / or for teaching those skilled in the art how to variously employ the teachings disclosed herein. Furthermore, the terminology used herein is used only for the purpose of describing particular embodiments of the invention and is not intended to be limiting in any way.

[0108] Although terms such as "first," "second," and "third" may be used herein to describe various elements, components, regions, layers, and / or sections, it will be understood that these elements, components, regions, layers, and / or sections are not limited by these terms. These terms are used only to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. Thus, a "first element," "component," "region," "layer," or "section" described below could be referred to as a second (or other) element, component, region, layer, or section without departing from the teachings herein.

[0109] The terms used herein are for the purpose of describing particular aspects only and are not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural, including "at least one," unless the content clearly dictates otherwise. "Or" means "and / or." As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. As used herein, the terms "comprise" and / or "comprising," or "includes" and / or "including," specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but it will be further understood that they do not exclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and groups thereof. The term "or combinations thereof" means combinations including at least one of the aforementioned elements.

[0110] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Terms as defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the relevant art and this disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly defined as such herein.

[0111] Throughout this application, where publications are referenced, the disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this disclosure pertains.

[0112] While embodiments of the invention have been illustrated and described, it is not intended that these embodiments illustrate and describe all possible forms of the invention. Rather, the words used herein are words of description rather than limitation, and it will be understood that various modifications and substitutions can be made thereto without departing from the spirit and scope of the invention.

Claims

1. 1. A method for preserving tissue at temperatures above cryogenic temperatures, comprising: providing a biological tissue containing at least one cell; The biological tissue is then vitrified with a vitrification agent and a support material (optionally a polymer support). contacting the vitrification agent with a vitrification medium containing a vitrification agent (vitrification agent) to form a vitrification mixture, wherein the vitrification agent is a substance that forms an amorphous structure or inhibits the crystal formation of the other material when the mixture of the vitrification agent and the other material is cooled or dried; A process of vitrifying the vitrification mixture, thereby obtaining a storage-stable sample. and generating a material.

2. The method of claim 1 , wherein the support material is a polymeric support material.

3. 3. The method of claim 2, wherein the polymeric support material is suitable for forming a hydrogel.

4. The method of claim 2 wherein the polymeric support material is polyethylene glycol.

5. 5. The method of claim 1, wherein the vitrification medium further comprises at least one adhesion agent.

6. 6. The method of claim 5, wherein the attachment agent is a boronic acid.

7. 5. The method of any one of claims 1 to 4, wherein the support material is a switchable support material, the method further comprising applying a stimulus to the vitrification mixture to convert the switchable support material to a gel state. A process comprising a method.

8. 5. The method of any one of claims 1 to 4, wherein the vitrification mixture further comprises a buffer.

9. 5. The method according to claim 1, wherein the vitrifying agent is selected from the group consisting of dimethyl sulfoxide, glycerol, sugars, polyhydric alcohols, methylamines, and betaines. , antifreeze proteins, synthetic antinuclear agents, polyvinyl alcohol, cyclohexanetriols, cyclohexanediols, inorganic salts, organic salts, ionic liquids, or combinations thereof.

10. 10. The method of claim 9, wherein the vitrification agent comprises trehalose.

11. The method of any one of claims 1 to 4, further comprising: enclosing the storage-stable sample in a water- and air-impermeable protective enclosure; storing the protective enclosure at a temperature between −196° C. and +60° C. for a storage period of up to or greater than 20 days; A method comprising:

12. 5. The method of claim 1, wherein the step of vitrifying the vitrification mixture comprises drying the vitrification mixture at a temperature greater than cryogenic temperatures until the vitrification mixture is in a glassy state.

13. 5. The method of claim 1, wherein the vitrification mixture is in one or more capillary channels.

14. 5. The method according to any one of claims 1 to 4, wherein the vitrifying step is carried out for a drying time of between 1 second and 1 hour.

15. The method of any one of claims 1 to 4, further comprising: Incubating the vitrification mixture for at least 5 minutes and not more than 30 minutes prior to vitrification.

16. The method of claim 15, wherein the culturing is carried out at a temperature of 18°C ​​or higher and 37°C or lower.

17. 5. The method of any one of claims 1 to 4, wherein the vitrification mixture further comprises a dissolving agent.

18. 18. The method of claim 17, wherein the lysing agent comprises sodium dodecyl sulfate (SDS), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonato) (CHAPS), guanidine hydrochloride, or a combination thereof.

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