Capillary-Assisted Vitrification Process and Materials for Preserving Biological Samples

Abstract

The present invention discloses a method for storing nucleic acids derived from a biological sample. The method includes providing a biological sample that contains one or more cells containing nucleic acids therein; contacting the biological sample with a vitrification medium containing a vitrification agent and a lysis agent to form a vitrified mixture; and vitrifying the vitrified mixture to produce a storage-stable sample. In various aspects, the storage-stable sample can be stored at a temperature above cryogenic temperature, for example, at room temperature, for 20 days or longer.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 982,856, filed on February 28, 2020, the entire content of which is incorporated herein by reference. Technical field

[0003] The present disclosure relates to the preservation of biological samples, particularly to the vitrification of biomaterials for preserving blood, saliva, or tissue samples or portions thereof. Background art

[0004] Vitrification is the process of direct transformation from a liquid to an amorphous glassy state, and is commonly used to preserve biomaterials by cooling the biomaterials to cryogenic temperatures at a high cooling rate. At cryogenic temperatures, vitrification techniques avoid the damaging effects of ice crystals known to form during conventional cryopreservation. However, to avoid ice nucleation during cooling, cryoprotective agents (CPAs) at extremely high and potentially toxic concentrations (6 - 8 M) are required. The most commonly used CPAs include dimethyl sulfoxide (DMSO), glycerol, ethylene glycol (EG), and 1,2 - propanediol (PROH). As a result, multiple steps and complex elaborate protocols are required to load and unload CPAs into and from cells.

[0005] Anhydrous vitrification at ambient temperature may be an alternative strategy for preserving biomaterials. In nature, a wide variety of organisms can survive extreme dehydration, which in many cases is associated with the accumulation of large amounts (up to 20% of their dry weight) of glass - forming sugars (such as trehalose and sucrose) within cells. However, the cumulative chemical stress that arises as the vitrification solution concentrates in the extracellular space can lead to the degradation of biomaterials, and thus dry preservation is severely limited for long - term storage. This results in irreversible damage to cells, including damage to proteins and nucleic acids, before the cells and the vitrification solution can reach a suitable low water content to become glassy. Therefore, improved vitrification methods are needed to preserve proteins and nucleic acids while vitrifying biomaterials through rapid drying. Summary of the invention

[0007] To facilitate understanding of the various aspects described herein, the following overview is provided, but it is not intended as a complete description. A full understanding of the various aspects can be obtained by taking the entire specification, claims, drawings, and abstract as a whole.

[0008] Multiple aspects of the present disclosure provide methods for storing cells, blood, saliva, tissue, or other samples from an organism, as well as proteins, particularly intracellular nucleic acids or other biological materials, in a biological sample. The method includes: providing a biological sample that includes one or more cells containing nucleic acids therein; contacting the biological sample with a vitrification medium that includes a cryoprotectant and a lysing agent to form a vitrified mixture; and vitrifying the vitrified mixture to produce a storage-stable sample. In multiple aspects, the storage-stable sample can be stored at a temperature above cryogenic temperatures, such as room temperature or higher, and optionally can be stored for 20 days or longer.

[0009] The present disclosure also provides methods for preserving tissue samples that provide structural support and do not introduce cell-damaging materials, thereby more effectively preserving cell structure for subsequent histological or other tissue studies. One method includes contacting a target biological tissue with a polymer, a cryoprotectant, and optionally a crosslinking agent and / or energy suitable for attaching the polymer to extracellular structures of cells in the tissue; vitrifying the tissue to form a vitrified tissue sample, and optionally slicing the tissue (before or after vitrification) into one or more thin tissue strips. The method optionally further includes rehydrating the vitrified tissue with a releasing agent and / or energy that releases the polymer from the tissue, thereby making the well-preserved living tissue available for subsequent analysis. Brief Description of the Drawings

[0011] The drawings are not necessarily to scale; certain features may be enlarged or minimized to show details of particular components. Accordingly, the specific structural and functional details disclosed herein should not be construed as limiting, but merely as a representative basis for teaching one of ordinary skill in the art to use the invention in various ways. Exemplary aspects can be more fully understood from the detailed description and the drawings, wherein:

[0012] Figure 1 An exemplary vitrification method that can be used in accordance with one or more aspects shown and described herein is schematically depicted;

[0013] Figure 2 Another exemplary vitrification method in accordance with one or more aspects shown and described herein is schematically depicted, which illustrates a biological sample of blood cells and a vitrification medium in a membrane for vitrification;

[0014] Figure 3 An exemplary method for extracting RNA from a storage-stable sample produced in accordance with one or more aspects shown and described herein is schematically depicted;

[0015] Figure 4 An exemplary system for separating desired materials and optionally separating contaminants such as bacteria or other unwanted organisms is described;

[0016] Figure 5 is an image of an electrophoresis gel showing the degradation of RNA extracted from Comparative Examples A - C (using conventional nucleic acid storage techniques) and Example 1 (using a nucleic acid storage method according to one or more aspects shown and described herein);

[0017] Figure 6A shows the GAPDH cycle threshold of Example B according to one or more aspects shown and described herein and Comparative Examples D and E;

[0018] Figure 6B shows the fold change in GAPDH cycle threshold of Example B according to one or more aspects shown and described herein and Comparative Examples D and E; and

[0019] Figure 6C shows the fold change in VEGF mRNA of Example B according to one or more aspects shown and described herein and Comparative Examples D and E. DETAILED DESCRIPTION OF THE INVENTION

[0021] The aspects described herein provide a method for preparing and storing cells, tissues, or cellular materials such as proteins (exemplary antibodies or other protein materials), nucleic acids, or other cellular materials using a vitrification medium comprising a vitrification agent and optionally a lysis agent. In aspects, a biological sample, including one or more cells containing nucleic acids, can be contacted with the vitrification medium and vitrified to produce a storage - stable sample. Specifically, aspects of the present invention allow for the preservation and storage of nucleic acids at temperatures above cryogenic temperatures, more specifically, at room temperature or higher, while maintaining robust, well - preserved biological materials. For example, these aspects can significantly reduce storage costs by eliminating the need for refrigeration or freezing and can simplify sample preparation for storage without adversely affecting the quality of nucleic acids (e.g., DNA and / or RNA). Thus, thereafter, after reconstitution, high - quality tissues, cells, proteins, nucleic acids, including RNA and DNA, can be used.

[0022] In addition, the aspects described herein can allow for the processing and storage of saliva, blood, tissue, or other biological samples with a minimal level of pretreatment, thereby allowing the sample to be used for any one of a variety of uses after storage. For example, a tissue sample of a tumor can be preserved for a long time and then processed according to a suitable protocol, such as for subsequent RNA mapping analysis. As another example, whole blood samples can be stored without first separating plasma from red and white blood cells, enabling any one or more components of the stored sample to be used later.

[0023] The following terms or phrases used herein have the exemplary meanings set forth below in connection with at least one aspect:

[0024] "Amorphous" or "glass" refers to an amorphous material in which there is no long-range order in the atomic positions, with an order parameter of 0.3 or less. At the glass transition temperature Tg, a liquid transforms into a glassy solid. In some aspects, the vitrification medium can be or form an amorphous material. In other aspects, a biomaterial can be an amorphous material.

[0025] "Glass transition temperature" means that above this temperature, the material behaves like a liquid; below this temperature, the material behaves like a solid phase and enters the amorphous / glass state. It is not a fixed temperature point but varies depending on the time scale of the measurement used. In some aspects, the glassy state can refer to the state that a biological composition enters when cooled below its glass transition temperature. In other aspects, the glassy state can refer to the state that a vitrification mixture and / or a vitrification agent enters when cooled below its glass transition temperature. In other aspects, the glassy state can have the mechanical rigidity of a crystal or a gel but have the random molecular disorder characteristic of a liquid.

[0026] "Crystal" refers to a three-dimensional atomic, ionic, or molecular structure composed of a specific ordered geometric array that is periodically repeated and called a lattice or unit cell.

[0027] "Crystallization" refers to a form of matter composed of components arranged in an ordered structure at the atomic level, which is different from glassy or amorphous. The solidification of a crystalline solid occurs at the crystallization temperature Tc.

[0028] As used herein, "vitrification" is the process of transforming a material into an amorphous material. The amorphous solid can be free of any crystalline structure.

[0029] As used herein, "vitrification mixture" refers to a heterogeneous mixture of (one or more) biomaterials and a vitrification medium comprising one or more vitrification agents, optionally a lysing agent, and optionally other materials.

[0030] As used herein, "biomaterial" or "biological sample" refers to a material that can be isolated or derived from a living organism. Examples of biomaterials include, but are not limited to, proteins, cells, tissues, organs, cell-based constructs, blood or its fractions, nucleic acids, or combinations thereof. In some aspects, the biomaterial can refer to mammalian cells. In other aspects, the biomaterial can refer to human mesenchymal stem cells, murine fibroblasts, white blood cells, red blood cells, platelets, bacteria, viruses, mammalian cells, liposomes, enzymes, tissues (such as intestine, liver, neurons, or others), or combinations thereof. In other aspects, the biomaterial can refer to germ cells, including sperm cells, spermatocytes, oocytes, eggs, embryos, blastocysts, or combinations thereof. In other aspects, the biomaterial can refer to whole blood, red blood cells, white blood cells, platelets, plasma, serum, algae, fungi, or combinations thereof.

[0031] As used herein, a "vitrification agent" is a material that forms an amorphous structure or inhibits crystal formation in other materials when a mixture of the vitrification agent and other materials is cooled or dried. The vitrification agent can also provide cryoprotection or otherwise enable cells to survive the dehydration process. In some aspects, the vitrification agent can be any water-soluble solution that produces an amorphous structure suitable for biomaterial storage. In other aspects, the vitrification agent can be absorbed within cells, tissues, or organs.

[0032] As used herein, "storable", "storage", or "storage stable" means that a biomaterial can be preserved and remain viable for subsequent use.

[0033] As used herein, "above cryogenic" refers to a temperature above -80°C. As used herein, room temperature refers to a temperature range from greater than or equal to 18°C to less than or equal to 37°C.

[0034] As used herein, "hydrophilic" refers to attracting water molecules or preferentially associating with water molecules. Hydrophilic materials with a specific affinity for water maximize contact with water and have a small contact angle with water.

[0035] As used herein, "hydrophobic" refers to a lack of affinity for water. Hydrophobic materials naturally repel water, resulting in the formation of droplets and having a small contact angle with water.

[0036] As used herein, "ambient temperature" refers to a temperature greater than or equal to about 16°C and less than or equal to about 30°C.

[0037] Multiple aspects described herein provide methods for storing biological materials from biological samples. According to various aspects, a biological sample containing one or more cells is contacted with a vitrification medium to form a vitrified mixture. As will be described in more detail below, the vitrification medium comprises at least a vitrification agent and optionally a lysing agent. The vitrified mixture is vitrified to produce a storage-stable sample that can be stored until further use. The storage-stable sample can then be rehydrated and processed to extract or characterize the biological material or a portion thereof, which can then be used for quantitative, qualitative, and / or clinical analysis.

[0038] In various aspects, the vitrification medium comprises at least a vitrification agent and a lysing agent. Exemplary examples of vitrification agents include, but are not limited to, dimethyl sulfoxide, glycerol, sugars (such as trehalose, etc.), polyols, methylamine compounds, betaine compounds, antifreeze proteins, synthetic anti-nucleating agents, polyvinyl alcohol, cyclohexanetriol, cyclohexanediol, inorganic salts, organic salts, ionic liquids, or combinations thereof. In some aspects, the vitrification medium includes 1, 2, 3, 4, or more vitrification agents.

[0039] The vitrification agent is included in the vitrification medium at a concentration depending on the characteristics of the vitrification agent. In some aspects, the concentration of the vitrification agent is below a concentration that is toxic to the biological sample being vitrified. As used herein, "toxic" means that functional or biological viability cannot be achieved upon subsequent sample use, or the biological sample is not suitable for subsequent analysis. In various aspects, the concentration of the vitrification agent is greater than or equal to 500 micromoles (μM) and less than or equal to 6 moles (M), or any value or range therebetween. As an example, trehalose is included at a concentration greater than or equal to 1 millimole (mM) and less than or equal to 6 M, optionally greater than or equal to 150 mM and less than or equal to 6 M in various aspects. In some aspects, when combined, the total concentration of all vitrification agents is greater than or equal to 1 mM and less than or equal to 6 M, optionally greater than or equal to 1 mM and less than or equal to 6 M.

[0040] The vitrification medium provided herein optionally includes a lysing agent. The lysing agent is included in the vitrification medium to cause partial or complete perforation of the cell membrane and optionally the cell nucleus or otherwise make it more porous, thereby facilitating the entry of the vitrification agent into the cell and the departure of water from the cell, thus achieving rapid vitrification. The lysing agent can be, for example but not limited to, a detergent. Suitable detergents can include sodium dodecyl sulfate (SDS), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (e.g., Triton X-100), CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), guanidine hydrochloride, other similar reagents, and combinations thereof. Other lysing agents are also possible, provided they do not interfere with the vitrification process or are non-toxic to the desired biological material. In various aspects, the lysing agent is present in the vitrification medium in an amount of 0.01 weight percent (wt%) to 5 wt% or greater. The specific amount of the lysing agent can vary depending on the specific aspect, and more specifically, the desired level of cell membrane permeability. For example, in some aspects, the lysing agent can completely lyse the cells, while in other aspects, the lysing agent can merely cause punctures or perforations in the cell membrane to facilitate improved transmembrane transfer of the vitrification agent.

[0041] It can be contemplated that, in some aspects, the vitrification medium can further include other components, such as but not limited to, water or other solvents, buffers, one or more salts, RNase or DNAse inhibitors, or combinations thereof. A buffer is any reagent having a pKa of 6 to 8.5 at 25°C. Illustrative examples of buffers include choline, betaine, HEPES, TRIS, PIPES, MOPS, etc. In some aspects, the buffer is a buffer containing large organic ions (greater than 120 kDa), such as choline, betaine, or HEPES. In aspects including a buffer, the buffer is provided at a concentration suitable for stabilizing the pH of the vitrification medium at the desired level.

[0042] The salt can include, for example but not limited to, sodium salts, potassium salts, chloride salts, or combinations thereof. When included in the vitrification medium, the salt can be provided at a concentration greater than or equal to 1 millimole (mM) to less than or equal to 500 mM. For example, the salt can be present at the following concentrations: greater than or equal to 1 mM to less than or equal to 500 mM, greater than or equal to 1 mM to less than or equal to 400 mM, greater than or equal to 1 mM to less than or equal to 300 mM, greater than or equal to 1 mM to less than or equal to 250 mM, greater than or equal to 1 mM to less than or equal to 200 mM, greater than or equal to 1 mM to less than or equal to 150 mM, greater than or equal to 1 mM to less than or equal to 100 mM, greater than or equal to 1 mM to less than or equal to 75 mM, greater than or equal to 1 mM to less than or equal to 50 mM, greater than or equal to 1 mM to less than or equal to 25 mM, greater than or equal to 25 mM to less than or equal to 500 mM, greater than or equal to 25 mM to less than or equal to 400 mM, greater than or equal to 25 mM to less than or equal to 300 mM, greater than or equal to 25 mM to less than or equal to 250 mM, greater than or equal to 25 mM to less than or equal to 200 mM, greater than or equal to 25 mM to less than or equal to 150 mM, greater than or equal to 25 mM to less than or equal to 100 mM, greater than or equal to 25 mM to less than or equal to 75 mM, greater than or equal to 25 mM to less than or equal to 50 mM, greater than or equal to 50 mM to less than or equal to 500 mM, greater than or equal to 50 mM to less than or equal to 400 mM, greater than or equal to 50 mM to less than or equal to 300 mM, greater than or equal to 50 mM to less than or equal to 250 mM, greater than or equal to 50 mM to less than or equal to 200 mM, greater than or equal to 50 mM to less than or equal to 150 mM, greater than or equal to 50 mM to less than or equal to 100 mM, greater than or equal to 50 mM to less than or equal to 75 mM, or any and all ranges or sub-ranges included therein.

[0043] In some aspects, an RNase and / or DNase inhibitor can be included in the vitrification medium to prevent nucleic acid degradation. Any known RNase and / or DNase inhibitor known and used in the art can be used, provided that they do not interfere with vitrification. However, it should be understood that in many aspects, an RNase and / or DNase inhibitor may not be necessary for preserving nucleic acids.

[0044] In some aspects, such as when the vitrification medium is to be used in conjunction with a blood sample, the vitrification medium can further include at least one anticoagulant. Alternatively, the blood sample can be collected into one or more anticoagulants and then contacted with the vitrification medium. Suitable anticoagulants can include, for example but not limited to, ethylenediaminetetraacetic acid (EDTA), oxalates, heparin, sodium citrate, sodium fluoride, and combinations thereof. When included, the anticoagulant is present in the vitrification medium or otherwise used in an amount of from 0.1 mg / mL to 5 mg / ml. In some aspects, the amount of anticoagulant varies depending on the specific anticoagulant selected. For example, EDTA can be included in an amount of 1 - 2 mg / mL of blood, heparin can be included in an amount of 0.2 mg / mL of blood, oxalate can be included in an amount of 1 - 2 mg / mL of blood, and sodium fluoride can be included in an amount of 2 mg / mL of blood. As another example, sodium citrate can be included in a ratio of 1:9, where 9 parts is blood and 1 part is sodium citrate. As will be understood by those skilled in the art, the anticoagulant can be present in other amounts to prevent the blood sample from clotting.

[0045] According to various aspects, a biological sample containing one or more biological materials is contacted with a vitrification medium to form a vitrified mixture. In some aspects, the vitrified mixture is incubated prior to vitrification. For example, the vitrified mixture can be incubated for greater than or equal to 5 minutes and less than or equal to 60 minutes, greater than or equal to 5 minutes and less than or equal to 45 minutes, greater than or equal to 5 minutes and less than or equal to 30 minutes, or greater than or equal to 5 minutes and less than or equal to 20 minutes. The incubation can be carried out at any suitable temperature, and in various aspects, can be at room temperature (i.e., from greater than or equal to 18°C to less than or equal to 37°C, optionally about 25°C). After incubation, the vitrified mixture, including the biological sample and the vitrification medium, is vitrified to produce a storage-stable sample. Vitrification can be carried out according to any known vitrification method.

[0046] Vitrified materials are typically prepared by rapidly cooling a liquid material or by directly immersing a small amount of biological material into liquid nitrogen. Cooling reduces the mobility of the molecules of the material, and then they can pack into a more thermodynamically favorable crystalline state. Additives that interfere with the crystallization ability of the main components can produce an amorphous / vitrified material. In the presence of a suitable glass former, biological materials can be stored in a vitrified matrix at a temperature above deep cryogenic temperatures, and vitrification can be achieved by dehydration.

[0047] Some animals and many plants can survive in a completely dehydrated state. This ability to survive in the dry (dehydrated) state depends on several complex intracellular physiological and genetic mechanisms. Among these mechanisms, sugars (such as carbohydrates, disaccharides, oligosaccharides) accumulate within the cell, and these sugars act as protectants during drying. Trehalose is an example of a disaccharide that is naturally produced in desiccation-tolerant organisms.

[0048] Sugars such as trehalose can provide protection to desiccation-tolerant organisms in several different ways. Due to the unique positions of the hydroxyl groups on the trehalose molecule, the trehalose molecule can effectively displace water molecules hydrogen-bonded to the surface of folded proteins without altering their conformational geometry and folding. The sugar molecule can also prevent cytoplasmic leakage during rehydration by binding to the phospholipid heads of the lipid bilayer. In addition, many sugars have a high glass transition temperature, which enables them to form a glass above deep cryogenic temperatures or a room-temperature glass at low water contents. The highly viscous "glass" state reduces the molecular mobility, thereby preventing degradative biochemical reactions that lead to cell function deterioration and death as well as protein and nucleic acid degradation.

[0049] In some aspects, vitrification of a biological sample involves dehydration in the presence of a glass-forming sugar - trehalose - as has been disclosed, for example, in N Chakraborty et al., Biopreservation and Biobanking, 2010, 8(2), 107 - 114. Refer Figure 1 As shown, system 10 is the most commonly used method for dehydrating biological materials. The sessile droplet 11 is placed on the substrate 12 and evaporated dry in a housing 16 having a low-humidity environment 13. The humidity, pressure, and temperature within the housing can be operably controlled by a control device 17. However, the dehydration drying of the sessile droplet by evaporation using system 10 is slow and non-uniform in nature. When the biological material is dried in a glass-forming medium, a glassy skin forms at the liquid / vapor interface 14 of the sample. This glassy skin slows down and ultimately prevents further drying of the sample beyond a certain drying level and results in significant spatial non-uniformity of water in the sample. As a result, the cells in the partially dried sample trapped under the glassy skin may not vitrify but instead undergo degradation due to high molecular mobility.

[0050] In other aspects, vitrification of a biological sample involves dehydration using capillary-assisted drying in the presence of the glass-forming sugar trehalose as has been disclosed, for example, in U.S. Patent No. 10,433,540. An example device for performing such a process is shown in Figure 2 shown.

[0051] The vitrification process can be carried out by vitrifying on or within a membrane, which can include one or more capillary channels, optionally continuous capillary channels. The capillary can provide an interface for rapid evaporation. The membrane can be formed by a plurality of capillary channels, optionally by a plurality of continuous capillary channels. The capillary network formed of a porous material such as a membrane can be made of a material that is non-toxic and non-reactive to biological materials or biological samples and does not chemically or physically react with the vitrification medium. The membrane can be made of a hydrophilic or a material that has been modified to be hydrophilic. In some aspects, optionally using a support structure as described below, the membrane can be partially soluble or have time / stimulus-dependent solubility in the vitrification mixture or reconstitution solution. The membrane material can be a suitable polymer, metal, ceramic, glass, or a combination thereof. In some aspects, the continuous capillary network is formed of materials such as polydimethylsiloxane (PDMS), polycarbonate, polyurethane, polyethersulfone (PES), polyester (e.g., polyethylene terephthalate), etc. Illustrative examples of capillary channel-containing membranes suitable as surfaces in the devices and methods provided herein include hydrophilic filter membranes, such as those sold by EMD Millipore, Bellerica, MA. In certain aspects, the porous material does not substantially bind or alter the components of the biological sample and / or the vitrification medium, or otherwise chemically or physically associate with it. The porous material is optionally not derivatized. Optionally, the capillary channels can be formed in a substrate (such as a dry chamber wall) having the desired material and thickness by PDMS forming techniques, laser drilling, or other drilling forming techniques known in the art.

[0052] In some aspects, the capillary network provided by the porous material includes pores having a cross-sectional dimension of about 100 μm or less, optionally 20 μm or less, whereby the pores can provide underlying capillaries to assist in vitrification. When the capillary network is used for vitrifying tissue, a larger pore size can optionally be used, such as a cross-sectional dimension of about 100 μm or less. When using cell samples (as opposed to tissue) in some aspects, a cross-sectional dimension of about 20 μm or less can be used. In some aspects, the average opening of the pores can be from about 100 μm to about 0.1 μm, including about 90, 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, and 0.2 μm. The capillary channels can have a length defined optionally by the thickness of the substrate forming the channels, or by the length of one or more individual channels themselves. The capillary channel length is optionally about one millimeter or less, but should not be construed as limited to these dimensions. Optionally, the capillary channel length is from about 0.1 micrometer to about 1000 micrometers, or any value or range therebetween. Optionally, the capillary channel length is from about 5 to about 100 micrometers, optionally from about 1 to about 200 micrometers, and / or optionally from about 1 to about 100 micrometers. The capillary channel length is optionally about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 micrometers. In some aspects, the length of the capillary channels varies among the plurality of capillary channels, optionally non-uniformly.

[0053] The cross-sectional area of the capillary channels can be about 8000 μm 2 or less, optionally 2000 μm 2 or less. Optionally, the cross-sectional area is from about 0.01 μm 2 to about 8000 μm 2 and optionally from about 100 μm 2 to about 2000 μm 2 or any value or range therebetween. Optionally, the cross-sectional area of the (one or more) capillary channels is about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000 μm 2 or less.

[0054] As Figure 2As shown, the capillary-assisted vitrification device 20 includes a capillary plate / membrane 22 placed within a housing 28. A biological sample is illustratively shown within the pores of the membrane. Optionally, in some aspects, the capillary membrane can be replaced by several filter layers or other membranes without a majority of distinct channels. In other aspects, a plurality of membranes can be used for the biological sample, where the biological sample is sandwiched between the membranes or otherwise contained within the plurality of membranes, and when the plurality of membranes are stacked together, they form a vitrification membrane suitable for vitrifying the biological sample. The plate / membrane 22 optionally includes a plurality of capillary channels, optionally substantially parallel capillary channels, each capillary channel having a first opening 23 and a second opening 25. A vitrification mixture 24 is placed on the first opening 23, and it can find its way into the capillary channels, and further, the surface of the vitrification mixture 24 is exposed to the surrounding atmosphere 29 through the second opening 25. In a plurality of aspects, the surrounding atmosphere 29 has a humidity lower than that of the vitrification mixture. Vitrification is accomplished by capillary action to dehydrate and dry the vitrification mixture until the vitrification mixture enters a vitrified state. The chemistry, humidity, pressure, and temperature within the housing 28 are controlled by one or more control mechanisms 21.

[0055] For illustrative purposes only, the control mechanism 21 is simplified; the control mechanism 21 can have multiple systems and mechanisms to obtain the most favorable conditions for drying and vitrification. In some aspects, the vitrification mixture 24 is optionally sandwiched between two plates / membranes similar to 22 to benefit from the capillary-assisted drying method at the upper and lower surfaces of the vitrification mixture 24.

[0056] In some aspects, a low-humidity (less than 30% relative humidity) gas flow is provided across the second opening 25 of the capillary plate / membrane or the opposite side of the vitrification medium of the membrane to enhance the capillary effect. An inert or relatively inert gas, such as nitrogen, argon, xenon, or other gases, can be used as the low-humidity gas. In some aspects, a reduced pressure or vacuum is maintained within the housing 28. In some aspects, suction / pressure is provided across the second opening 25 to achieve an increased drying rate. It should be noted that maintaining a low-humidity environment (optionally 5% relative humidity or lower) is crucial for preventing rehydration after drying. More details regarding capillary-assisted drying can be found in U.S. Patent No. 10,433,540.

[0057] The vitrification method can be carried out at a temperature ranging from -80°C to +60°C. The temperature range is optionally such that the mobility of water molecules in the sample is high and the temperature does not damage the health and vitality of the biological material. This can vary depending on the material and the composition of the vitrification medium. In some aspects, the vitrification temperature is from 0.1°C to 40°C. Optionally, the vitrification temperature is from 4°C to 26°C. Optionally, the vitrification temperature is about 25°C.

[0058] 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. In some aspects, the humidity level of the environment, such as the environment on the second side of the capillary, is 30% or lower relative humidity, optionally 20% or lower, optionally 10% or lower, optionally 5% or lower. The humidity of the dry environment is optionally between 1% and 30% or any value or range therebetween, optionally 1% to 5%.

[0059] The vitrification method can be carried out in a low-pressure environment (less than 1 atm (760 mmHg)). The low-pressure environment will have a favorable effect on the vitrification rate. The environmental pressure can be selected as 100 mmHg or 0.1 atm. Optionally, the environmental pressure is 10 mmHg to 760 mmHg, or any value or range therebetween. Optionally, the environmental pressure is 10 mmHg to 200 mmHg.

[0060] The vitrification method can be carried out with a drying time. The drying time is the time sufficient to promote suitable drying to vitrify the vitrification medium. The drying time is optionally 1 second to 1 hour. Optionally, the drying time is 1 second to 50 minutes, optionally 5 seconds to 60 minutes. The drying time can vary depending on the sample type or physical properties and the details of the capillary channel.

[0061] In other aspects, vitrification can be carried out on or between a membrane or filter paper. Depending on the specific aspect, other vitrification methods can be used.

[0062] After vitrification, the sample is storage-stable and can be stored at a temperature higher than cryogenic while remaining viable and not undergoing significant degradation for subsequent use. In some aspects, after vitrification, the vitrification mixture can be encapsulated in an airtight and watertight protective casing in a glassy state and stored. In some aspects, the storage-stable sample, before use, can be stored at a temperature greater than or equal to -196°C to less than or equal to +60°C or higher, greater than or equal to 16°C to less than or equal to 60°C or higher, or greater than or equal to 18°C to less than or equal to 60°C or higher, for a period of time. In some aspects, the storage time is greater than or equal to 1 day, greater than or equal to 5 days, greater than or equal to 10 days, greater than or equal to 20 days, greater than or equal to 30 days, greater than or equal to 45 days, greater than or equal to 60 days, or longer.

[0063] In multiple aspects, when the stored stable sample is ready for use, it is rehydrated (or reconstituted) and processed according to a specific protocol for using the sample. In some aspects, a rehydration solution for precipitating proteins or one or more types of nucleic acids can be used to rehydrate the stored stable sample. In some aspects, the stored stable sample is reconstituted using a lysis buffer, for example, a lysis buffer can be included in an extraction and / or purification kit for processing the stored stable sample to completely lyse the cell material.

[0064] Although the sample can be used for multiple protocols, in some aspects, the sample is processed to extract nucleic acids, such as DNA and / or RNA. For example, after rehydration, DNA and / or RNA can be precipitated, bound, washed, eluted, dried, and / or dissolved according to the specific extraction method employed.

[0065] In some aspects, RNA is extracted according to a GITC-based method. Thus, the stored stable sample is rehydrated, phase separation is performed, and isopropanol is added to the supernatant. Then the mixture is centrifuged to form a precipitate containing RNA. Next, the RNA precipitate is washed, dried, and dissolved for analysis.

[0066] In some aspects, RNA is extracted according to the TRIspin method. Thus, the stored stable sample is rehydrated, phase separation is performed, and ethanol is added to the supernatant. RNA is bound, washed, and eluted and can then be used for analysis.

[0067] In some aspects, a column-based method is used to extract RNA. In this regard, the stored stable sample is rehydrated and ethanol is added. RNA is bound, washed, and eluted and can then be used for analysis.

[0068] Other extraction methods are under consideration. Thus, regardless of the specific extraction method employed, in some aspects, the cell lysis step (which is typically the first step in any such extraction method) can be performed during the vitrification process as described above, and the remaining steps can be performed after rehydration. Thus, the aspects described herein can achieve faster vitrification while providing a stored stable sample that can be used for any of a variety of processes after storage.

[0069] In some aspects, DNA or RNA can be extracted from vitrified cells. For example, any of many commercially available DNA or RNA extraction kits or similar methods can be used to extract DNA or RNA from the stored stable sample. In multiple aspects, the lysis buffer used in the extraction kit can be used to reconstitute the sample.

[0070] In addition, the various aspects described herein may allow whole blood or portions thereof to be preserved and stored at temperatures above deep hypothermia. For example, whole blood, serum, plasma, red blood cells, platelets, and / or lymphocytes can be collected in a tube (optionally using an anticoagulant) or on a membrane, contacted with a vitrification medium, incubated at ambient temperature for 5 - 20 minutes, and vitrified. The sample can be stored at ambient temperature until use. Extraction of DNA, RNA, and / or protein can be performed by treating the sample with trizol. Thus, storage costs can be reduced, and the stored blood sample can be used for any one of a variety of processes after storage, providing greater flexibility for the sample. For example, the storage-stable sample can be rehydrated and a leukocyte lysate can be extracted in lysis buffer. The lysate can be transferred to a spin column with ethanol and washed with wash buffer. Total RNA can then be eluted with RNase-free water, and the RNA can be subjected to quantitative, qualitative, and clinical analyses.

[0071] In some aspects, whole blood can be collected, centrifuged, and one or more layers (e.g., the plasma layer, the buffy coat layer, or the red blood cell layer) can be transferred into a matrix of the vitrification medium and vitrified. After storage, the sample can be reconstituted with a diluent (e.g., PBS liquid, with or without protease inhibitors) and subjected to qualitative and / or quantitative analysis.

[0072] While the various aspects have been described herein in terms of the use of biological samples in the form of whole blood, the use of other types of biological samples can be further contemplated, as generally described herein. In some aspects, the biological sample can be in the form of tissue. In some such aspects, a lysis solution can be used to homogenize, pulverize, or enzymatically digest the tissue. In alternative aspects, the tissue can be subjected to cryosectioning methods as used according to the various aspects.

[0073] In some aspects, the biological sample is heterogeneous. A heterogeneous sample is a sample that contains one or more foreign organisms (non-test subject biological origin), which also includes nucleic acids that may be stored concurrently with the biological sample and may contaminate the downstream analysis results of the sample. Thus, additional preparation steps may be required to selectively isolate the desired biological sample components relative to the undesired contaminants. As a non-limiting example, the biological sample can be saliva. It is well known that saliva includes both cells from the host organism and contaminating bacteria or viruses.

[0074] Heterogeneous samples can be subjected to a pre-treatment step to remove or reduce the amount of foreign organisms (optionally bacteria, viruses, yeast or others) in the sample. The pre-treatment step can be combined with the vitrification step, but in some aspects, the pre-treatment step occurs prior to actual vitrification. The pre-treatment step can be to contact the biological sample with a separation medium, which is optionally in the form of particles or a membrane, and which comprises a molecule selective for one or more foreign organisms (optionally bacteria, viruses, yeast and / or other non-test organisms) or which binds to such a molecule. In some aspects, the sample is placed in contact with the surface of a separation medium that includes a separating agent specific for one or more non-test organisms, optionally a mannose-binding lectin (MBL), optionally having the NCBI reference sequence: NP_000233. MBL is a C-type lectin that binds to N-acetylglucosamine residues and mannose on bacteria, yeast and some parasites and viruses. By contacting the biological sample with a surface comprising MBL, non-test organisms can be selectively separated from the test cells, thereby enhancing the ability of the method to selectively isolate and optionally vitrify the test cells in the biological sample.

[0075] Now referring to Figure 4 , in some aspects, the sample is applied to a membrane system where at least one separation membrane 32 is used to remove foreign organisms. Figure 4 A plurality of particles 36 are shown, which include a separating agent bound to their surface. The particles can be incubated with the vitrification mixture and / or the biological sample. The particles selectively bind to foreign organisms, where one or more separating agents, optionally a mannose-binding lectin (MBL), are bound to the particles. The sample is placed on a separation membrane 32 having a sufficient pore size to allow the biological sample to pass through while retaining the particles on its surface.

[0076] Alternatively or additionally, the separation membrane itself includes one or more separating agents bound thereto, whereby the biological sample or the vitrification mixture is contacted with the separation membrane and the foreign organisms bind to the separation membrane while the desired materials in the biological sample pass through the separation membrane for subsequent vitrification of the biological sample in contact with a vitrification membrane. The separation membrane can be any membrane system that is substantially porous, whereby cell material can pass through the membrane without binding to the separating agent therein or thereon.

[0077] The separation medium can be a suitable polymer (e.g., polyvinylidene fluoride (PVDF)), metal, ceramic, glass, or a combination thereof. In some aspects, the separation medium can be made of PVDF, cellulose ester, nitrocellulose, or other desired materials. One or more separation agents can be combined with or otherwise associated with a suitable separation medium. When a biological sample is then contacted with the separation medium, foreign cells / organisms can selectively bind to the separation agent, whereby the test cells can be captured or collected through the system and subsequently vitrified, as provided elsewhere herein.

[0078] As provided elsewhere herein, a separation membrane (optionally with a separation agent) can be laminated on a vitrification membrane suitable for vitrification of a biological sample. The vitrification membrane can be any material that includes or defines a capillary network. Optionally, such a porous material membrane can be made of a material that is non-toxic and non-reactive with respect to biological materials or biological samples and does not chemically or physically react with the vitrification medium. The material can be a suitable polymer, metal, ceramic, glass, or a combination thereof. In some aspects, a continuous capillary network is formed of materials such as polydimethylsiloxane (PDMS), polycarbonate, polyurethane, polyethersulfone (PES), polyester (e.g., polyethylene terephthalate), etc. Illustrative examples of membranes containing capillary channels suitable as surfaces in the devices and methods provided herein include hydrophilic filter membranes such as those sold by EMD Millipore, Bellerica, MA. In certain aspects, the porous material does not substantially bind or alter the components of the biological sample and / or the vitrification medium, or otherwise chemically or physically associate therewith. Optionally, the porous material is not derivatized. Optionally, the capillary channels can be formed in a substrate (e.g., a dry chamber wall) having the desired material and thickness by PDMS forming techniques, laser drilling, or other drilling forming techniques known in the art.

[0079] After the biological sample has passed through the separation membrane with the separation agent to separate or remove non-test cell material, the remaining cell material is collected within or on the membrane for subsequent vitrification and optional storage. The separation membrane can be laminated on top of the vitrification membrane. After contact with the biological sample, the separation membrane can be removed for analysis or discarded and the remaining biological sample is vitrified on or within the vitrification membrane by the methods described herein. Alternatively, the separation membrane can remain associated with the vitrification membrane and the entire membrane system is vitrified as described herein. The separation membrane can then be peeled off or otherwise removed from the vitrification membrane before the biological material in the reconstituted vitrification membrane is accessed. The result of the pretreatment step is the substantial removal of non-test cell material, thereby improving the separation and storage of the test biological sample material.

[0080] Optionally, the separating agent can be bound to beads or particles. In some aspects, prior to pouring the vitrification mixture onto the membrane system, the beads with the bound separating agent can be mixed with the biological sample and the vitrification mixture. It will be appreciated that this step will provide sufficient contact time between the beads and the vitrification mixture to capture the desired contaminating pathogens. Now referring to Figure 4 , when the vitrification mixture is poured onto the membrane system, the beads with the captured pathogens will separate from the vitrification mixture and remain on the upper surface of the membrane system. To achieve this, the pore size of the membrane system should be smaller than the bead size and larger than the size of the cells that must enter the vitrification membrane. The bead size can be between 6 microns and 500 microns, optionally between 10 microns and 500 microns. The beads can be made of a polymer, such as polystyrene or iron oxide, such as magnetite (Fe3O4) functionalized to bind the separating agent.

[0081] In a number of aspects also provided herein, the biological sample can be a tissue or other part of an organism. Storing tissue samples under non-cryogenic conditions is generally difficult. Simple vitrification of tissue samples cannot achieve sufficient stability of tissue materials due to non-uniform drying or lack of maintenance of tissue structure. Accordingly, methods for vitrifying tissue are provided that not only preserve the molecular material of the tissue, but also maintain the structure and other properties of the entire tissue itself, thereby significantly enhancing the effectiveness and ability to perform subsequent tissue analysis. Tissues that can be used as biological samples in the methods provided herein include, but are not limited to, tissues from the following sources: neurons, liver, heart, kidney, blood vessels, kidney, lung, larynx, stomach, esophagus, pancreas, thyroid, muscle, epithelium, hair, or any other recognized type of biological tissue.

[0082] In the vitrification process provided herein, the tissue sample can be permeated with a vitrification agent (optionally a sugar) and contacted with a polymer or polymer former (such as a PEG hydrogel). For example, a trehalose-PEG hydrogel precursor and an attachment agent suitable for associating the tissue or a part thereof with the polymer covalently, ionically, or otherwise can be injected into the tissue.

[0083] The polymer can be any molecule that can be used as or produce a polymer suitable for storing tissues at temperatures above cryogenic temperatures, including but not limited to: polyalkyl alcohols and diols, such as polyethylene oxide or polyethylene oxide derivatives; neopentyl glycol diacrylate (NPGDA), polyethylene oxide (PEO), polyacrylamide (PAAm), poly(2-hydroxyethyl 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, polysiloxane, rubber, agar, carboxyvinyl copolymer, polydioxolane, polyacrylic acetate, polyvinyl chloride, maleic anhydride, styrene and styrene polymers; dextran; heparin and heparin polymers; polypeptides of glutamic acid, aspartic acid or combinations thereof.

[0084] The polymer is optionally linear, branched, cross-linked or a combination thereof. The polymer is optionally homopolymeric or heteropolymeric. Exemplary examples of polymer moieties include one or more molecules of carbohydrates or polyethylene oxide (also known as polyethylene glycol or "PEG").

[0085] The polymer is optionally polyethylene glycol. The polyethylene glycol optionally includes 2 to 20,000 ethylene glycol units. Optionally, the number of ethylene glycol units is 2 to 10,000, optionally 2 to 5,000, optionally 2 to 2,000. In various aspects, the polyethylene glycol (PEG) is a derivative of polyethylene glycol, including 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 2-, 4-, 6-, 8- or other arm PEG molecule.

[0086] In some aspects, a method further includes adding a crosslinking agent with a polymer former. A crosslinking agent is any reagent suitable for linking two or more monomers / polymers together. The crosslinking agent optionally has one or more acrylate or methacrylate functional groups. Exemplary crosslinking agents include, but are not limited to, 2-hydroxyethyl methacrylate (HEMA), acrylic acid and methacrylic acid, adipic dihydrazide diacrylamide, 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, isobornyl (meth)acrylate, cyclohexyl (meth)acrylate, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, 2-hydroxypropyl acrylate, 2-hydroxypropyl methacrylate, 2-hydroxybutyl acrylate, N-(2-hydroxyethyl)acrylamide [N-(2-hydroxyethyl)acrylamide], N-methylacrylamide, N-butoxymethylacrylamide, N-methoxymethylacrylamide, N-methoxymethylmethacrylamide, 2-acrylamido glycolic acid and 2-carboxyethyl 2-carboxyethyl acrylate, 2-hydroxy-5-methoxyacetophenone, 2-hydroxyethyl cellulose, and 2-hydroxyethyl disulfide, etc., or combinations thereof.

[0087] A method optionally further includes the presence of one or more adhesion agents in the vitrification mixture. Optionally, the adhesion agent is boric acid. Without being limited to a particular theory, it is believed that boric acid can covalently bind the polymer to the membrane proteins of tissue cells. Thus, the hydrogel forms between the cells of the tissue and provides support for the cells and tissue to prevent shrinkage or collapse during vitrification and sectioning. After the hydrogel is formed, the tissue can be vitrified. If desired, the vitrified sample can then be effectively sectioned. After the tissue is sectioned, a mixture of glucose and water can be added to decouple the boric acid, thereby decoupling the hydrogel from the membrane proteins. Then the boric acid and the hydrogel can be washed away, leaving the tissue sample, including its original structure preserved during vitrification.

[0088] These aspects enable histological analysis of the sample. Conventional tissue processing for histology involves injecting low melting point paraffin or agarose into the tissue and subsequently cryo-preserving or vitrifying the sample before sectioning the tissue sample. Although paraffin provides the hardness required to maintain cell structure, it causes tissue contamination. Agarose affects RNA extraction and adheres to the tissue walls. Thus, the use of trehalose-PEG hydrogel (as an example provided herein) enables vitrification of the tissue while providing support for the cell structure without contamination or adverse impact on downstream processing as the hydrogel can be eluted and washed away by a glucose-water wash step. Other advantages will be apparent to those skilled in the art.

[0089] Optionally, the vitrification medium includes a convertible support material that is capable of transitioning between a relatively high viscosity state and a low viscosity state triggered by two different stimuli. Before application of the stimuli, the convertible material will behave as a low viscosity liquid with excellent fluidity, allowing in situ perfusion into the tissue matrix by the methods provided herein. Once the first stimulus is applied, the material will transition from the low viscosity liquid (sol state) to a yield stress fluid (gel state), thereby providing sufficient rigidity and support to substantially maintain the conformation of the tissue and / or membrane structure during vitrification. When the second stimulus is applied, the support material will transition back from the rigid material to the low viscosity liquid, allowing for easy removal.

[0090] In some aspects, light of different wavelengths can be used as stimuli. Optionally, ultraviolet (UV) and visible light will be used as conversion stimuli. In some aspects, the photo-convertible hydrogel support will exhibit rigidity under visible light but will be able to completely dissociate into a solution state under UV radiation. Exemplary photo-convertible materials are supramolecular hydrogels formed from azobenzene (azo) and cyclodextrin (CD) host-guest complexes, 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 cyclodextrins with different cavity sizes: α-CD, β-CD, and γ-CD. They consist of six, seven, and eight linked glucopyranose subunits, respectively, forming a conical structure. The hydroxyl groups are located outside the cone, making it hydrophilic. However, the interior of the cone is hydrophobic, so it can accommodate hydrophobic molecules such as azobenzene in an aqueous solution to form a host-guest complex. Azobenzene (azo) is a well-known photo-responsive molecule. Under visible light (~520 nm), it is in a thermodynamically stable trans state. When irradiated with ultraviolet light (~375 nm), it can be photo-isomerized to the cis isomer. The geometric structure of trans-azo allows it to enter the CD cavity in an aqueous solution (driven by hydrophobic interactions) to form a host-guest complex. When photo-isomerized to cis-azo, its geometry is no longer suitable for accommodation in the CD cone, resulting in the dissociation of the complex. Using the photo-reversible azo-CD complex as a cross-linker, a photo-convertible hydrogel that gels under visible light but easily decomposes into a liquid (sol) under UV radiation can be prepared. This solution-gel conversion is reversible and can be completed within two minutes. Other methods and materials for forming the convertible support material can be found in Koopmans and Ritter, Macromolecules, 2008; 41:7418-7422.

[0091] Accordingly, the methods provided herein optionally further include a convertible support material within a vitrification medium, and applying a stimulus to convert the convertible support material into a viscous state, optionally followed by subjecting the vitrification medium to the vacuum vitrification process provided herein, thereby storing the tissue or other cellular material in a vitrified state with sufficient support to maintain other physical and / or chemical properties of the biological sample. Examples

[0092] The following examples are provided to illustrate various aspects but are not intended to limit the scope of the claims. Approximate properties, characteristics, parameters, etc. are provided below for various working examples, comparative examples, and the materials used in the working and comparative examples.

[0093] Example 1

[0094] Cell culture: LINTERNA Jurkat T cells (stably expressing tGFP and having a G418 resistance gene) were obtained from Innoprot, Spain. The 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. The cultures were maintained at 37 °C in 25 cm 2 T-flasks (Corning Incorporated, NY) and equilibrated with 10% CO2–90% air. Fresh medium was changed every three days to maintain the cells.

[0095] For Example A, a sample of 5X10 6 Jurkat T cells was incubated in 250 μl of vitrification medium containing 600 mM trehalose, 5% glycerol, and 0.01% Triton X-100 for 10 - 15 minutes. The sample was then sandwiched between two PES membrane supports with a pore size of 1.2 microns and vitrified in a vacuum of -29 mmHg for approximately 6 minutes to achieve a moisture residue rate (MRR) of 0.01. The sample was then stored at 25 °C or 55 °C for 3 days before RNA extraction.

[0096] RNA was extracted using the PureLink RNA Mini Kit (Invitrogen). The 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 homogenized by passing it through a 21-gauge syringe needle 10 times. An equal volume of 70% ethanol was added to the lysate. Then, the lysate / supernatant was extracted by centrifugation (12,000 x g for 15 seconds at room temperature) and passed through an RNA-binding centrifugation column by centrifugation at 12,000 x g for 15 seconds at room temperature. Next, DNA contamination was removed by DNase digestion on the column (PureLink DNase Set, Invitrogen), and then contaminants and inhibitors were removed by two-step washing (centrifugation at 12,000 × g for 15 seconds) with 700 μl of wash buffer-I and 500 μl of wash buffer-II and ethanol. The centrifugation column bound with RNA was dried for 2 minutes, and then 50 μl of RNase-free water was added to the centrifugation column and incubated for 2 minutes. The pure RNA was eluted into a new tube by centrifugation at 12,000 × g at room temperature.

[0097] As a control, RNA was extracted from a sample of fresh cells with the same number using the same technique as for the vitrified cells (Comparative Example A).

[0098] For Comparative Example B, samples containing the same number of cells were cryopreserved and stored at -80 °C for 2 hours. The cells were thawed, and RNA was extracted using the same RNA extraction method as in Example A.

[0099] For Comparative Example C, 5X 10 6 Jurkat T cells were incubated in 250 μL of vitrification medium containing 600 mM trehalose and 5% glycerol, which did not include Triton X-100, for 10 - 15 minutes. 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 technique as for vitrified cells.

[0100] The same vitrification protocol used for Example A and Comparative Example C was implemented, and the cells were stored at 25 °C or 55 °C for three days, and then RNA was extracted.

[0101] For all samples, 5 μg of RNA per well was loaded onto a 1.2% agarose gel (Native Sybr-safe) and electrophoresed to achieve separation. The gel images are as Figure 5 shown. Intact total RNA run on a denaturing gel will have distinct 28S and 18S rRNA bands (eukaryotic samples). The intensity of the 28S rRNA band should be approximately twice that of the 18S rRNA band. This 2:1 ratio (28S:18S) is a good indicator of RNA integrity. Partially degraded RNA will exhibit a diffuse appearance, lack distinct rRNA bands, or not show the 2:1 ratio, as seen in Comparative Example A (lane 1) and Comparative Example C (lane 3). Completely degraded RNA will appear as a diffuse smear of very low molecular weight (Comparative Example B; lane 2). As Figure 5 shown, Example A vitrified and stored according to various aspects described herein, stored at 55 °C for 1 week (lane 4), has distinct 28S and 18S rRNA bands with a good intensity ratio, indicating that the RNA is intact after vitrification and room temperature storage.

[0102] Similar results were obtained by RNA quantification. The RNA prepared as above for each sample was quantified spectrophotometrically, and the results are shown in Table 1.

[0103] Table 1:

[0104] Sample Name 260 / 280 ng / μL Comparative Example A Fresh Jurkat cells 2.07 378.353 Comparative Example B Jurkat cells [1 - 2h @ -80°C] 1.889 22.833 Comparative Example C Cells + VM vitrification 2.111 122.273 Example A Cells + VM + TRITON vitrification 2.113 202.033

[0105] Vitrification in the presence of the lysis agent Triton-X100 showed a significant improvement in RNA integrity compared to cells vitrified in the absence of the lysis agent. Comparative Example B was almost completely degraded. When the vitrification medium contained the lysis agent, improved RNA quality was observed both when stored at 25 °C and when stored at the elevated temperature of 55 °C for 3 days, demonstrating the robust storage ability of the cell samples prepared as described herein.

[0106] Example 2

[0107] LINTERNA Jurkat T cells were obtained and cultured as in Example 1.

[0108] 5X10 6 Jurkat T cell samples were incubated in 250 μl of vitrification medium containing 600 mM trehalose, 5% glycerol, 0.01% Triton X-100 for 10 - 15 minutes. The samples were then vitrified for approximately 6 minutes to achieve an MRR of 0.01. The samples were then stored at 25 °C or 55 °C for 3 days before RNA extraction. RNA extraction was performed as in Example 1.

[0109] As a comparison, RNA was extracted from fresh cell samples that were stored at 4 °C before RNA extraction and analysis in the same manner as the vitrified cells.

[0110] RT-PCR was then performed on the RNA extracted from each sample using RNA templates for VEGF (a member of the PDGF / VEGF growth factor family) or GAPDH. Figure 6A The cycle threshold (Ct) of GADPH mRNA from cells of various preparation and storage techniques was shown. Figure 6B The fold change in Ct values of GADPH mRNA from cells of various preparation and storage techniques was shown. Figure 6C The fold change in VEGF mRNA from cells of various preparation and storage techniques was shown. As Figure 6A and 6B shown, for the GAPDH gene, there was no significant change between the Ct values of fresh and vitrified cells at the two storage temperatures. Additionally, robust VEGF mRNA ( Figure 6C ) was observed. Therefore, it was concluded that the vitrification method described herein can be used to vitrify and store cells without degrading VEGF mRNA.

[0111] Example 3:

[0112] Various animal tissues were obtained and vitrified. Samples of mouse intestine and liver were humanely collected through an approved animal protocol. The tissues were cut into small pieces (about 1 mm thick, weighing 15 mg) and incubated in a vitrification medium containing 600 mM trehalose, 5 wt% glycerol, and 0.5 wt% Triton X-100 for 20 minutes. The samples were vitrified for 5 minutes according to Example 1 to achieve an MRR of 0.01 and stored at 25 °C or 55 °C for one week.

[0113] A second group of samples was vitrified in the same vitrification medium further comprising a trehalose polymer. The trehalose polymer was synthesized by dissolving azobisisobutyronitrile (AIBN) (5.28 mg, 3.22 x 10 -2 mmol) and styrenyl ether trehalose monomer (634 mg, 1.38 mmol) in a mixture of dimethylformamide (DMF) (2.31 mL) and H2O (4.61 mL). Oxygen was removed by three freeze-pump-thaw cycles and polymerization was initiated at 75 °C. Polymerization was stopped after 8.5 hours by immersing the vial in liquid nitrogen.

[0114] A third group of samples was vitrified in a vitrification medium comprising 600 mM trehalose, 5 wt% glycerol, and 0.5 wt% Triton X-100 as described above, further comprising 8-arm polyethylene glycol conjugated with boric acid. The polymer was synthesized by dissolving 8-arm PEG amine (400 mg, 10 kDa, 4 x 10 -2 mmol) and 4-formylphenylboronic acid (96 mg, 6.40 x 10 -1 mmol) in 2.8 ml of MeOH. Then NaBH3CN (37.7 mg, 6.00 x 10 -1 mmol) was added and stirred at 25 °C. The remaining vitrification and storage protocols were the same.

[0115] After storage, the tissue samples were reconstituted in lysis buffer and mRNA was isolated as described in Example 1. RNA was quantified and analyzed spectroscopically by using a Take3 Plate (BioTek Instrument) of Synergy H1 Hybrid MF. Briefly, 2 μl / well of each sample was added to Take3 and RNase-free ultrapure water was used as a blank. The total RNA concentration was calculated based on the A260 reading using Gen5 software (1.0 corresponds to ~40 μg / ml ssRNA), and the A260 / A280 ratio was used for RNA quality (an A260 / A280 ratio of 1.8 - 2.1+ indicates highly purified RNA). The results shown in Table 2 demonstrate a robust yield of intact mRNA.

[0116] Table 2:

[0117] Sample 260 / 280 ratio ng / μl Total yield (ng) Liver 2.168 97.12 6318 Small intestine 2.099 108.48 7051

[0118] Samples incubated with polyethylene glycol or trehalose polymers conjugated to boric acid are expected to give more reliable results.

[0119] Example 4:

[0120] When storing biological samples obtained from a non-sterile environment, the possibility of contamination from non-donor biological sources always exists. To address this issue, a protocol was developed to selectively isolate organism samples and exclude unwanted bacterial contamination. Two layers of 8-micron nitrocellulose membranes were placed above the PES membrane used for vitrification in Example 1 and assembled into a three-layer system, where the nitrocellulose membrane was conjugated with mannose-binding protein (MBL) or used as supplied.

[0121] To test the ability of the assembled system to selectively isolate desired nucleic acids from bacterial nucleic acids, three samples with different bacterial strains were formed. Escherichia coli (E. coli), Staphylococcus epidermidis, and Pseudomonas aeruginosa were tested. Each bacterium was diluted to 10 4 colony-forming units (CFUs) in the medium. 100 μL of bacterial material was added to each precipitate containing 10 6 of each bacterium, and the bacterial precipitate was resuspended. The resuspension was then added to the surface of the nitrocellulose membrane of the assembled three-layer membrane system and incubated at room temperature for 30 minutes. Then the three-layer membrane was separated and washed with PBS. The washing solution was then counted for cells and plated on agar plates, which were then incubated at 37 °C for two days. The results in Table 3 are the average of two experiments for E. coli, with each experiment performed in triplicate.

[0122] Table 3:

[0123] Sample %CFU StDev SEM MBL coating 35.69 11.47 8.11 Negative control 93.08 2.34 1.66 No filtration 100 0 0

[0124] The results in Table 4 are the average of two experiments for Staphylococcus epidermidis, with each experiment performed in triplicate.

[0125] Table 4:

[0126] Sample %CFU StDev SEM MBL coating 21.57 7.22 5.11 Negative control 48.89 27.42 19.39 No filtration 100 0 0

[0127] The results showed that the filter conjugated with MBL selectively and robustly bound bacteria from the samples, indicating that the system was able to selectively remove bacterial contamination from samples containing mRNA.

[0128] Cell counts in the PES membranes were also analyzed. The PES membranes were washed in PBS and the cells were counted. The results in Table 5 are the average of two experiments performed in triplicate for each experiment.

[0129] Table 5:

[0130]

[0131] In all cases, the PES membranes included the initially loaded cells with a recovery rate greater than 84%, and the number of Jurkat cells was independent of the presence or absence of MBL on the nitrocellulose layer. These results indicate a robust filtration system that is selective for bacterial cells and can be used for the selective storage of biological cells.

[0132] Other embodiments

[0133] 1. A method for storing a biological sample, the method comprising:

[0134] providing a biological sample containing one or more cells;

[0135] contacting the biological sample with a vitrification medium containing a vitrification agent and a lysing agent to form a vitrification mixture;

[0136] vitrifying the vitrification mixture to produce a storage-stable sample.

[0137] 2. The method according to embodiment 1, further comprising:

[0138] storing the storage-stable sample at a temperature greater than or equal to 16 °C to less than or equal to 30 °C, optionally greater than 30 °C, optionally greater than 50 °C.

[0139] 3. The method of any one of embodiments 1-2, wherein the biological sample contains nucleic acid, optionally RNA.

[0140] 4. The method of any one of embodiments 1-3, wherein the biological sample includes whole blood, plasma or serum.

[0141] 5. The method according to embodiment 4, wherein the biological sample contains whole blood, serum or plasma, and wherein the vitrification mixture further contains an anticoagulant.

[0142] 6. The method of any one of embodiments 1-5, wherein the vitrification mixture further contains a buffer.

[0143] 7. The method according to any one of embodiments 1-6, wherein the vitrification agent includes dimethyl sulfoxide, glycerol, sugar, polyol, methylamine compound, betaine compound, antifreeze protein, synthetic anti-nucleating agent, polyvinyl alcohol, cyclohexanetriol, cyclohexanediol, inorganic salt, organic salt, ionic liquid or a combination thereof.

[0144] 8. The method according to embodiment 7, wherein the vitrification agent comprises trehalose.

[0145] 9. The method according to any one of embodiments 1-8, wherein the lysis agent comprises a detergent.

[0146] 10. The method according to embodiment 9, wherein the detergent comprises sodium dodecyl sulfate (SDS), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (e.g., Triton X-100), CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), guanidine hydrochloride, or a combination thereof.

[0147] 11. The method according to any one of embodiments 1-10, further comprising: encapsulating the storage-stable sample in a protective housing impermeable to water and air, and storing the protective housing at a temperature between -196 °C and +60 °C for a storage time of 20 days or longer.

[0148] 12. The method according to any one of embodiments 1-11, wherein vitrifying the vitrification mixture comprises drying the vitrification mixture at a temperature above cryogenic temperature until the vitrification mixture enters a glassy state.

[0149] 13. The method according to embodiment 12, wherein the vitrification medium is within one or a plurality of capillary channels.

[0150] 14. The method according to any one of embodiments 1-13, wherein the vitrification is performed for a drying time of 1 second to 1 hour, optionally 10 minutes or less.

[0151] 15. The method according to any one of embodiments 1-14, further comprising incubating the vitrification mixture for greater than or equal to 5 minutes and less than or equal to 30 minutes before vitrification.

[0152] 16. The method according to embodiment 15, wherein the incubation is performed at a temperature greater than or equal to 18 °C and less than or equal to 37 °C.

[0153] 17. The method according to any one of embodiments 1-16, wherein the biological sample is pretreated by contacting it with a separation medium comprising one or more separation agents.

[0154] 18. The method according to embodiment 17, wherein the separation agent is a mannose-binding lectin.

[0155] 19. The method according to embodiment 17, wherein the separation medium comprises nitrocellulose or polyvinylidene difluoride.

[0156] 20. The method according to any one of embodiments 1-19, wherein the biological sample comprises tissue and wherein the vitrification mixture further comprises a support material, optionally a polymeric support material.

[0157] 21. The method according to embodiment 20, wherein the polymer is suitable for forming a hydrogel.

[0158] 22. The method according to embodiment 20, wherein the polymer is polyethylene glycol.

[0159] 23. The method according to any one of embodiments 20-22, wherein the vitrification medium further comprises one or more adhesion agents.

[0160] 24. The method according to embodiment 23, wherein the adhesion agent is boric acid.

[0161] 25. The method according to any one of embodiments 20-24, wherein the support material is a convertible support material, and the method further comprises subjecting the vitrification mixture to a stimulus to convert the convertible support material into a gel state.

[0162] Various aspects are disclosed herein; however, it should be understood that the disclosed aspects are merely examples of the invention, and the invention may be embodied in various and alternative forms. Accordingly, the specific details disclosed herein should not be construed as limiting, but merely as a representative basis for any aspect of the invention and / or as a representative basis for teaching one of ordinary skill in the art to employ the teachings disclosed herein in various manners. Additionally, the terms used herein are for the purpose of describing particular aspects of the invention only and are not intended to be limiting in any way.

[0163] It should be understood that although the terms "first", "second", "third", etc. may be used herein to describe various elements, components, regions, layers, and / or sections, these elements, components, regions, layers, and / or sections should not be limited by these terms. These terms are only used 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" discussed below may be referred to as a second (or other) element, component, region, layer, or section without departing from the teachings herein.

[0164] 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 forms, including "at least one", unless the context 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. It should also be understood that when used in this specification, the terms "comprises" and / or "comprising" refer to the presence of the stated features, regions, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof. The term "or combinations thereof" refers to a combination including at least one of the foregoing elements.

[0165] 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 pertains. It should also be understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and this disclosure, and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0166] Throughout this application, where publications are referenced, the disclosures of these publications are hereby incorporated by reference in their entirety into this application to more fully describe the state of the art to which this disclosure pertains.

[0167] Although aspects of the invention have been illustrated and described, these aspects are not intended to illustrate and describe all possible forms of the invention. On the contrary, the words used in the specification are descriptive words rather than limiting words, and it should be understood that various modifications and substitutions can be made therein without departing from the spirit and scope of the invention.

Claims

1. A method for storing a biological sample, the method comprising: Providing a biological sample containing one or more cells; Contacting the biological sample with a vitrification medium containing a vitrification agent and a lysing agent to form a vitrified mixture; Vitrifying the vitrified mixture to produce a storage-stable sample, wherein vitrifying the vitrified mixture includes, at a temperature above cryogenic temperatures, drying the vitrified mixture until the vitrified mixture enters a vitreous state, wherein the vitrification agent includes glycerol, trehalose, or a combination thereof; and wherein the lysing agent includes 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton X-100), CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), or a combination thereof.

2. The method according to claim 1, further comprising: Storing the storage-stable sample at a temperature greater than or equal to 16 °C and less than or equal to 50 °C.

3. The method according to claim 1, wherein the biological sample contains nucleic acids.

4. The method according to claim 1, wherein the biological sample includes whole blood, plasma, or serum.

5. The method according to claim 4, wherein the biological sample contains whole blood, and the vitrified mixture further contains an anticoagulant.

6. The method according to claim 1, wherein the vitrified mixture further contains a buffer.

7. The method according to claim 2, wherein the vitrification agent includes glycerol and trehalose.

8. The method according to claim 2, wherein the vitrification agent contains trehalose.

9. The method according to claim 7, wherein the lysing agent contains 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton X-100).

10. The method according to claim 1, wherein the lysing agent is 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton X-100), and the vitrification agent contains glycerol and trehalose.

11. The method according to any one of claims 1-8, further comprising: Encapsulating the storage-stable sample in a protective housing impermeable to water and air, and storing the protective housing at a temperature between -196 °C and +60 °C for a storage time of 20 days or longer.

12. The method according to claim 1, wherein the vitrification medium is within one or more capillary channels.

13. The method according to claim 1, wherein the vitrification is carried out for a drying time of 1 second to 1 hour.

14. The method according to any one of claims 1-8, further comprising incubating the vitrified mixture for greater than or equal to 5 minutes and less than or equal to 30 minutes before vitrification.

15. The method according to claim 14, wherein the incubation is carried out at a temperature greater than or equal to 18 °C and less than or equal to 37 °C.

16. The method according to any one of claims 1-8, wherein the biological sample is pretreated by contacting it with a separation medium containing one or more separating agents.

17. The method according to claim 16, wherein the separating agent is a mannose-binding lectin.

18. The method according to claim 16, wherein the separation medium includes nitrocellulose or polyvinylidene difluoride.

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