Systems, devices, and methods for generating magnetoporation for delivery of extracellular material into cells

Abstract

Disclosed herein as systems, devices and methods for introducing cryoprotective agents into cells or membrane-bound cell fragments using magnetoporation, and methods of using same to prepare cells or membrane-bound cell fragments for lyophilization or cryopreservation. The cells can be blood cells, such as red blood cells or platelets.

Description

SYSTEMS, DEVICES, AND METHODS FOR GENERATING MAGNETOPORATION FOR DELIVERY OF EXTRACELLULAR MATERIAL INTO CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63 / 686,979, filed August 26, 2024, titled “SYSTEMS, DEVICES, AND METHODS FOR GENERATING MAGNETOPORATION FOR DELIVERY OF EXTRACELLULAR MATERIAL INTO CELLS,” and U.S. Provisional Patent Application No. 63 / 651,343, filed May 23, 2024, titled “SYSTEMS, DEVICES, AND METHODS FOR GENERATING MAGNETOPORATION FOR DELIVERY OF EXTRACELLULAR MATERIAL INTO CELLS,” the disclosures of each of which are incorporated herein by reference.BACKGROUND

[0002] A stable supply of blood is a fundamental component of any healthcare setting. The current blood supply depends on a steady stream of healthy donors, as well as complex coldchain transportation and storage systems, substantial weight and space capacity, and systems for warming at points of delivery. Cryopreservation and lyophilization (also known as freeze drying) of blood products can increase longevity of blood products and simplify collection, storage, and delivery. There thus exists a need for systems, devices and methods for lyophilizing blood products.SUMMARY

[0003] The disclosure provides methods of introducing a cryoprotective agent into a cell or a membrane-bound cell fragment, comprising: (a) contacting the cell or membrane-bound cell fragment with the cryoprotective agent; and (b) applying a transient magnetic field to the cell or membrane-bound cell fragment under conditions sufficient to introduce the cryoprotective agent into the cell or membrane-bound cell fragment.

[0004] In some embodiments of the methods of the disclosure, the cryoprotective agent comprises a sugar. In some embodiments, the sugar comprises trehalose.

[0005] In some embodiments of the methods of the disclosure, the cell comprises a blood cell. In some embodiments, the blood cell comprises a red blood cell, neutrophil, monocyte orlymphocyte. In some embodiments, the cell comprises a red blood cell. In some embodiments, the membrane-bound cell fragment comprises a platelet.

[0006] In some embodiments, the cell comprises a red blood cell, and the methods comprise contacting a plurality of red blood cells with trehalose. In some embodiments, the trehalose is at a concentration of between about 100 mM and about 1,000 mM. In some embodiments, the trehalose is at a concentration of between about 200 mM and 600 mM. In some embodiments, the trehalose is at a concentration of between about 100 mM and 400 mM. In some embodiments, the trehalose is at a concentration of about 200 mM. In some embodiments, the trehalose is at a concentration of about 300 mM. In some embodiments, the trehalose is at a concentration of about 400 mM. In some embodiments, the trehalose is at a concentration of about 500 mM.

[0007] In some embodiments, the plurality of red blood cells is at a concentration of between about IxlO5cells per pL (cells / pL) and about lxl08cells / pL. In some embodiments, the plurality of red blood cells is at a concentration of between about 5xl06cells / pL and about 5xl07cells / pL. In some embodiments, the plurality of red blood cells is at a concentration of between about IxlO7cells / pL and about 5xl07cells / pL. In some embodiments, the plurality of red blood cells is at a concentration of about IxlO6cells / pL to about 5xl06cells / pL. In some embodiments, the plurality of red blood cells is at a concentration of about 5xl06cells / pL. In some embodiments, the plurality of red blood cells is at a concentration of about 3.36xl06cells / pL.

[0008] In some embodiments, the methods comprise, prior to step (a), (i) centrifuging whole blood at about 1400 g for about 5 minutes, thereby collecting the plurality of red blood cells; (ii) washing the plurality of red blood cells with Dulbecco’s Phosphate Buffered Saline (DPBS); and (3) suspending the cells in DBPS. In some embodiments, the cells are suspended at a concentration of at a concentration of between about IxlO5cells / pL and about IxlO8cells / pL. In some embodiments, the cells are suspended at a concentration of at a concentration of about IxlO7cells / pL and about IxlO8cells / pL.

[0009] In some embodiments, the membrane-bound cell fragment comprises a platelet, and the method comprises contacting a plurality of platelets with trehalose. In some embodiments, the trehalose is at a concentration of between about 100 mM and about 1,000 mM. In some embodiments, the trehalose is at a concentration of between about 200 mM and about 600 mM. In some embodiments, the trehalose is at a concentration of between about 100 mM and 400 mM. In some embodiments, the trehalose is at a concentration of about 200 mM. In some embodiments, the trehalose is at a concentration of about 300 mM. In someembodiments, the trehalose is at a concentration of about 400 mM. In some embodiments, the trehalose is at a concentration of about 500 mM.

[0010] In some embodiments, the plurality of platelets is at a concentration of between about IxlO5and about IxlO8platelets per pL (platelets / pL). In some embodiments, the plurality of platelets is at a concentration of between about IxlO6platelets / pL and about 5xl06platelets / pL. In some embodiments, the plurality of platelets cells is at a concentration of about 5xl06platelets / pL. In some embodiments, the plurality of platelets is at a concentration of about 3.36xl06platelets / pL.

[0011] In some embodiments, the methods comprise incubating the plurality of red blood cells in cell culture medium following application of the magnetic field for a recovery period. In some embodiments, the recovery period comprises incubating the plurality of red blood cells in complete RPMI for about 8 to about 24 hours. In some embodiments, the recovery period comprises 8 to 24 hours. In some embodiments, the recovery period comprises 8 to 18 hours.

[0012] In some embodiments, the methods comprise incubating the plurality of platelets in cell culture medium following application of the magnetic field for a recovery period. In some embodiments, the recovery period comprises incubating the plurality of platelets in complete RPMI for about 8 to about 24 hours. In some embodiments, the recovery period comprises 8 to 24 hours. In some embodiments, the recovery period comprises 8 to 18 hours.

[0013] In some embodiments, the magnetic field is pulsed. In some embodiments, the magnetic field comprises at least 50 pulses, at least 100 pulses, at least 300 pulses, at least 500 pulses, at least 700 pulses, at least 1,000 pulses, at least 12,00 pulses, at least 1,500 pulses, at least 2,000 pulses, at least 2,500 pulses, at least 3,000 pulses, at least 4,000 pulses, at least 5,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000 at least 90,000 or at least 100,000 pulses. In some embodiments, the magnetic field comprises an interval of between about 0.1 second and about 1 second between pulses or between trains of pulses. In some embodiments, the magnetic field comprises, the magnetic field comprises between about 50 and about 100,000 pulses, between about 100 and about 50,000 pulses, between about 1,000 and about 30,000 pulses, between about 1,500 and about 15,000 pulses, or between about 5,000 and about 10,000 pulses.

[0014] In some embodiments, contacting the cell or membrane-bound cell fragment with the cryoprotective agent occurs in a volume of about 2 pL to about 1000 pL, about 5 pL to about 500 pL, about 5 pL to about 50 pL, about 10 pL to about 50 pL, or about 10 pL to about 30pL. In some embodiments, contacting the cell or membrane-bound cell fragment with the cryoprotective agent occurs in a volume of between about 1 mL and about 500 mL, between about 10 mL and about IL, between about 50 mL and about 5L, between IL and about 10,000L, between about 5L and about 500L, between about 10L and about 500L, between about 10L and about 100L, between about 10L and about 50L, between about 100L and about 10,000L, between about 100L and about lOOOL, or between about 100L and about 500L.

[0015] In some embodiments of the methods of the disclosure, the methods comprise cry opreserving the cell or membrane-bound cell fragment after step (b). In some embodiments, cryopreserving the cell or membrane-bound cell fragment comprises reducing the temperature of the cell or membrane-bound cell fragment to a temperature of -20 °C or less. In some embodiments, cryopreserving the cell or membrane-bound cell fragment comprises reducing the temperature of the cell or membrane-bound cell fragment to a temperature of about -15 °C to about -200 °C, about -30 °C to about -180 °C, about -60 °C to about -160 °C, or about -80 °C to about -140 °C.

[0016] In some embodiments, introducing the cryoprotective agent into the cell or the membrane-bound cell fragment increases a percentage of viable cells or membrane-bound cell fragments after cry opreservation and thawing when compared to a percentage of viable cells or membrane-bound cell fragments which have been subject to cry opreservation and thawing without the cryoprotective agent. In some embodiments, introducing the cryoprotective agent increases the percentage of viable membrane-bound cell fragments by at least about 5%, about 10%, about 15% or about 20%. In some embodiments, introducing the cryoprotective agent increases the percentage of viable cells by at least about 30%, about 40%, about 50%, about 60% or about 70%.

[0017] In some embodiments, the methods comprise incubating the plurality platelets in cell culture medium following magnetoporation for a recovery period. In some embodiments, the recovery period comprises incubating the plurality of platelets in complete RPMI for about 8 to about 24 hours.

[0018] In some embodiments of the methods of the disclosure, the methods further comprise lyophilizing the cell or membrane-bound cell fragment after step (b). In some embodiments, the lyophilizing comprises: (i) contacting the cell or membrane-bound cell fragment with a lyophilization buffer; (ii) cooling the cell or membrane-bound cell fragment to less than at least -60 °C; and (iii) drying the cell or membrane bound cell fragment. In some embodiments, drying the cell or membrane bound cell fragment comprises: (1) drying the cellor membrane-bound cell fragment at about -45 °C and about 200 mTorr for about 15 hours; and (2) drying the cell or membrane-bound cell fragment at about 15 °C for about 10 hours. In some embodiments, the lyophilization buffer comprises: glucose, adenine, NaCl, mannitol, KC1, dextran, bovine serum albumin (BSA) and / or sodium citrate.

[0019] The disclosure provides pharmaceutical compositions comprising a cryopreserved cell or membrane-bound cell fragment produced by the methods of the disclosure.

[0020] The disclosure provides pharmaceutical compositions comprising a lyophilized cell or membrane-bound cell fragment produced by the methods of the disclosure.

[0021] The disclosure provides kits, comprising the pharmaceutical compositions of the disclosure, and instructions for use. In some embodiments, the kits comprise a rehydration buffer.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0023] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0024] FIG. 1A is a series of fluorescence activated cell sorting (FACS) histogram plots, gated on intact RBCs, of FITC positivity for untreated red blood cells (RBCs, left), RBCs + FITC-trehalose without magnetoporation (middle) and RBCs + FITC-trehalose with magnetoporation (right).

[0025] FIG. IB is a series of fluorescence microscopy images showing untreated RBCs(left), RBCs + FITC-trehalose without magnetoporation (middle) and RBCs + FITC-trehalose with magnetoporation (right).

[0026] FIG. 2 is a plot showing intracellular trehalose concentration (y-axis, in millimolar, or mM), of untreated RBCs (left), RBCs + trehalose without magnetoporation (middle) and RBCs + trehalose with magnetoporation (right).

[0027] FIG. 3 is a series of images showing morphology of human RBCs that were magnetoporated with extracellular trehalose at 30 minutes, 2 hours, and 24 hours postmagnetoporation, as indicated.

[0028] FIG. 4 is a pair of histogram plots, gated on intact RBCs, which show mean fluorescent intensity (MFI). Percentage reduction in MFI compared to untreated RBCs is calculated as [MFIcontroi-MFIsampie] / MFIControix 100 and correlates with defects in membrane integrity.

[0029] FIG. 5A is a pair of plots showing hemoglobin leakage post magnetoporation from RBCs from two subjects.

[0030] FIG. 5B is a pair of plots showing that RBCs retain hemoglobin post magnetoporation.

[0031] FIG. 6 is a plot showing RBC intracellular trehalose concentration.

[0032] FIG. 7 is a plot showing the total number of live RBCs after treatment with experimental conditions 1-7 as described in Example 11. After magnetoporation with reghalose, cells were incubated in RPMI medium overnight, and after 24 hours were analyzed for live / dead cell counts.

[0033] FIG. 8 is a plot showing femtograms of trehalose per RBC for RBCs treated with the experimental conditions described in Example 11. Intracellular trehalose in femtograms is shown in the x-axis, and the experimental conditions are shown on the y-axis.

[0034] FIG. 9 is a plot showing RBC viability 1 hour post magnetoporation with 500 mM trehalose (left) versus after magnetoporation with 500 mM trehalose followed by freezing the cell pellet overnight and thawing it at room temperature (right), as described in Example 11. Each of the two left bars show RBCs treated with 500 mM trehalose without magnetoporation (condition #3), while the two right bars show RBCs treated with 500 mM trehalose and magnetoporation at 170 V and 150 Hz (condition #5).

[0035] FIG. 10 is a plot showing the total number of viable platelets after treatment with experimental conditions 1-8 as described in Example 11.

[0036] FIG. 11 is a plot showing femtograms of trehalose per platelet for platelets treated with the experimental conditions described in Example 11. Intracellular trehalose in femtograms is shown in the x-axis, and the experimental conditions are shown on the y-axis.DETAILED DESCRIPTIONIntroduction

[0037] A stable supply of blood is a fundamental component of any healthcare setting, and is essential for traumatic injuries, surgeries, chronic illnesses, and cancer care. In the United States, approximately 29,000 units of red blood cells (RBCs), 5,000 units of platelets, and 6,500 units of plasma are needed daily. This demand increases exponentially during publicemergencies, such as pandemics, natural disasters, or mass casualty events, and during times of war. The current blood supply depends on a steady stream of healthy donors. Unfortunately, the number of donors has declined over the last two decades, leading to critically low levels and declaration of an emergency blood shortage by the American Red Cross in January 2024. Exacerbating the supply issue are the inherent constraints of current blood products: structural and functional instability and limited shelf-lives as well as the need for complex cold-chain transportation and storage systems, substantial weight and space capacity, and systems for warming at points of delivery. These factors contribute to wastage of 200,000 to 1 million units of blood per year and are of particular consequence in remote areas where infrastructure is limited. Lyophilization (also known as freeze drying) of blood products offers a method for both increasing longevity of blood products and simplifying the system of collection, storage, and delivery, providing a solution to the blood supply shortage and a revolutionary advancement for the U.S. blood supply chain. The disclosure provides a platform for stabilizing cellular blood products in preparation for lyophilization. By enabling long-term, simple, and compact storage, lyophilized blood products support banking of blood during non-crisis periods, simplifying blood storage and distribution infrastructure, minimizing wastage, and allowing for easy transport to far-reaching sites of need.Cry opreservation of blood products also can increase longevity and stability, reducing wastage in the blood supply.

[0038] Blood for resuscitation can be in the form of whole blood or blood components, with clinical outcomes comparable between transfusion of whole blood and replacement of components (red blood cells [RBCs], fresh frozen plasma [FFP], and platelets) in a 1 :1 : 1 ratio. FDA-approved whole blood needs to be collected and tested for transfusion transmitted disease by a licensed blood donor center and is stored up to 35 days at 1-6°C with an anticoagulant (CPD or CPDA-1). Fresh whole blood can be collected emergently (e.g., at “walking blood banks”) for use within 24 hours at room temperature but comes with increased risk of disease transmission and is not FDA-approvable. Both fresh and stored whole blood require complex screening (e.g., group O donors with low anti-A / -B titers) to prevent blood group mismatch and hemolysis. Additional disadvantages of stored whole blood include the need for refrigeration and decreased hemostatic capability beyond two weeks.

[0039] Use of individual blood components allows for less complex screening as well as precision resuscitation based on need but also has disadvantages. FFP requires storage at - 30°C for preservation of constituent coagulation factors although it can be maintained for upto 1 year. Platelets require room temperature storage to maintain membrane integrity, which necessitates a brief shelf-life of 5 days to avoid microbial contamination. RBC biopreservation is the most complex due to propensity toward cell damage. Refrigeration at 1-6°C is the most common mode of storage, with an FDA-approved shelf-life of up to 42 days; but cold storage of RBCs causes accumulation of known biochemical, oxidative, and biomechanical damages (i.e., “storage lesion”) as well as increased extravascular hemolysis over time. Cry opreservation, or storage at ultra-low temperatures of -80°C to -140°C, halts cellular metabolism, thereby minimizing RBC degradation and allowing for longer-term storage up to 10 years. However, cry opreservation requires the addition of a cryoprotectant (i.e., glycerol) to minimize freeze damage to the RBCs and in turn requires careful thawing followed by deglycerolization in order to avoid swelling and lysis of transfused RBCs. Deglycerolization itself can cause osmotic stress and cellular loss up to 20%. In addition to these limitations, all components have substantial weight and space requirements and necessitate an infrastructure for warming or thawing + / - deglycerolization at the site of transfusion, while RBCs and FFP require additional complex cold-chain transportation and storage systems.

[0040] Due to the limitations outlined above, current forms of blood products are unable to meet worldwide demand, are slow to deploy and utilize, and are impractical for large-scale and far-reaching needs. Lyophilization of blood products can circumvent many of these limitations, supporting worldwide accessibility, scenarios of high demand, and streamlining of a complex blood supply chain system. Lyophilization, or freeze-drying, is a process by which water is removed from a product after freezing. The material first undergoes supercooling, then a vacuum-induced pressure differential drives sublimation from solid to vapor phase. Without water, most biological processes cease, even at room temperature. Lyophilization has many advantages. It minimizes oxidative stresses that accumulate in cold storage, maintains cellular structure and function, and allows for stable long-term storage at room temperature. It lowers product weight considerably (RBCs are -64% water and plasma is -92% water), facilitating transport. Reconstitution is rapid and easy, requiring only sterile water that should be readily obtainable in even austere circumstances. Lyophilized plasma (freeze-dried plasma, FDP) has been successfully employed since World War II, while lyophilized platelets have been actively investigated in animal models for decades. In contrast, lyophilization of RBCs remains a challenge. There thus exists a need for systems, devices and methods to create lyophilized whole blood product, or a component replacement. However, our initial focus will be on lyophilization of RBCs. RBCs are most critical duringsevere hemorrhage for oxygen carrying capacity to the brain and vital organs. And they are the most commonly transfused among blood components, with about 15 million units transfused annually in the U.S. and 85 million units transfused annually worldwide. Realization of a lyophilized RBC product will also contribute to the creation of a lyophilized whole blood product. However, unlike plasma, cellular blood products such as RBCs and platelets poorly tolerate current lyophilization methods known in the art.Definitions

[0041] Unless defined otherwise, technical and scientific terms as used herein have the same meaning as commonly understood by one of ordinary skill in the art. One skilled in the art will recognize many methods can be used in the practice of the present disclosure. Indeed, the present disclosure is in no way limited to the methods and materials described. For purposes of the present disclosure, the following terms are defined below.

[0042] As used herein, the term “patient” or “subject” refers a mammal in need of a medical procedure receiving a blood product. The subject can be, inter alia, a human, pig, mouse, rat monkey and the like.

[0043] As used herein, “blood product” includes separated platelets, plasma, or white blood cells.

[0044] “Reconstituted whole blood” refers to products that provide platelets, RBC, and plasma in parallel to a patient during transfusion.

[0045] As used herein, “whole blood” includes white blood cells (WBCs), platelets suspended in plasma, red blood cells, and electrolytes, hormones, vitamins, antibodies, etc. Whole blood is collected from a blood donor, and is usually combined with an anticoagulant. A “unit” of blood is about 450-500 ml including anticoagulant. Suitable anticoagulants include CPD, CPDA1, ACD, and ACD-A.

[0046] As used herein, “red blood cells” (RBCs) refers to red blood cells in whole blood, or after purification from whole blood. Red blood cells contain hemoglobin, the iron-containing protein that carries oxygen throughout the body and gives red blood its color. The percentage of blood volume composed of red blood cells is called the hematocrit.

[0047] “Platelets” are small, membrane-bound cell fragments formed from megakaryocytes and are present in the blood and spleen, and help form blood clots.

[0048] As used herein, the term “cryoprotective agent,” “cryoprotectant,” “cryopreservative” and the like refers to a molecule which, when introduced into a cell or membrane-bound cell fragment, prevents or reduces damages to cellular components, including, but not limited to,damage to cellular membranes, membrane lipids, and preventing or reducing oxidative stress when temperature is reduced.

[0049] As used herein “cryopreservation” refers to the use of very low temperatures (below freezing, i.e., 0 °C) to preserve living cells and tissues.

[0050] Lyophilization, also known as freeze drying or cryodessication, refers to low- temperature processes used to remove water from and preserve perishable materials. Typical lyophilization processes involve freezing the material and lowering atmospheric pressure, thereby removing water by sublimation.

[0051] As used herein, the terms “about,” “approximately,” and “substantially” refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, e.g., the limitations of the measurement system. For example, “about,” “approximately,” or “substantially” can mean within one or more than one standard deviation per the practice in the art. Alternatively, “about” or “approximately” can mean a range of up to 10% (e.g., ±10%) or more depending on the limitations of the measurement system. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5- fold of a value. When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of “about,” “approximately,” and “substantially” can be assumed to be within an acceptable error range for that particular value or composition. Also, where ranges, or subranges, or combinations thereof, of values are provided, the ranges, or subranges, or combinations thereof, can include the endpoints of the ranges, or subranges, or combinations thereof.

[0052] The terms “comprises,” “comprising,” “includes,” “including,” “having,” and their conjugates mean “including but not limited to.”

[0053] The term “consisting of’ means “including and limited to.”

[0054] The term “consisting essentially of’ means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0055] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.

[0056] Throughout this application, various aspects of this disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as “from 1 to 6” should be considered to have specifically disclosed subranges such as “from 1 to 3,” “from 1 to 4,” “from 1 to 5,” “from 2 to 4,” “from 2 to 6,” “from 3 to 6,” etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0057] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0058] Throughout this application various publications, patents, and / or patent applications are referenced. The disclosures of the publications, patents and / or patent applications are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art to which this disclosure pertains. The contents of U.S. Patent No. 11,442,117 are incorporated by reference in their entirety herein.Systems and Devices

[0059] The disclosure provides systems and devices for delivery of cryoprotective agents to the interior of cells or membrane-bound cellular fragments using magnetoporation.

[0060] Magnetoporation can be used for clinically translatable intracellular delivery. Magnetoporation offers a gentle and efficient method for introducing cryoprotective agents such as trehalose into cellular products, such as RBCs and platelets. One hurdle in harnessing the cryoprotective effects of cryoprotective agents such as trehalose is delivering the molecule intracellularly at sufficient concentrations. The Cryoprotective agent, such as trehalose must exist on both sides of the cell membrane to impart its protective effects. However, mammalian cells lack transporters necessary for efficient intracellular uptake of trehalose. A number of different approaches have attempted to deliver trehalose into cells, with variable success. Passive loading via fluid-phase endocytosis can occur for platelets but for RBCs is complicated by oxidation of hemoglobin during prolonged incubation at 37°C.Liposome-mediated and nanoparticle-mediated transfer have proven unsuccessful due to long processing times and low intracellular trehalose yields, while electroporation-based delivery comes at the expense of structural compromise and high rates of hemolysis. Sonoporationbased technology may be used to deliver trehalose intracellularly for lyophilized RBCs. However, while able to process high volumes quickly, in vivo safety and efficacy of sonoporation-based methods are yet to be determined. In contrast, magnetoporation offers a number of advantages, including scalability, ease of use, transportability, and minimal regulatory burden.

[0061] Exemplary magnetoporation protocols use external, pulsed magnetic fields to introduce foreign molecules into cells efficiently and gently. Magnetoporation can be quick and require only a few simple consumables, which minimizes costs, labor, and regulatory burden. In addition, the process can be fine-tuned to the physical characteristics of a particular cell, minimizing manipulation and physical stressors to the cell, and has shown high efficiency of intracellular transfer of varying payload (i.e., DNA, RNA, and polysaccharides) into a variety of immortalized and primary cell types including RBCs. The circuitry dynamics of the magnetoporation process are suited to scalability: as sample volume increases, the uniformity of the magnetic field and the efficiency by which it interacts with the sample increases.

[0062] Magnetoporation utilizes rapidly changing magnetic fields to generate electric current, with rate of change of the magnetic field being a large determinant of transfer efficiency. Cells are placed inside a cuvette that is then situated within a fixed and cooled coil. Magnetic fields generated by pulsed power are transmitted through the coil and coupled to the sample volume by a ferrite core within the cuvette. This core amplifies the magnetic field and also acts as a type of reservoir when saturated, wherein the channeling of the magnetic field is temporarily lost. The core's passage into and out of saturation causes the magnetic permeability of the volume around the core to rapidly change, which in turn causes a higher rate of change of the local magnetic field. The transient electric currents induced by these rapidly changing fields result in transient poration of cell membranes and entry of extracellular material.

[0063] Scalability is an asset of magnetoporation and a significant differentiator from competitive technologies. Electroporation employs a point-to-point current of high voltage, which passes through cells, requires a conductive buffer, and subjects cells to electrical arcing. Although it can accomplish high transfer efficiencies, this is offset by high cellular death. With electroporation, larger sample volumes have higher resistance and require highervoltages to pass current across them, which leads to lower transfer efficiency, higher thermal stress, and more cell death with scale. With sonoporation, higher volume processing appears to increase cellular stress leading to low cell (e.g., RBC) recovery following lyophilization. In contrast, with magnetoporation, an increase in transfer efficiency with higher processing volumes is an inherent part of its electromagnetic circuitry dynamics and occurs without additional stress on cells. Specifically, electric fields are induced on the cells as a result of the rapidly changing applied magnetic field. As the radial dimension around the axis of the magnetic coil increases, inductance inside the treated volume increases proportionally with cross sectional area of the sample volume (as radius squared) while the electrical resistance increases with circumference (linearly with radius). In other words, as the volume increases, the ratio of inductance to electrical resistance increases. Furthermore, the efficiency by which the magnetic field interacts with the sample (i.e., magnetic coupling) also increases with sample volume, as its cross-sectional area relative to coil radius increases. These nonlinear changes result in the electric field being applied to the sample volume for higher durations and voltages, which is expected to shorten processing times and simplify circuitry while avoiding additional stress on cells.

[0064] Suitable systems and devices for generating magnetic fields and magnetoporating cells are known in the art, and are described, for example, in U.S. Patent No. 11,442,117, the contents of which are incorporated by reference in their entirety herein.

[0065] In some embodiments, the methods comprise applying a transient magnetic field to a sample of cells or membrane-bound cell fragments. In some embodiments, the transient magnetic field is applied to a sample with a volume of between about 2 pL and about 50 mL, about 0.5 mL and about 30 mL, about 1 mL and about 20 mL, about 2 pL and about 1000 pL, about 5 pL and about 500 pL, about 5 pL and about 50 pL, about 10 pL and about 50 pL, or about 10 pL and about 30 pL. In some embodiments, the sample volume is about 5 pL, about 10 pL, about 20 pL, about 50 pL, about 100 pL, about 1 mL, about 5 mL, about 10 mL, about 20 mL, or about 50 mL. In some embodiments, the transient magnetic field is applied to a sample with a volume of between about 1 mL and about 500 mL, between about 10 mL and about IL, between about 50 mL and about 5L, between about IL and about 10,000L, between about 5L and about 500L, between about 10L and about 500L, between about 10L and about 100L, between about 10L and about 50L, between about 100L and about 10,000L, between about 100L and about lOOOL, or between about 100L and about 500L.

[0066] In some embodiments, the transient magnetic field is applied to the sample in multiple trains of pulses, with intervals between the trains of pulses in which the transient magneticfield is not applied. In some embodiments, the transient magnetic field is applied to the sample in 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 trains of pulses. In some embodiments, the transient magnetic field is applied to the sample in 4 trains of pulses. In some embodiments, the transient magnetic field is applied to the sample in 5 trains of pulses. In some embodiments, the transient magnetic field is applied to the sample in 6 trains of pulses. In some embodiments, the transient magnetic field is applied to the sample in 7 trains of pulses. In some embodiments, the transient magnetic field is applied to the sample in 8 trains of pulses. In some embodiments, the transient magnetic field is applied to the sample in 9 trains of pulses. In some embodiments, the transient magnetic field is applied to the sample in 10 trains of pulses. In some embodiments, an individual train of pulses comprises between about 1 thousand and about 50 thousand pulses, between about 2 thousand and about 25 thousand pulses, or between about 2 thousand and about 15 thousand pulses. In some embodiments, an individual train of pulses comprises about 5 thousand pulses. In some embodiments, an individual train of pulses comprises about 10 thousand pulses. In some embodiments, an individual train of pulses comprises about 15 thousand pulses. In some embodiments, an individual train of pulses comprises about 20 thousand pulses. In some embodiments, the time between trains of pulses is about 0.1 seconds, about 0.2 seconds, about 0.5 seconds, or about 1 second. In some embodiments, the time between trains of pulses is about 0.2 seconds. In some embodiments, the time between trains of pulses is about 0.5 seconds. In some embodiments, the time between trains of pulses is about 1 second.

[0067] In some embodiments, the transient magnetic field is applied to the sample at a pulse strength of between about 75 Volts (V) to about 250 V. In some embodiments, the transient magnetic field is applied to the sample at a pulse strength of between about 100 V to about 200 V. In some embodiments, the transient magnetic field is applied to the sample at a pulse strength of between about 150 V to about 200 V. In some embodiments, the transient magnetic field is applied to the sample at a pulse strength of about 150 V, 160 V, 170 V, 180 V, 190 V or 200 V. In some embodiments, the transient magnetic field is applied at a frequency of between about 100 Hertz (Hz) to about 200 Hz. In some embodiments, the transient magnetic field is applied at a frequency of between about 120 Hz to about 150 Hz. In some embodiments, the transient magnetic field is applied at a frequency of about 150 Hz to about 175 Hz. In some embodiments, the transient magnetic field is applied at a pulse strength of about 170 V and a frequency of about 150 Hz to about 175 Hz. In some embodiments, the transient magnetic field is applied at a pulse strength of about 180 V and a frequency of about 120 Hz to about 150 Hz.Methods

[0068] The disclosure provides methods of introducing cryoprotective agents into cells or membrane-bound cell fragments using the devices and systems disclosed herein. In some embodiments, the methods comprise (a) contacting the cell or membrane-bound cell fragment with the cryoprotective agent; and (b) applying a magnetic field to the cell or membranebound cell fragment under conditions sufficient to introduce the cryoprotective agent into the cell or membrane-bound cell fragment.

[0069] In some embodiments the cell comprises a red blood cell, and the cryoprotective agent comprises trehalose. In some embodiments, the methods comprise contacting a plurality of red blood cells with trehalose and applying a magnetic field. In some embodiments, the methods comprise, prior to applying the magnetic field, centrifuging whole blood at about 1400 g for about 5 minutes thereby collecting the plurality of red blood cells; washing the plurality of red blood cells with Dulbecco’s Phosphate Buffered Saline (DPBS); and suspending the cells in DBPS at a concentration of IxlO7cells per pL.

[0070] In some embodiments the membrane-bound cell fragment comprises a platelet, and the cryoprotective agent comprises trehalose. In some embodiments, the methods comprise contacting a plurality of platelets with trehalose and applying a magnetic field.Cryoprotective Agents

[0071] The disclosure provides cryoprotective agents, for introduction into cells or membrane-bound cell fragments using the devices, systems and methods described herein.

[0072] For cells or cell fragments to remain viable, membrane porosity, structural integrity, and thermodynamic stability must be maintained throughout the freezing, drying, and rehydration steps. A number of cryoprotective compounds have been shown to offer such biostability. Exemplary cryoprotective agents are described in US 2023-0381338, the contents of which are incorporated by reference herein.

[0073] In some embodiments, the cryoprotective agent comprises glycerol, dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, sucrose, trehalose, polyvinylpyrrolidone (PVP), hydroxyethyl starch (HES), L-proline, human serum albumin (HSA), epigallocatechin gallate (EGCG), hydroxypropyl-P-cyclodextrin, polysorbate (e.g polysorbate 80, polysorbate 20, and combinations thereof), inulin, CAHS D, isoleucine, 3- hydroxynonanoic acid, deoxycarnitine, spermidine, gamma-glutamylalaine, malate, tyrosine, leucine, X - 23650, sebacate, gamma-glutamylvaline, gamma-glutamylthreonine, myristate, l-palmitoyl-2-oleoyl-GPE, ornithine, N-acetylasaparagine, mal onate, UDP-glucose, UDP-galactose, 3 -Hydroxy octanoate, gamma-glutamylglutamine, 2 -hydroxy decanote, L-proline, dimethlyglycine, sugars (sucrose, trehalose), polyols (sorbitol, glycerol, mannitol, mannosyl- glyceramide, mannosyl-glycerol), N-acetylated diamino acids (N-acetylglutaminylglutamine amide), betaines (betaine, glycine, and deritatives), and amino acids and derivatives thereof (e.g., proline, glutamate, glutamine, alanine, ectoine, and hydroxy ectoine), and combinations thereof.

[0074] In some embodiments, the cryoprotective agent comprises a polyhydroxy compound such as a sugar (e.g., a mono-, di-, or polysaccharide), polyalcohol, or a derivative thereof, glycerol, or polyethyleneglycol. In some embodiments, the cryoprotective agent comprises trehalose, maltose, sucrose, glucose, lactose, dextran, glycerol, or aminoglycoside. In some embodiments, the cryoprotective agent comprises mannitol, trehalose, sorbitol, or sucrose.

[0075] In some embodiments, the cryoprotective agent comprises a sugar, such as trehalose, maltose, sucrose, glucose or lactose.

[0076] In some embodiments, the cryoprotective agent comprises trehalose. Trehalose is one such cryoprotective compound, given its excellent protective effects upon freezing and dehydration. Widely occurring among bacteria, fungi, plants, and animals, trehalose is a nontoxic and biocompatible disaccharide sugar that stabilizes cells throughout the transition from hydrated state to a fully frozen or desiccated state and back. Trehalose contains multiple hydroxyl groups which bind the hydrophilic surfaces of cellular proteins and the plasma membrane. The presence of trehalose reduces the formation of ice crystals by interfering with hydrogen bond formation and slows the dynamics of water molecules close to biomolecules, preventing water from crystalizing next to them during cooling. During dehydration, trehalose begins to replace water altogether as the stabilizer at these hydrophilic surfaces. In the completely desiccated state, trehalose vitrifies into a glass; this protective encasement prevents formation of intra- and extracellular ice crystals as well as limits movement intracellularly to reduce enzymatic degradation. Trehalose has been approved as safe by all international and national food regulatory authorities, including the FDA, and is already used in foods and vaccines.

[0077] In some embodiments, the cells or membrane-bound cell fragments are contacted with the trehalose at a concentration of between about 100 mM and about 1,000 mM. In some embodiments, the cells or membrane-bound cell fragments are contacted with the trehalose at a concentration of between about 200 mM and about 600 mM. In some embodiments, the cells or membrane-bound cell fragments are contacted with the trehalose at a concentration of between about 100 mM and about 400 mM. In some embodiments, the cells or membrane-bound cell fragments are contacted with the trehalose at a concentration of between about 100 mM and about 300 mM. In some embodiments, the cells or membrane-bound cell fragments are contacted with the trehalose at a concentration of about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM or about 500 mM. In some embodiments, the cells or membrane-bound cell fragments are contacted with the trehalose at a concentration of about 100 mM. In some embodiments, the cells or membrane bound cell fragments are contacted with the trehalose at a concentration of about 200 mM. In some embodiments, the cells or membrane-bound cell fragments are contacted with the trehalose at a concentration of about 300 mM. In some embodiments, the cells or membrane-bound cell fragments are contacted with the trehalose at a concentration of about 400 mM. In some embodiments, the cells or membrane-bound cell fragments are contacted with the trehalose at a concentration of about 500 mM. In some embodiments, the cells or membrane-bound cell fragments are contacted with the trehalose at a concentration of about 600 mM. In some embodiments, the cells or membrane-bound cell fragments are contacted with the trehalose at a concentration of about 700 mM. In some embodiments, the cells or membrane-bound cell fragments are contacted with the trehalose at a concentration of about 800 mM. In some embodiments, the cells or membrane-bound cell fragments are contacted with the trehalose at a concentration of about 900 mM. In some embodiments, the cells or membrane-bound cell fragments are contacted with the trehalose at a concentration of about 1,000 mM. In some embodiments, the cells or membrane-bound cell fragments are contacted with the trehalose immediately prior (i.e., less than about 1 minute, 2, minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, or 30 minutes) prior to application of the transient magnetic field.

[0078] Cryoprotective agents can be classed as “penetrating” or “non-penetrating” cryoprotective agents. Without wishing to be bound by theory, it is thought that penetrating and non-penetrating cryoprotective agents facilitate cryopreservation by different mechanisms. Penetrating agents are thought to reduce cellular water content and mitigate the potentially damaging effects of concentrated solutes in cells or membrane-bound cell fragments at lower temperatures, while non-penetrating agents osmotically remove water from cells during the initial phases of freezing.

[0079] In some embodiments, the cryoprotective agent is a penetrating cryoprotective agent. In some embodiments, the penetrating cryoprotective agent comprises glycerol, ethylene glycol, dimethyl sulfoxide, or propylene glycol.

[0080] In some embodiments, the cryoprotective agent is a non-penetrating cryoprotective agent. In some embodiments, the non-penetrating cryoprotective agent comprises sucrose, trehalose, polyvinylpyrrolidone, hydroxyethyl starch, L-proline, human serum albumin, epigallocatechin gallate, hydroxypropyl-P-cyclodextrin, polysorbate 80, inulin, CAHS D, isoleucine, 3-hydroxynonanoic acid, spermidine, gamma-glutamylalaine, malate, tyrosine, leucine, X-23650, sebacate, gamma-glutamylvaline, gamma-glutamylthreonine, myristate, 1- palmitoyl-2-oleoyl-GPE, ornithine, N-acetylasaparagine, malonate, UDP -glucose, UDP- galactose, 3 -hydroxy octanoate, gamma-glutamylglutamine or 2-hydroxydecanote.

[0081] Exemplary cryoprotective agents are shown in Table 1 below.Table 1. Cryoprotective agents and usesCells and Membrane-Bound Cell Fragments

[0082] The disclosure provides systems and devices, and methods of using same, to introduce cryoprotective agents into cells or membrane-bound cell fragments. Any suitable cell type, or cell fragment type, is envisaged as within the scope of the instant disclosure. In some embodiments, the cells are mammalian cells, such as human, rodent or non-human primate cells. In some embodiments, the cells are human cells. In some embodiments, the cells are fungal, yeast, parasitic or bacterial cells. In some embodiments, the cells are bacterial cells, fungal cells, parasite cells, or plant cells. Exemplary cells include, but are not limited to, stem cells, hematopoietic (blood-forming) stem cells (HSC), skeletal muscle stem cells, mesenchymal stem cells, lymphocytes, dendritic cells, and pancreatic islet cells.

[0083] In some embodiments, the cells are mammalian cells. In some embodiments, the mammalian cells are selected from the group consisting of heart cells, liver cells, kidney cells, skin cells, brain cells, bladder cells, testes cells, ovary cells, uterus cells, eye cells, pancreatic cells, fallopian tube cells, vaginal cells, teste cells, prostate cells, placenta cells, large intestine cells, small intestine cells, colon cells, cancer cells, muscle cells, epithelial cells, connective tissue cells, nerve cells, blood cells, white blood cells, red blood cells, T cells, B cells, lymphocytes, antigen presenting cells, platelets, macrophages, monocytes,granulocytes neutrophils, eosinophils, basophils, and cancer cells. In some embodiments, the cells are red blood cells, stem cells, oocytes, embryonic cells or sperm.

[0084] In some embodiments, the cell comprises a blood cell. In some embodiments, the blood cell comprises a red blood cell or a white blood cell.

[0085] Membrane-bound cell fragments include, inter alia, platelets, lysosomes, exosomes and the like. In some embodiments, the membrane-bound cell fragment comprises a platelet. In some embodiments, the membrane-bound cell fragment comprises a platelet.

[0086] In some embodiments, the blood cell comprises a red blood cell. Red blood cells (RBCs), also referred to as erythrocytes are the most common type of blood cell and the principal means of delivering oxygen (O2) to tissues in vertebrates. Red blood cells lack a cell nucleus, but include a plasma membrane filled with the oxygen carrier hemoglobin. In some embodiments, the cell are contacted with the cryoprotective agent, e.g. trehalose, and are at a concentration of between about IxlO4and IxlO8cells per pL, between about IxlO5and IxlO8cells per pL, between about IxlO5and IxlO7cells per pL, between about IxlO5and 5xl06cells per pL, or between about IxlO5and IxlO6cells per pL. In some embodiments, the cell are contacted with the cryoprotective agent, e.g. trehalose, and are at a concentration of about IxlO4cells per pL, about 5xl04cells per pL, about IxlO5cells per pL, about 5xl05cells per pL, about IxlO6cells per pL, about 5xl06cells per pL, about IxlO7cells per pL, about 5xl07cells per pL, about IxlO8cells per pL or about 5xl08cells per pL.

[0087] In some embodiments, the blood cell comprises a white blood cell. White blood cells, also termed leukocytes, immune cells, or immunocytes, are cells of the immune system. Exemplary white blood cells envisaged as within the scope of the disclosure include, but are not limited to, granulocytes (e.g., neutrophils, eosinophils, and basophils), and agranulocytes (monocytes, and lymphocytes such as T cells and B cells).

[0088] The cells and / or membrane bound cell fragments can be suspending in any suitable buffer known in the art, including but not limited to, Dulbecco’s Phosphate Buffered Saline (DPBS), HEPES, MOPS, Tris-based buffers and the like.

[0089] In some embodiments, the cells and / or membrane bound cell fragments undergo a recovery period after magnetoporation and prior to downstream applications, such as lyophilization or cryopreservation. An exemplary recovery period comprises transferring the cells to a cell culture medium, and incubating the cells or membrane bound cell fragments under physiologically suitable conditions (e.g., 37°C, 5% CO2) for between about 8 and 18 hours, e.g. 8, 9, 10, 11, 12 13, 14, 15, 16, 17 or 18 hours, or any range therebetween. In some embodiments, the recovery period comprises between 8 and 18 hours. In some embodiments,the recovery period comprises between 8 and 24 hours. Suitable cell culture media will be known to persons of ordinary skill in the art, and include, inter alia, Roswell Park Memorial Institute (RPMI) complete medium, Dulbecco’s Modified Eagle Medium (DMEM) and the like.Lyophilization or Cry opreservation

[0090] In some embodiments, the methods of the disclosure comprise lyophilizing the cells or membrane-bound cell fragments following introduction of the cryoprotective agent into the cells or membrane-bound cell fragments.

[0091] Lyophilization (or freeze-drying) is a potentially simple solution to blood supply shortage, yielding products that can be easily stocked, stored long term, and transported to even far-reaching sites of need. When lyophilized, material is supercooled then sublimated from solid to vapor phase. The removal of water not only decreases the material’s mass considerably but also halts cellular biological processes, which in turn maintains cellular structure and function and allows long-term storage at room temperature. Preparation of a transfusable product at the bedside would be rapid and easy, requiring only reconstitution with sterile water. Lyophilized blood products would make large reserves readily and continuously available for healthcare settings, and moreover would address scenarios of high demand, allow for a more streamlined blood supply chain system, and even support worldwide accessibility.

[0092] Any suitable lyophilization method is envisaged as within the scope of the instant disclosure. In some embodiments, lyophilization comprises contacting the cell or membranebound cell fragment with a lyophilization buffer; cooling the cell or membrane-bound cell fragment to less than at least -40 °C (e.g., less than at least -45 °C, at least -50 °C, at least -60 °C or at least -65 °C); drying the cell or membrane-bound cell fragment at less than -45 °C and about 50 to 500 milliTorr (mTorr) for about 10-20 hours; and drying the cell or membrane-bound cell fragment at 10-20 °C for about 5-20 hours. In some embodiments, lyophilization comprises contacting the cell or membrane-bound cell fragment with a lyophilization buffer; cooling the cell or membrane-bound cell fragment to less than at least - 60 °C; drying the cell or membrane-bound cell fragment at -45 °C and 200 mTorr for about 15 hours; and drying the cell or membrane-bound cell fragment at 15 °C for about 10 hours. Suitable low temperature vacuum freeze dryers are known in the art, and include, e.g., the Labconco Freezone Freeze Dry System and the Lyovapor™ L-300.

[0093] Any suitable lyophilization buffer may be used in the methods disclosed herein. An exemplary lyophilization buffer can include bulking agents that provide bulk to the formulation (e.g., mannitol, glycerine, sucrose and the like), buffers that control pH during the lyophilization process (Tris, HC1, histidine, phosphate and the like), structural modifiers that modify the properties of the lyophilized product (e.g., disaccharides), tonicity adjusters that yield an isotonic solution and control osmotic pressure, such as salts, stabilizers such as glucose, dextran, sucrose, trehalose, lactose, mannitol and / or alanine, agents that increase the collapse temperature of the cells or membrane-cell fragments to enable lyophilization at higher temperatures (glucose, dextran, maltose, maltotriose, maltotetrose and the like), excipients such as sugars or polyols, polymers, surfactants such as polysorbate 20 or polysorbate 80, and / or chelating complexes.

[0094] In some embodiments, the lyophilization buffer comprises a sugar. In some embodiments, the sugar comprises sucrose. In some embodiments, the sugar comprises lactose. In some embodiments, the sugar comprises glucose. In some embodiments, the sugar comprises trehalose. In some embodiments, the sugar comprises maltose. In some embodiments, the lyophilization buffer comprises between 5 mM and 800 mM of the sugar. In some embodiments, the lyophilization buffer comprises between 50 mM and 800 mM of the sugar. In some embodiments, the lyophilization buffer comprises between 50 mM and 500 mM of the sugar. In some embodiments, the lyophilization buffer comprises between 500 mM and 800 mM of the sugar. In some embodiments, the lyophilization buffer comprises between 10 mM and 200 mM of the sugar. In some embodiments, the lyophilization buffer comprises between 10 mM and 50 mM of the sugar. In some embodiments, the lyophilization buffer comprises between 15 mM and 30 mM of the sugar. In some embodiments, the lyophilization buffer comprises between 20 mM and 25 mM of the sugar. In some embodiments, the lyophilization buffer comprises about 5 mM, about 10 mM , about 15 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 32 mM, about 35 mM or about 40 mM of the sugar. In some embodiments, the lyophilization buffer comprises no more than about 500 mM of the sugar.

[0095] In some embodiments, the lyophilization buffer comprises an nucleobase, for example adenine, cytosine, guanine, thymine and / or uracil. In some embodiments, the nucleobase comprises guanine. In some embodiments, the nucleobase comprises adenine. In some embodiments, the lyophilization buffer comprises between about 0.10 mM and 1.0 mM of the nucleobase. In some embodiments, the lyophilization buffer comprises between about 0.20mM and 0.60 mM of the nucleobase. In some embodiments, the lyophilization buffer comprises between about 0.30 mM and about 0.50 mM of the nucleobase. In some embodiments, the lyophilization buffer comprises about 0.10 mM, about 0.20 mM, about 0.30 mM, about 0.35 mM, about 0.40 mM, about 0.41 mM, about 0.42 mM, about 0.43 mM, about 0.44 mM, about 0.45 mM, about 0.46 mM, about 0.47 mM, about 0.48 mM, about 0.49 mM, about 0.50 mM, about 0.52 mM, about 0.55 mM or about 0.60 mM of the nucleobase.

[0096] In some embodiments, the lyophilization buffer comprises a salt. Exemplary salts include, but are not limited to, NaCl, CaCh, KC1, MgCh and MgSC In some embodiments, the salt comprises NaCl. In some embodiments, the salt comprises KC1. In some embodiments, the salt comprises CaCh. In some embodiments, the lyophilization buffer comprises between about 1 mM and about 15 mM of the salt. In some embodiments, the lyophilization buffer comprises between about 2 mM and about 10 mM of the salt. In some embodiments, the lyophilization buffer comprises between about 4 mM and about 8 mM of the salt. In some embodiments, the lyophilization buffer comprises between about 5 mM and about 100 mM of the salt. In some embodiments, the lyophilization buffer comprises between about 10 mM and about 80 mM of the salt. In some embodiments, the lyophilization buffer comprises between about 20 mM and about 50 mM of the salt. In some embodiments, the lyophilization buffer comprises between about 25 mM and about 40 mM of the salt. In some embodiments, the lyophilization buffer comprises between about 25 mM and about 35 mM of the salt. In some embodiments, the lyophilization buffer comprises about 5 mM, about 7 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 35 mM, about 37 mM, about 40 mM, about 42 mM, about 43 mM, about 45 mM, about 47 mM or about 50 mM of the salt. In some embodiments, the lyophilization buffer comprises a combination of salts, e.g. two or more salts selected from the group consisting of NaCl, CaCh, KC1, MgCh and MgSC In some embodiments, the lyophilization buffer comprises NaCl and CaCh. In some embodiments, the lyophilization buffer comprises KC1 and MgSCN. In some embodiments, the lyophilization buffer comprises KC1 and NaCl.

[0097] In some embodiments, the lyophilization buffer comprises a stabilizer. In some embodiments, the stabilizer comprises a polysaccharide. In some embodiments, the stabilizer comprise glucose, dextran, sucrose, trehalose, lactose or mannitol. In some embodiments, the lyophilization buffer comprises glucose. In some embodiments, the lyophilization buffer comprises dextran. In some embodiments, the lyophilization buffer comprises sucrose. In some embodiments, the lyophilization buffer comprises trehalose. In some embodiments, thelyophilization buffer comprises lactose. In some embodiments, the lyophilization buffer comprises mannitol. In some embodiments, the stabilizer is at a concentration of between 0.1 mM and 30 mM. In some embodiments, the stabilizer is at a concentration of between 1 mM and 20 mM. In some embodiments, the stabilizer is at a concentration of between 5 mM and 15 mM. In some embodiments, the stabilizer is at a concentration of between 7 mM and 12 mM. In some embodiments, the stabilizer is at a concentration of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, or about 12 mM.

[0098] In some embodiments, the lyophilization buffer comprises a polysaccharide. In some embodiments, the polysaccharide is selected from the group consisting of dextran, hydroxyethyl starch, glycogen, pectin, chitin, chitosan, hyaluronic acid, inulin and trehalose. In some embodiments, the lyophilization buffer comprises the polysaccharide at between about 2% and about 30% weight per volume (w / v), between about 5% and about 20% w / v, between about 10% and about 17% w / v, or between about 12% and about 15% w / v. In some embodiments, the lyophilization buffer comprises the polysaccharide at about 5% w / v, about 7% w / v, about 10% w / v, about 12% w / v, about 15% w / v, about 17% w / v, about 20% w / v, about 22% w / v, about 25% w / v, about 27% w / v or about 30% w / v.

[0099] In some embodiments, the lyophilization buffer comprises bovine serum albumin (BSA). In some embodiments, the lyophilization buffer comprises about 0.1% w / v, about 0.5% w / v, about 1.0% w / v, about 1.5% w / v, about 2.0% w / v, about 2.5% w / v, about 3.0% w / v or about 3.5% w / v BSA. In some embodiments, the lyophilization buffer does not include BSA or any other animal derived protein.

[0100] In some embodiments, the lyophilization solution comprises a buffering agent. In some embodiments, the buffering agent is selected from the group consisting of histidine, sodium acetate, sodium phosphate, aspartate, sodium citrate and Tris. In some embodiments, the buffering agent comprises histidine. In some embodiments, the buffering agent comprises Tris-HCl. In some embodiments, the buffering agent comprises sodium acetate. In some embodiments, the buffering agent comprises sodium citrate. In some embodiments, the lyophilization comprises between about 0.10% w / v and about 20 % w / v, between about 0.50% w / v and about 10% w / v, between about 1.0% w / v and about 15% w / v, or between about 1% w / v and about 5% w / v buffering agent. In some embodiments, the lyophilization buffer comprises about 0.10% w / v, about 0.50% w / v, about 0.70% w / v, about 1.0% w / v, about 1.5% w / v, about 2.0% w / v, about 2.5% w / v, about 2.7% w / v, about 3.0% w / v, about3.2% w / v, about 3.5% w / v, about 3.7% w / v, about 4.0% w / v, or about 4.5% w / v buffering agent.

[0101] In some embodiments, the lyophilization buffer comprises sucrose, inosine, NaCl, mannitol, KC1 dextran, and / or sodium acetate.

[0102] In some embodiments, the lyophilization buffer comprises glycogen, guanine, NaCl, mannitol, dextran, and / or sodium citrate.

[0103] In some embodiments, the lyophilization buffer comprises glucose, adenine, NaCl, mannitol, KC1, dextran, bovine serum albumin (BSA), and / or sodium citrate.

[0104] In some embodiments, the lyophilization buffer comprises glucose, adenine, KC1 dextran, bovine serum albumin (BSA), and / or sodium acetate.

[0105] In some embodiments, the methods of the disclosure comprise cry opreserving the cells or membrane-bound cell fragments following introduction of the cryoprotective agent into the cells or membrane-bound cell fragments.

[0106] Any suitable method for cryopreserving cells or membrane-bound cell fragments can be used with the methods of introducing a cryoprotective agent into cells or membrane-bound cell fragments described herein. As an example, the cells or membrane-bound cell fragments can be frozen by gradually reducing the temperature at a rate of about 1 °C per minute until the target temperature is reached. In some embodiments, the target temperature is less than about -20 °C or about -30 °C, for example between about -30 °C to -180 °C, about -60 °C to about -160 °C, or about -80 °C to about -140 °C. In some embodiments, the target temperate is between about -20 °C to about -200 °C, for example about -196 °C, the temperature of liquid nitrogen. In some embodiments, the target temperature is between about -70 °C to about -90 °C. In some embodiments, the target temperature is between about -120 °C to about -180 °C. In some embodiments, the target temperature is about -15 °C, -20 °C, about -30 °C, about -80 °C, -about -100 °C, about -120 °C, about -150 °C, about -200 °C, or any range therebetween. In some embodiments, the target temperature is about -15 °C. In some embodiments, the target temperature is about -20 °C. In some embodiments, the target temperature is about -30 °C. In some embodiments, the target temperature is about -80 °C. In some embodiments, the target temperature is about -196 °C. In some embodiments, the target temperature is about -200 °C.

[0107] Cells or membrane-bound cell fragments can be cryopreserved in any suitable cryopreservation medium, which typically protects the cells or membrane-bound cell fragments from stress during the free-thaw process. Exemplary cry opreservation media include CryoStor® available from Stemcell Technologies, and the like.Pharmaceutical Compositions

[0108] The disclosure provides pharmaceutical composition comprising cells or membranebound cell fragments comprising the cryoprotective agents disclosed herein, and lyophilized by the methods disclosed herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, for example the lyophilization buffers described herein.

[0109] In general, the pharmaceutical compositions comprising cells or membrane bound cell fragments provided herein can be formulated for administration to a patient by any of the accepted modes of administration. Various formulations and drug delivery systems are available in the art. See, e.g., Gennaro, A. R., ed. (1995) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.

[0110] The pharmaceutical compositions disclosed herein can be solid or semi-solid (e.g., after lyophilization) or in liquid form (e.g., following rehydration of lyophilized cells or membrane-bound cell fragments, or following thawing of cryopreserved cells or membranebound cell fragments).[OHl] In general, pharmaceutical compositions provided herein will be suitable for administration by parenteral (e.g., intramuscular, intravenous or subcutaneous) administration, optionally following rehydration, if the pharmaceutical composition comprises lyophilized cells or membrane-bound cell fragments.

[0112] The pharmaceutical compositions described herein include, in general, at least one pharmaceutically acceptable excipient. Acceptable excipients are non-toxic, aid administration, and do not adversely affect the therapeutic benefit of the cells or membranebound cell fragments. Such excipient may be any solid, liquid, or semi-solid that is generally available to one of skill in the art and described herein.

[0113] The pharmaceutical compositions may, if desired, be presented in a pack or dispenser device containing one or more unit dosage forms containing the cells or membrane bound cell fragments. Such a pack or device may, for example, comprise metal or plastic foil, such as a blister pack, or glass, and rubber stoppers such as in vials. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising the cells or membrane bound cell fragments of the disclosure formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container and labeled for treatment of an indicated condition.

[0114] In some embodiments, the pharmaceutical composition comprises the lyophilized cells described herein, after rehydration.

[0115] Any suitable rehydration medium can be used to rehydrate the lyophilized cells or membrane-bound cell fragments of the disclosure. In some embodiments, the rehydration medium comprises water (e.g., sterile water). In some embodiments, the rehydration medium comprises a rehydration buffer.

[0116] In some embodiments, the rehydration buffer comprises a sugar, for example trehalose, sucrose, glucose and the like. In some embodiments, the rehydration buffer comprises glucose. In some embodiments, the rehydration buffer comprises sucrose. In some embodiments, the rehydration buffer comprises trehalose. In some embodiments, the rehydration buffer comprises between about 50 mM and about 500 mM, between about 100 mM and about 200 mM or between about 125 mM and about 150 mM of the sugar.

[0117] In some embodiments, the rehydration comprises a buffering agent such as histidine, sodium acetate, sodium phosphate, aspartate, Tris and / or ascorbate and the like. In some embodiments, the rehydration buffer comprises about 0.5 mM to about 50 mM, about 1 mM to about 30 mM, about 20 mM to about 30 mM, about 2 mM to about 10 mM, or about 3 mM to about 7 mM of the buffering agent.

[0118] In some embodiments, the rehydration buffer comprises one or more salts. In some embodiments, the one or more salts are selected from the group consisting of NaCl, CaCh, KC1, MgCh, KH2PO4, Na2HPO4 and MgSCh. In some embodiments, the one or more salts comprise NaCl and KC1. In some embodiments, the one or more salts comprise NaCl, KH2PO4, and KC1. In some embodiments, the one or more salts comprise NaCl, KH2PO4, Na2HPO4 and KC1. In some embodiments, the one or more salts comprise CaCh, KH2PO4, Na2HPO4 and KC1. In some embodiments, the rehydration buffer comprises between about 10 mM about 20 mM of the one or more salts. In some embodiments, the one or more salts comprises NaCl, and the rehydration buffer comprises between about 5 mM and about 100 mM, between about 10 mM and about 70 mM, between about 20 mM and about 50 mM, or between about 30 mM and about 45 mM NaCl. In some embodiments, the one or more salts comprises KC1, and the rehydration buffer comprises between about 10 mM and about 500 mM, between about 20 mM and about 200 mM, between about 30 mM and about 150 mM, or between about 50 mM and about 130 mM KC1. In some embodiments, the one or more salts comprises KH2PO4, and the rehydration buffer comprises between about 0.01 mM and about 5 mM, between about 0.10 mM and about 2 mM, between about 0.15 mM and about 0.50 mM, or between about 0.20 mM and about 0.35 mM KH2PO4. In some embodiments, the oneor more salts comprises Na2HPO4, and the rehydration buffer comprises between about 0.10 mM and about 20 mM, between about 0.50 mM and about 10 mM, between about 0.75 mM and about 5.0 mM, or between about 1.0 mM and about 3.0 mM Na2HPO4.

[0119] In some embodiments, the rehydration buffer comprises a polysaccharide. In some embodiments, the polysaccharide is selected from the group consisting of dextran, hydroxyethyl starch, glycogen, pectin, chitin, chitosan, hyaluronic acid, inulin and trehalose. In some embodiments, the rehydration buffer comprises the polysaccharide at between about 2% and about 30% weight per volume (w / v), between about 5% and about 20% w / v, between about 10% and about 17% w / v, or between about 12% and about 15% w / v. In some embodiments, the lyophilization buffer comprises the polysaccharide at about 5% w / v, about 7% w / v, about 10% w / v, about 12% w / v, about 15% w / v, about 17% w / v, about 20% w / v, about 22% w / v, about 25% w / v, about 27% w / v or about 30% w / v.

[0120] In some embodiments, the rehydration buffer comprises bovine serum albumin (BSA). In some embodiments, the rehydration buffer comprises about 0.1% w / v, about 0.5% w / v, about 1.0% w / v, about 1.5% w / v, about 1. 7% w / v, about 1.9% w / v, about 2.0% w / v, about 2.5% w / v, about 3.0% w / v or about 3.5% w / v BSA. In some embodiments, the rehydration buffer does not include BSA or any other animal derived protein.

[0121] In some embodiments, the rehydration buffer comprises a nucleobase, for example adenine, cytosine, guanine, thymine and / or uracil. In some embodiments, the nucleoside comprises adenine. In some embodiments, the nucleobase comprises guanine. In some embodiments, the nucleobase comprises thymine. In some embodiments, the rehydration buffer comprises between about 0.10 mM and 20.0 mM of the nucleobase. In some embodiments, the rehydration buffer comprises between about 1.0 mM and 10.0 mM of the nucleobase. In some embodiments, the rehydration buffer comprises between about 3.0 mM and about 7.0 mM of the nucleobase. In some embodiments, the rehydration buffer comprises about 1.0 mM, about 2.0 mM, about 3.0 mM, about 4.0 mM, about 5.0 mM, about 6.0 mM, about 7.0 mM, about 8.0 mM, about 9.0 mM, or about 10.0 mM of the nucleobase.

[0122] In some embodiments, the rehydration buffer comprises a nucleoside, for example adenosine, guanosine or inosine. In some embodiments, the rehydration buffer comprises between about 50 mM and about 500 mM, between about 20 mM and about 200 mM or between about 50 mM and about 150 mM of the nucleoside.

[0123] An exemplary rehydration buffer comprises trehalose, ascorbate, NaCl, KH2PO4, Na2HPO4, hydroxy ethyl starch, BSA, KC1, inosine, adenine and combinations thereof. A further exemplary rehydration buffer comprises sucrose, ascorbate, NaCl, KH2PO4, glycogen,KC1, inosine, adenine and combinations thereof. A still further exemplary rehydration buffer comprises trehalose, acetate, NaCl, KH2PO4, Na2HPO4, hydroxy ethyl starch, BSA, KC1, adenosine and combinations thereof.

[0124] In some embodiments, the pharmaceutical composition comprises cryopreserved cells or membrane-bound cell fragments as described herein, and a suitable cry opreservation medium. In some embodiments, the composition comprising cryopreserved cells or membrane-bound cell fragments is suitable for administration immediately after thawing. In alternative embodiments, the composition comprising cryopreserved cells or membranebound cell fragments can be mixed with one or more suitable pharmaceutically acceptable carriers or diluents after thawing and prior to administration to the subject.Kits and Articles of Manufacture

[0125] The disclosure provides kits comprising the lyophilized cells, membrane-bound cell fragments, cryopreserved cells and membrane-bound cell fragments, and pharmaceutical compositions described herein. In some embodiments, the kit comprises containers (e.g., vials, tubes and the like), and instructions for use. In some embodiments, the kit comprises a rehydration buffer as described herein.EXAMPLESExample 1: Protocol for Magnetoporation of Primary Human Erythrocytes with Trehalose Definitions and Abbreviations1. Erythrocytes (red blood cells, RBC)2. RPMI 1640 with L-glutamine (RPMI)3. Fetal Bovine Serum (FBS, heat inactivated)4. RPMI 1640 with L-glutamine, 10% FBS (Complete RPMI)5. Glucose-free RPMI 1640 with L-glutamine, 10% FBS (Glucose-free Complete RPMI)6. IX Dulbecco’s Phosphate Buffered Saline (DPBS)7. Fluorescence-activated cell sorting (FACS)8. Magnetoporation (Mag)Equipment / Materials1. Laminar flow hood or Biosafety Cabinet (workspace capable of maintaining a clean environment)2. 5% CO2 Incubator set at 37°C3. Water bath set for 37°C + / -1.0C°C (for prewarming media)4. Pipet-aid and set of pipettemans5. Individually sterile wrapped pipettes (5mL, lOmL, and 25mL)6. 0.20 micron filter pipette tips: 1 Opl, 20pl, 200 pL, 1000 pL7. Sterile 15mL and 50mL centrifuge tubes or equivalent8. Sterile 5mL snap cap tubes (must be polypropylene for Magnetoporation)9. Sterile 1.5 / 1.6 mL eppendorf tubes10. Sterile Pasteur pipettes for aspiration and aspiration pump11. Nexcelom Cellometer Auto 200012. Cellometer Disposable Counting Chambers, 20pl13. Centrifuge with insert adaptors to hold 5mL snap cap tubes, 15mL centrifuges tubes, and 50mL centrifuge tubes14. Sigma Genetics Magnetoporator15. Beckman Coulter CytoFLEX S (4 laser - blue laser required)16. Flow Jo software package for analyzing flow cytometry data17. Molecular Devices SpectraMax® plate readerReagents18. Fetal Bovine Serum, heat inactivated (FBS)19. RPMI 1640 with L-glutamine (RPMI)20. IX Dulbecco’s Phosphate Buffered Saline (DPBS, sterile)21. Disinfectant (such as 70% ethanol, must not be denatured)22. Purified, tissue culture grade trehalose23. FITC-Trehalose24. Filter-sterilized trypan blueProcedurePreparation of Culture Medium1. Transfer 45 mL of RPMI 1640 medium into a 50mL conical tube.2. Add 5 mL heat inactivated FBS.3. Prewarm at 37° C .Preparation of Tissue-Grade Trehalose Stock4. Weigh out 3.7833g tissue-grade trehalose and transfer to a 50 mL conical tube.5. Add 7 mL DPBS, vortex and warm slightly to resuspend.6. Measure volume and top up to 10 mL total DPBS for a 1 M stock solution.7. Filter sterilize into 15 mL conical tube for easier handling.8. Dilute 1 M stock solution to a 400 mM working concentration by adding 400 pL of 1 M stock solution to 600 pL DPBS in an eppendorf tube. Vortex well.Preparation of FACS Buffer9. In a 50mL conical tube, place 49 mL IX DPBS and 1 mL FBS.Preparation of Lyophilization Buffer10. Weigh out the indicated amounts of each buffer component and transfer to a 50 mL conical tube.Lyophilization Buffer glucose (180.18 g / mol MW) adenine (135.15 g / mol MW)NaCl (58.44 g / mol MW) mannitol (182.20 g / mol MW)KC1 (74.55 g / mol MW) dextranBSA sodium citrate11. Add 25 mL DPBS, vortex to resuspend.12. Measure volume and top up to 50 mL total DPBS for stock solution; filter sterilize.Preparation of Rehydration Buffer13. Weigh out the indicated amounts of each buffer component and transfer to 50mL conical.Rehydration Buffer trehalose (342.30 g / mol MW) ascorbate (176.13 g / mol MW)NaCl (58.44 g / mol MW)KH2PO4 (136.09 g / mol MW)Na2HPO4 (141.96 g / mol MW) hydroxyethyl starch (HES)BSAKC1 (74.55 g / mol MW) inosine (268.23 g / mol MW) adenine (135.15 g / mol MW)14. Add 25 mL DPBS, vortex to resuspend.15. Measure volume and top up to 50 mL total DPBS for stock solution; filter sterilize.Cell Preparation16. Collect whole blood into purple capped tubes (containing EDTA, ethylene diamine tetra-acetic acid).17. Gently mix whole blood and centrifuge RBC at 1400 g for 5 minutes.18. Through aseptic aspiration, discard supernatant, buffy coat, and upper layer of RBC.19. Wash RBC three times (3x) with isotonic PBS (DPBS); centrifuge at 1400 g for 5 minutes each wash.20. Following the 3rdwash, adjust cell suspension to 50% hematocrit with DPBS.21. Dilute RBC 1 : 10 in a 15 mL conical tube: 9 mL DPBS + 1 mL RBC, for final hematocrit of approximately 5%.22. Count 10 pL RBC in 990 pL DPBS; adjust final concentration of RBC to 10A6 RBC per 10 pL in 10 mL DPBS.23. Transfer 1 mL to a 1.5 mL eppendorf tube; count again for final concentration of working RBC.24. Place working concentration RBC into 37°C water bath until ready for use. Magnetoporation25. Prewarm s 50mL conical tube of complete RPMI media in 37°C water bath.26. Aliquot 980 pL of pre-warmed complete RPMI into 24-well tissue culture plates, one sample per well. Prewarm in 37°C, 5% CO2 incubator.27. Remove the eppendorf tube of RBC from water bath, and resuspend RBC gently with 200 pL pipetteman.28. Using the 20 pL pipetteman, pipette 10 pL of RBC into a 5 mL polypropylene tube for magnetoporation.29. Using the 20 pL pipetteman, pipette 10 pL of 400mM working concentration trehalose into the tube containing RBC (in above step), and gently pipette up and down 5 times to mix well.30. If including FITC-trehalose in the assay, using the 20 pL pipetteman, pipette 10 pL of FITC-trehalose into the tube containing RBC (in above step), and gently pipette up and down 5 times to mix well.31. Include the following control groups: no mag / no trehalose (cells alone); mag / no trehalose; no mag + trehalose [for background uptake levels],32. Magnetoporate samples at 170V / 150Hz (6x 10,000 pulses; 0.5 seconds between pulses).33. Let the sample sit at room temperature (RT) for 30 minutes post magnetoporation and prior to transfer to a 24-well plate containing prewarmed complete RPMI. Wash core with complete RPMI from well; wash tube with complete RPMI from well; gently pipette up and down to distribute RBC evenly in well. Return plate to 37°C, 5% CO2 incubator.34. Following overnight incubation, measure the volume of media in each well; top to 1 mL total volume with pre-warmed complete RPMI. Extract 50 pL for cell counts; utilize the remainder for analysis by flow cytometry, Megazyme Trehalose Assay Kit, or for lyophilization.Flow Cytometry35. Bring FACS buffer to room temperature.36. Centrifuge RBC in eppendorf tubes at 1400 rpm, RT, 5 min. Cells will form a pellet at bottom as long as the tube is handled gently.37. Aspirate supernatant with tube in an inverted position allowing all liquid to be removed.38. Add 1 mL DPBS, resuspend RBC gently, and centrifuge at 1400 rpm, RT, 5 minutes.39. Add 450 pL FACS buffer to each tube and gently pipette up and down.40. Place 50 uL into a 48-well plate containing 250-300 pL complete RPMI. Let cells settle before image analysis on Revolve microscope.41. Analyze for GFP expression on Beckman Coulter CytoFLEX S (4 laser - blue laser required). Signal will be picked up with the blue laser.42. Analyze flow cytometry data using FlowJo software package. Trehalose Assay (Megazyme Kit)43. Centrifuge RBC in eppendorf tubes at 1400 rpm, RT, 5 min.44. Wash cell pellet once (lx) with DPBS, centrifuge at 1400 g for 5 minutes.45. Remove all wash supernatant. If freezing for another day, place cell pellet in a prechilled rack at -80°C until ready to assay. If not, proceed to the addition of HEPES buffer.46. Thaw cells at RT.47. Add 10 mM HEPES to cell pellet for 100 pl final volume (begin with the addition of 70 pl HEPES, resuspend pellet, then measure volume. Top to 100 pl with additional HEPES buffer).48. Freeze / thaw 3x using liquid nitrogen (dry ice / ethanol bath if no liquid nitrogen available).49. Centrifuge at 14,000 rpm for 10 minutes at 4°C.50. Harvest supernatant (cell extract) into new pre-chilled (on ice) tubes and use immediately or store at -80°C until use (thaw on ice).51. Follow manufacturer’ s instructions for Megazyme Kit (for 96-well plate).52. For standard curve, use 0 pl, 1 pL, 2 pL, 4 pL, 8 pL, 16 pL, 32 pL of trehalose stock (200 pg / mL; 0.2 pg / pL). Adjust H2O volume accordingly. All wells will have 254 pL final volume after all solutions are added.53. Add 20 pL sample per well.54. Take plate reading at 340 nm BEFORE adding suspension 4 (trehalase). This is the baseline / background.55. Add 2 pL trehalase to each well, mix plate well by shaking on plate reader, and take reading.56. Wait 5 minutes, take reading.57. Starting at 5 minutes after the addition of trehalase, read plate every 2 minutes, saving data after every reading. Take plate reading every 2 minutes until color intensity no longer increases (stays consistent value).58. During calculations, note that l / 5thof the total sample was used per well when calculating trehalose concentration / cell.Lyophilization59. Centrifuge RBC in eppendorf tubes at 1400 rpm, RT, 5 minutes.60. Mix magnetoporated RBC with lyophilization buffer at a 1 :4 ratio.61. Pre-cool samples to -60° prior to lyophilization; primary dry at -45°C and 3 Pa for 15 hours, secondary dry at 15°C for 10 hours.62. Store the lyophilized RBC at 4°C.Rehydration and Recovery63. Rehydrate lyophilized RBC by adding 2 ml of preheated (37°C) rehydration buffer and gently shaking until fully dissolved.64. Evaluate numerical recovery by counting RBC before and after rehydration using a hemocytometer.Additional MethodsRBC Extraction1. Collect whole blood into CPDA-1 anticoagulant.2. Centrifuge at 1400 g for 5 minutes to separate layers.3. Remove the plasma (top layer) and huffy coat, collect RBC from the bottom.4. Wash RBC three times with isotonic Phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KC1, 1.18 mM KH2PO4, 20 mM Na2HPO4, pH 7.4).5. Adjust the hematocrit of the cell suspension to 50-60% using isotonic PBS.6. Store at 4°C and use within two days for electroporation and lyophilization. Cell Harvest and Freeze / Thaw1. Magnetoporated RBC are harvested after overnight culture in complete media.2. 50 uL of each sample is counted on the Cellometer, while the remainder of the RBC are centrifuged.3. Cell pellets washed once with DPBS and supernatants removed.4. HEPES buffer is added to RBC pellets, and pellets are freeze / thawed three times in liquid nitrogen.5. The cell extract is harvested by high centrifugation and used in the Megazyme trehalose assay.Loading Trehalose by Electroporation1. Prepare Electroporation Buffer: Dissolve trehalose (290-800 mM) and 5 mM KC1 in double-distilled water.2. Mix washed RBC with Electroporation Buffer in a 1 : 1.5 (v / v) ratio.3. Transfer 400 pl of cell suspension to a 2 mm-gap electroporation cuvette.4. Electroporate using an ECM 830 BTX Electro Square Porator. Parameters: field strength (0.5-1.5 kV / cm), pulse length (0.01-1 ms), frequency (4 pulses / min to 4 pulses / 15 min).5. Incubate the cells at 37°C for 1 hour post-electroporation to allow resealing. Lyophilization1. Prepare Freeze-Drying Buffer: 24 mM glucose, 0.43 mM adenine, 33 mM NaCl, 8.9 mM mannitol, 6.6 mM KC1, 15% dextran, 2.5% BSA, 3% sodium citrate.2. Mix electroporated RBC with Freeze-Drying Buffer at a 1 :4 ratio.3. Freeze-dry using a lyophilizer: Cool to -60°C, primary dry at -45°C and 3 Pa for 15 hours, secondary dry at 15°C for 10 hours.4. Store the lyophilized RBC at 4°C.Rehydration and Recovery1. Prepare Rehydration Buffer: 141 mM trehalose, 5 mM ascorbate, 38.5 mM NaCl, 0.265 mM KH2PO4, 1.4 mM Na2HPO4, 12.5% hydroxyethyl starch, 1.9% BSA, 90 mM KC1, 100 mM inosine, 5 mM adenine.2. Rehydrate lyophilized RBC by adding 2 ml of preheated (37°C) Rehydration Buffer and gently shaking until fully dissolved.3. Evaluate numerical recovery by counting RBC before and after rehydration using a hemocytometer.Trehalose Loading - MagnetoporationMaterials:• Fresh RBC• Trehalose (various concentrations)• Isotonic buffer (e.g., phosphate-buffered saline, PBS)• Hypotonic buffer (e.g., 0.1% NaCl)• Centrifuge• Microcentrifuge tubes (20 pl capacity)• Magnetoporation device (as available)• SpectrophotometerProtocol:1. Sample Preparation:• Collect fresh blood in an anticoagulant (e.g., EDTA) and separate RBC by centrifugation at 600 x g for 5 minutes.• Wash the RBC 3 times with isotonic buffer to remove plasma.2. Trehalose Loading via Magnetoporation:• Resuspend RBC in trehalose solution with the desired concentration in a microcentrifuge tube.• Apply the magnetoporation technique according to standard instructions.• Incubate the treated RBC in isotonic buffer to allow recovery.3. Hemolysis Assay Setup:• Prepare control samples: untreated RBC and RBC treated only with magnetoporation.• Aliquot 20 pl of each treated and control sample into separate microcentrifuge tubes.• Add 80 pl of isotonic buffer to each tube.• Prepare one tube with hypotonic buffer (0.1% NaCl) for positive control (complete hemolysis).4. Incubation:• Incubate the tubes at 37°C for 30 minutes.5. Centrifugation:• Centrifuge the tubes at 600 * g for 5 minutes to pellet the intact RBC.6. Supernatant Collection:• Carefully collect the supernatant from each tube.7. Hemoglobin Measurement:• Measure the absorbance of the supernatant at 540 nm using a spectrophotometer.• Use the positive control (hypotonic buffer) to set 100% hemolysis.8. Data Analysis:• Calculate the percentage of hemolysis using the formula:Percentage of Hemolysis = (Absorbance of sample / Absorbance of positive control) x 100• Compare the percentage of hemolysis across the different conditions to determine the effect of trehalose and magnetoporation.Example 2: Delivery of Trehalose into Red Blood Cells Using Magnetoporation

[0126] FITC-tagged trehalose was used to determine whether and to what extent magnetoporation was able to transfer trehalose into RBCs. FITC-trehalose (at a concentration of 1 pg / pL) was transferred into RBCs at an average efficiency of 98.2% [standard error, or SE = 0.21], measured as percent of RBCs with trehalose uptake on flow cytometry and confirmed by fluorescent imaging (FIGS. 1 A-1B). In FIGS. 1 A-B, human RBCs were washed in Dulbecco's Phosphate-Buffered Saline (DPBS) and magnetoporated at 1 x 106cells in 20 pL DPBS with 20 pg of FITC-trehalose. Cells were incubated at 1 x 106cells in RPMI for up to 18 hours at 37°C prior to flow cytometry. These experiments defined magnetoporation parameters, as well as cell densities, media formulations, and other experimental variables for efficient trehalose delivery.

[0127] These experiments confirmed the efficient transfer of FITC-trehalose into RBCs.

[0128] Next, the effect of experimental and magnetoporation variables on intracellular trehalose concentration was assayed. It was possible to achieve intracellular trehalose concentrations up to 108.0 mM [SE = 8.3], which surpassed the presumptive cryoprotectiveconcentration of 100 mM (FIG. 2). In FIG. 2, human RBCs were washed in DPBS and magnetoporated at 1 x 106cells in 20 pL DPBS with 200 mM extracellular trehalose. Cells were incubated at in RPMI for up to 18 hours at 37°C prior measurement of average intracellular trehalose concentration by Megazyme assay.Example 3: RBC Viability and Structural Integrity

[0129] The viability and integrity of magnetoporated RBCs was also determined. Human RBCs were magnetoporated at 1 x 106cells in 20 pL DPBS with 200 mM extracellular trehalose. Cells were incubated for 30 minutes and 2 hours at room temperature, or at 1 x 106cells / mL in RPMI for 24 hours at 37°C. Blood smears were taken at precise time points post magnetoporation, and the results are shown in FIG. 3.

[0130] Across a range of magnetoporation settings, viability counts have consistently shown minimal cell death following magnetoporation, with survival rates similar to controls, in the range of -90%. Morphologically, at 30 minutes post-magnetoporation, there is a notable increase in acanthocytes, a reflection of membrane instability. This occurs both in the absence or presence of trehalose, although the latter cells appear less shriveled. However, cells started to recover their normal morphology by 2 hours post-magnetoporation and regain completely normal morphology by 24 hours post-magnetoporation (FIG. 3).

[0131] As a more sensitive survey of RBC membrane health, a eosin-5’-mal eimide (EMA) binding assay was also used. This assay is based on covalent binding of the EMA fluorescent dye to intact band 3 protein on the RBC membrane. RBCs were magnetoporated at 1 x 106cells in 20 pL DPBS with 200 mM extracellular trehalose. Cells were incubated for 30 minutes and 2 hours at room temperature, then stained with eosin-5’-mal eimide (EMA) fluorescent dye for 1 hour in the dark prior to flow cytometry. The results are shown in FIG.4. If the RBC membrane compromised, mean fluorescent intensity (MFI) will shift to the left indicating decreased amount of protein detected. At 30 minutes, no shift was detected between testing conditions. At 2 hours, a slight shift was detected in the magnetoporated control (RBCs magnetoporated without trehalose). RBC sample magnetoporated in the presence of 200mM trehalose did not shift. This is indicative that any potential damage magnetoporation may cause to RBCs is offset by the protective effects of trehalose.

[0132] In parallel with the acanthocytosis observed at 30 minutes following magnetoporation, there appears to be a reduction in mean fluorescent intensity (MFI) of magnetoporated cells at this early time point (FIG. 4). This shift is very small (4.5% reduction in MFI for magnetoporated cells compared to controls, measured as MFIcontrol-MFIsample / MFIcontrol x 100) and appears to normalize by 2 hours post-magnetoporation (1.0% MFI reduction for magnetoporated cells compared to controls), supporting that magnetoporation itself is a gentle process for RBCs. As with morphology, the presence of trehalose appears to protect against the early shift caused by magnetoporation (1.9% MFI reduction for magnetoporation with trehalose vs. 4.5% MFI reduction for magnetoporation without trehalose, as seen in FIG. 4).

[0133] In summary, magnetoporation can deliver trehalose into human RBCs with up to 98% efficiency and intracellular trehalose concentrations surpassing 100 mM. Cell survival and morphology are indicators of RBC health.Example 4: RBC Viability and Function

[0134] For each analysis, post-magnetoporation samples are compared with premagnetoporation RBCs from the same initial RBC source batch to account for inter-batch variability. Samples are blinded and shipped, and refrigerated overnight and stored at 4°C until analyses within 1-2 days. RBC integrity (hemolysis, deformability, and morphology) and metabolic activity, prior to magnetoporation and / or lyophilization, is evaluated using the following analyses.

[0135] Predisposition to hemolysis is determined by 3 high-throughput hemolysis assays:(1) Spontaneous hemolysis, as reflected in cell-free hemoglobin relative to total hemoglobin, is calculated using the equation [(100-Hct) x Hgbsup] / Hgbsampie. Het is the sample hematocrit measured by hematology analyzer. Hemoglobin is determined by the Drabkin’s method, for both the intact sample (HgBsampie) and the supernatant after centrifugation at 1500 * g for 10 minutes at 18°C (Hgbsup).(2) Oxidative hemolysis tests the RBC’s ability to withstand oxidative stress. RBC samples are washed 3 times with DPBS and incubated in 150 mM of 2,2'-azobis-2-methyl- propanimidamide dihydrochloride (AAPH) at 37°C to induce lipid peroxidation-mediated hemolysis. AAPH-induced oxidative hemolysis is calculated using the equation (HgbAAPH- Hgbcontroi) / Hgbtotaix100. HgbAAPH and Hgbcontroi refer to hemoglobin of supernatant after centrifugation of AAPH-treated RBCs and untreated RBCs, respectively; Hgbtotai refers to the total amount of hemoglobin of each sample.(3) Osmotic hemolysis tests the RBC’s ability to withstand osmotic stress and will be determined by a modified “pink test.” RBC samples are washed 3 times with DPBS and incubated in “pink test” buffer at 22°C for 4 hours. Osmotic hemolysis is calculated using the equation Hgbosmotic / Hgbtotaix100. Hgbosmotic refers to hemoglobin content of the supernatantafter centrifugation of pink test-treated RBCs; Hgbtotai refers to the total amount of hemoglobin of each sample.

[0136] RBC deformability is measured by the Laser Optical Rotational Red Cell Analyzer (Lorrca; Warwick, RI) ektacytometer according to the manufacturer's protocol

[0020] , Samples are run on the osmotic resistance test (Osmoscan) module, which provides multiple measurements of RBC deformability across a wide spectrum of osmolarities (50 to 500 mOsm / kg). The endpoints of interest are the maximal elongation index (Elmax), which represents maximal RBC deformability at the isotonic point (-300 mOsm / kg), and osmotic hyper (Ohyper), which evaluates cellular hydration and cytoplasmatic viscosity at the hypertonic region (-350 to 500 mOsm / kg).

[0137] Morphologic changes are evaluated using light microscopy and quantified by a validated scoring system of disc-to-discoid morphology changes. Glutaraldehyde is used as a fixative because of its preservation of the fine structural detail and reduction of enzyme activity. 100 RBCs are assigned a morphologic factor correlating with 6 different morphologies from smooth disc (1.0) to smooth sphere (0.0), and morphology score is reported as a percentage. Morphologic RBC indices, including mean cellular volume (MCV), will also be measured using a hematology analyzer (XN-1000BB; Sysmex, Kobe, Japan).

[0138] Oxygen carrying functionality of RBCs will be reflected in oxygen affinity (p50) measured by a blood gas analyzer (ABL90 Flex; Radiometer, Copenhagen, Denmark). The p50 measurement is the traditional way to measure oxygen affinity and represents the partial pressure of oxygen at which hemoglobin is 50% saturated with oxygen. This measurement is calculated using the standard hemoglobin dissociation curve, and it is expressed in units of mmHg. In addition, the device can report a p50(ST) measurement which is adjusted for the temperature and pH of the blood sample.Example 5: Evaluation of Trehalose-RBCs through Lyophilization and Rehydration

[0139] Magnetoporated RBCs as resuscitative blood product lies must undergo and survive lyophilization, prolonged storage, and rehydration. Factors that affect RBC viability and biostability at these stages - e.g., cellular environment, intracellular trehalose concentration, and electrical parameters - are determined.

[0140] Trehalose-loaded RBCs are evaluated following lyophilization and rehydration. Cells are concentrated and the medium adjusted for freeze-drying, then lyophilized in a Revo R&D Freeze Dryer (Millrock Technology, Kingston, NY). After completion of lyophilization andreturn to room temperature, samples are rehydrated in a rehydration medium. Following rehydration, cells are evaluated for viability and biostability.

[0141] Tests are run on a Linkam FDCS196 lyophilization stage. This platform allows realtime light microscopic visualization of RBC morphologic changes as temperature and pressure are adjusted, yielding a range of drying parameters.

[0142] RBC health following rehydration is also evaluated. To allow for the most versatility in application, the RBCs should be infusion-ready after rehydration, which means that the lyophilized extracellular material must also be ready for immediate infusion. Additives to the medium and / or diluent that might minimize cell damage during lyophilization; such additives include trehalose, glucose, adenine, ascorbate, inosine, mannitol, and dextran, are also evaluated. Although rehydration would likely require only sterile water, electrolytes or other additives might enhance the rehydration process or end product. This includes evaluating whether lyophilized packed RBCs can be reconstituted in plasma diluted with extra water to create instant "whole blood", as this may be gentler on cells than direct reconstitution with sterile water, and would simulate sterile water being added first to lyophilized plasma followed by addition of the mixture to lyophilized RBCs in the field.

[0143] Rehydrated trehalose-loaded RBCs are compared to pre-lyophilized trehalose-loaded RBCs from the same magnetoporation run as well as pre-magnetoporated RBCs, as described in Example 4, all from the same initial RBC source batch to account for inter-batch variability. Samples are provided in a blinded fashion. Pre-magnetoporated and prelyophilized samples are shipped refrigerated overnight and stored at 4°C until analyses, while lyophilized samples are shipped at room temperature and rehydrated at just prior to analyses.

[0144] Formulations that demonstrate high viability after immediate rehydration are be evaluated for stability in lyophilized form, either by storing the cells at room temperature for up to 6 months or incubating them at 50°C to accelerate cellular breakdown. Cells are then rehydrated and quality measured as above. Means of storing, and ultimately delivering, a final commercial product are also assayed. Specifically, various plastic alternatives and vessel types that support sterile, compact, long-term storage, along with tubing and other ancillary components to facilitate rapid reconstitution and transfusion are evaluated.Example 6: Evaluation of Trehalose-Stabilized Rat RBCs in a Normovolemic Rat Model

[0145] In vivo studies are carried out in Sprague-Dawley rats (Rattus norvegicus) given their similar baseline and disease physiology to humans. Trehalose-loaded RBC formulations found to be maximally stabilized following rehydration are evaluated upon transfusion intohealthy adult Sprague-Dawley rats. Initial testing will utilize a normovolemic rat model (i.e., equivolume replacement). Anesthetized animals have their femoral artery and vein cannulated for blood removal and return, respectively. 6 mL / kg (approximately 10% of total blood volume) of blood is removed and separated; plasma is set aside and RBCs quantified. Experimental (FITC-trehalose-loaded and lyophilized) rat RBCs are rehydrated, mixed with native plasma to a volume and concentration equivalent to that removed, then returned to the animal. A range of experimental RBC formulations are tested. Control rats receive their native (autologous) RBCs reconstituted in native plasma. After 5 minutes to allow intravascular mixing, the process is repeated twice, resulting in replacement of approximately 27% of circulating RBCs with experimental RBCs. Arterial blood samples are taken before blood removal (baseline) and at t=5 minutes after completion of exchange (to allow for mixing) and t=l, 2, 3, and 4 hours.

[0146] Retention of the exogenous trehalose-stabilized RBCs in the circulation is evaluated by measuring relative trehalose amounts among the circulating RBCs over time in a given animal. Blood samples are centrifuged at 600 x g for 10 minutes at 4°C; plasma is removed and set aside. Cells are washed with DPBS, counted, and reconstituted to set RBC concentration. These samples are subjected to the trehalose assay to determine trehalose amount. Loss of exogenous RBCs is reported as percentage drop in trehalose amount compared to time 0.

[0147] Intravascular hemolysis is assessed by 3 parameters, using plasma from the centrifuged blood samples. Release of Hgb and RBC-specific lactate dehydrogenase (LDH) isoenzymes into the circulation is measured, as well as depletion of circulating haptoglobin. Total LDH is measured via the Lactate Dehydrogenase Activity Assay Kit (Sigma-Aldrich, St. Louis, MO), measuring production of reduced NADH on spectrophotometry. Specific LDH isoenzymes are measured using the Spife LD Isoenzyme Procedure (Helena Laboratories, Beaumont, TX), in which isoenzymes are separated by agarose gel electrophoresis and isoenzyme level is determined by colorimetric detection of a reduced dye. RBC-specific LDH is reported as the relative percentage of total LDH. Circulating cell-free Hgb is measured via spectrophotometry. Haptoglobin, whose function is to bind to free circulating Hgb for removal, is measured via Rat Haptoglobin ELISA Kit (Novus Biologicals).

[0148] Finally, blood oxygenation is measured by standard arterial blood gas analysis immediately upon blood draw via blood gas analyzer. RBC oxygen carrying capacity (Hgb), as well as cell count, Het, and morphologic indices, is measured via hematology analyzer.Example 7: Evaluation of Trehalose-Stabilized Rat RBCs in a Rat Model of Hemorrhagic Shock

[0149] Magnetoporated and lyophilized RBCs are to serve as resuscitation in the setting of blood loss, whether traumatic or related to disease. Acute hemorrhage is a critical scenario for blood resuscitation, and one with clearly definable measures of success - i.e., RBC retention, HgB restoration, and tissue oxygenation. The resuscitative capacity of magnetoporated and lyophilized RBCs to address hemorrhage in a rat model is evaluated.

[0150] Trehalose-stabilized RBC formulations are evaluated for therapeutic potential in a hemorrhagic shock rat model. These rats undergo carotid artery catheterization to allow for continuous blood pressure and heart rate monitoring, while controlled hemorrhage and blood sampling will be accomplished via femoral artery catheter. Controlled hemorrhage will proceed until the mean arterial pressure (MAP) drops to 35 mmHg (i.e., the start of shock and t = 0 hour), then continue at a slower rate to keep a MAP of 35 mmHg until total blood loss of 24 mL / kg (approximately 40% of total blood volume), after which pressure is allowed to recover. At t = 1 hour, simulating delays in resuscitation on the battlefield, animals are resuscitated with reconstituted experimental rat RBCs, prepared as above and at a volume (and concentration) comparable to that lost.

[0151] Control rats receive their native (autologous) RBCs reconstituted in native plasma. Arterial blood samples are taken before hemorrhage (baseline) and at t=l hour (immediately before resuscitation), t=l hour 5 minutes (after infusion of RBCs), and t=2, 3, and 4 hours. The following primary endpoints are evaluated: 1) retention of RBCs within the circulation, 2) evidence of intravascular hemolysis, and 3) oxygen saturation and carrying capacity. Retention of the exogenous trehalose-stabilized RBCs in the circulation is evaluated as described above. Intravascular hemolysis, blood oxygenation, and HgB is also evaluated as described above.

[0152] Blood samples are analyzed for hemolysis and oxygenation as described above. Since shock itself can cause hemolysis, the criteria for success is non-inferiority to controls. Additional primary endpoints for the hemorrhagic shock model include mortality and hemodynamic stability (blood pressure and heart rate). Secondary endpoints evaluated may include pathologic analysis of the spleen and / or evidence of end organ stress in lungs, liver, and kidneys.Example 8: Evaluation of Leakage of Hemoglobin from RBCs

[0153] RBCs, with and without serum, from two subjects underwent magnetoporation, with and without 200 mM extracellular trehalose, using the protocol described in Example 1. In addition, a subset of cells were subject to three freeze-thaw cycles, as described in Example 1. Hemoglobin leakage post magnetoporation, and post free-thaw, was assayed. The results are shown in FIG. 5 A.

[0154] Hemoglobin in cells prior to magnetoporation, and in serum prior to magnetoporation, as well as after magnetoporation (with and without trehalose), and after freeze / thaw, was also assayed by looking at absorbance at 405 nanometers (nm) (left bars) and 420 nm (right bars). The results are shown in FIG. 5B.Example 9: Evaluation of Intracellular Trehalose in RBCs

[0155] RBCs were magnetoporated with trehalose, under various magnetoporation conditions shown in FIG. 6, following the protocol described in Example 1. Magnetoporated RBCs were harvested after overnight culture in complete media. 50 uL of each sample was counted on the Cellometer, while the remainder of the RBC were centrifuged. Cell pellets washed once with DPBS and supernatants removed. HEPES buffer was added to RBC pellets, and pellets are freeze / thawed three times in liquid nitrogen. The cell extract was harvested by high speed centrifugation, and used in the Megazyme trehalose assay described in Example 1. A 96 well plate was used to analyze the intracellular trehalose concentration within the RBC cell extracts, as compared to a trehalose standard curve. The results are shown in FIG. 6.Example 10: Lyophilization of RBCs with Intracellular Trehalose

[0156] Magnetoporated RBCs were harvested after overnight culture in complete media, as described in Example 1. 50 uL of each sample was counted on the Cellometer, while the remainder of the RBCs were centrifuged and the supernatants removed and discarded. Cells were resuspended in lyophilization buffer and are frozen at -80 °C for a minimum of two hours. Cells were transferred to a pre-cooled lyophilizer on dry ice. Once the chamber of the lyophilizer reached -80 °C, the samples were subjected to vacuum pressure of 200 mTorr for 18-24 hours. Once samples dried, they were rehydrated with rehydration buffer, and counted to determine viability.Example 11: Trehalose Delivery to RBCs and Platelets

[0157] Trehalose was introduced into RBCs and platelets via magnetoporation with the experimental conditions shown in Tables 11-1 and 11-2 below. For all conditions, the starting number of cells or platelets was 3.36 x 107cells in 10 pL of DPBS. Copper cup height settings in the magnetoporation device were kept constant across all experimental conditions.Table 11-1. Experimental Conditions for RBCsTable 11-2. Experimental Conditions for Platelets

[0158] Live RBCs for each of the experimental conditions shown in Table 11-1 were determined using a hemocytometer after staining with trypan blue, and the results are shown in Table 11-3 below, and FIG. 7. In FIG. 7, the cells were magnetoporated (except for no magnetoporation controls), and then incubated overnight in RPMI medium. After 24 hours, the numbers of live and dead cells were counted. For Table 11-3, cells were magnetoporated with trehalose, frozen at -80 °C, thawed, and then subjected to staining and cell counting for live / dead analysis. In addition, as shown in FIG. 8, femtograms of trehalose per RBC was calculated as described in Example 9. RBC viability, comparing 1 hour post-magnetoporationversus after overnight freezing and thawing of the cell pellet, is shown in FIG. 9. As shown in FIG. 9, magnetoporation with 500 mM trehalose (bars on right) resulted significantly more viable RBCS following freeze-thaw of the cell pellet when compared to RBCs treated with trehalose without magnetoporation (bars on left).

[0159] Live platelets for each of the experimental conditions shown in Table 11-2 were determined using a hemocytometer after staining with trypan blue. The results are shown in Table 11-4 and FIG. 10. In FIG. 10, the platelets were magnetoporated (except for no magnetoporation controls), and then incubated overnight in RPMI medium. After 24 hours, the numbers of live and dead platelets were counted. For Table 11-4, platelets were magnetoporated with trehalose, frozen at -80 °C, thawed, and then subjected to staining and counting for live / dead analysis. In addition, as shown in FIG. 11, femtograms of trehalose per platelet was calculated as described in Example 9. As shown in FIG. 11, magnetoporation with 180V, and 125, 1560 or 175 Hz significantly increased the amount of trehalose per platelet.

[0160] Cell counts, live cells, and cell sizes after freezing the pellet of RBCs or platelets magnetoporated with trehalose, experimental conditions 7 and 8 respectively, are provided in tables 11-3 and 11-4 below. For both RBCs and platelets, three samples treated with the indicated magnetoporation conditions and trehalose were compared to control cells in which the cell pellet was frozen and thawed without trehalose treatment.Table 11-3. RBC Properties after Magnetoporation with Trehalose, Followed by Freezing andThawingTable 11-4. Platelet Properties after Magnetoporation with Trehalose, Followed by Freezing and Thawing

[0161] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment s) without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.

[0162] Also, various concepts may be embodied as one or more methods, of which an example has been provided. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.

[0163] As used herein, the terms “about” and / or “approximately” when used in conjunction with numerical values and / or ranges generally refer to those numerical values and / or ranges near to a recited numerical value and / or range. In some instances, the terms “about” and “approximately” may mean within ± 10% of the recited value. For example, in some instances, “about 100 [units]” may mean within ± 10% of 100 (e.g., from 90 to 110). The terms “about” and “approximately” may be used interchangeably.

[0164] Some embodiments described herein relate to a computer storage product with a non- transitory computer-readable medium (also may be referred to as a non-transitory processor- readable medium) having instructions or computer code thereon for performing various computer-implemented operations. The computer-readable medium (or processor-readable medium) is non-transitory in the sense that it does not include transitory propagating signals per se (e.g., a propagating electromagnetic wave carrying information on a transmission medium such as space or a cable). The media and computer code (also may be referred to ascode or algorithm) may be those designed and constructed for the specific purpose or purposes. Examples of non-transitory computer-readable media include, but are not limited to, magnetic storage media such as hard disks, floppy disks, and magnetic tape; optical storage media such as Compact Disc / Digital Video Discs (CD / DVDs), Compact Disc-Read Only Memories (CD-ROMs), and holographic devices; magneto-optical storage media such as optical disks; carrier wave signal processing modules; and hardware devices that are specially configured to store and execute program code, such as Application-Specific Integrated Circuits (ASICs), Programmable Logic Devices (PLDs), Read-Only Memory (ROM) and Random-Access Memory (RAM) devices. Other embodiments described herein relate to a computer program product, which may include, for example, the instructions and / or computer code disclosed herein.

[0165] The systems, devices, and / or methods described herein may be performed by software (executed on hardware), hardware, or a combination thereof. Hardware modules may include, for example, a general-purpose processor (or microprocessor or microcontroller), a field programmable gate array (FPGA), and / or an application specific integrated circuit (ASIC). Software modules (executed on hardware) may be expressed in a variety of software languages (e.g., computer code), including C, C++, Java®, Ruby, Visual Basic®, and / or other object-oriented, procedural, or other programming language and development tools. Examples of computer code include, but are not limited to, micro-code or micro-instructions, machine instructions, such as produced by a compiler, code used to produce a web service, and files containing higher-level instructions that are executed by a computer using an interpreter. Additional examples of computer code include, but are not limited to, control signals, encrypted code, and compressed code.

Claims

CLAIMSWhat is claimed is:

1. A method introducing a cryoprotective agent into a cell or a membrane-bound cell fragment, comprising: a. contacting the cell or membrane-bound cell fragment with the cryoprotective agent; and b. applying a transient magnetic field to the cell or membrane-bound cell fragment under conditions sufficient to introduce the cryoprotective agent into the cell or membrane-bound cell fragment.

2. The method of claim 1, wherein the cryoprotective agent comprises a sugar.

3. The method of claim 2, wherein the sugar comprises trehalose.

4. The method of any one of claims 1-3, wherein the cell comprises a blood cell.

5. The method of claim 4, wherein the blood cell comprises a red blood cell, neutrophil, monocyte or lymphocyte.

6. The method of claim 4, wherein the cell comprises a red blood cell.

7. The method of any one of claims 1-3, wherein the membrane-bound cell fragment comprises a platelet.

8. The method of any one of claims 1-5, wherein the cell comprises a red blood cell, and the method comprises contacting a plurality of red blood cells with trehalose.

9. The method of claim 8, wherein the trehalose is at a concentration of between about 100 mM and 1,000 mM.

10. The method of claim 8, wherein the trehalose is at a concentration of between about 200 mM and 600 mM.

11. The method of any one of claims 8-10, wherein the plurality of red blood cells is at a concentration of between about IxlO5and about IxlO8cells per pL (cells / pL).

12. The method of any one of claims 8-10 wherein the plurality of red blood cells is at a concentration of between about IxlO6cells / pL and about 5xl06cells / pL.

13. The method of any one of claims 8-12, comprising, prior to step (a), i. centrifuging whole blood at about 1400 g for about 5 minutes, thereby collecting the plurality of red blood cells; ii. washing the plurality of red blood cells with Dulbecco’s Phosphate Buffered Saline (DPBS); and iii. suspending the cells in DBPS at a concentration of between about IxlO7cells per pL and IxlO8cells per pL.

14. The method of any one of claims 8-13, comprising incubating the plurality of red blood cells in cell culture medium following magnetoporation for a recovery period.

15. The method of claim 14, wherein the recovery period comprises incubating the plurality of red blood cells in complete RPMI for about 8 to about 24 hours.

16. The method of any one of claims 1-5, wherein the membrane-bound cell fragment comprises a platelet, and the method comprises contacting a plurality of platelets trehalose.

17. The method of claim 16, wherein the trehalose is at a concentration of between about 100 mM and about 1,000 mM.

18. The method of claim 16, wherein the trehalose is at a concentration of between about 200 mM and about 600 mM.

19. The method of any one of claims 16-18, wherein the plurality of platelets is at a concentration of between about IxlO5and about IxlO8platelets per pL (platelets / pL).

20. The method of any one of claims 16-18, wherein the plurality of platelets is at a concentration of between about IxlO6platelets / pL and about 5xl06platelets / pL.

21. The method of any one of claims 16-20, comprising incubating the plurality platelets in cell culture medium following magnetoporation for a recovery period.

22. The method of claim 21, wherein the recovery period comprises incubating the plurality of platelets in complete RPMI for about 8 to about 24 hours.

23. The method of any one of claims 1-22, wherein the magnetic field is pulsed.

24. The method of claim 23, wherein the magnetic field comprises at least 50 pulses, at least 100 pulses, at least 300 pulses, at least 500 pulses, at least 700 pulses, at least 1,000 pulses, at least 12,00 pulses, at least 1,500 pulses, at least 2,000 pulses, at least 2,500 pulses, at least 3,000 pulses, at least 4,000 pulses, at least 5,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000 at least 90,000 or at least 100,000 pulses.

25. The method of claim 23 or 24, wherein the magnetic field comprises an interval of between about 0.1 second and about 1 second between pulses or between trains of pulses.

26. The method of any one of claims 1-25, wherein contacting the cell or membrane-bound cell fragment with the cryoprotective agent occurs in a volume of about 2 pL to about 1000 pL, about 5 pL to about 500 pL, about 5 pL to about 50 pL, about 10 pL to about 50 pL, or about 10 pL to about 30 pL.

27. The method of any one of claims 1-25, wherein contacting the cell or membrane-bound cell fragment with the cryoprotective agent occurs in a volume of between about 1 mLand about 500 mL, between about 10 mL and about IL, between about 50 mL and about 5L, between about IL and about 10,000L, between about 5L and about 500L, between about 10L and about 500L, between about 10L and about 100L, between about 10L and about 50L, between about 100L and about 10,000L, between about 100L and about lOOOL, or between about 100L and about 500L.

28. The method of any one of claims 1-27, comprising cryopreserving the cell or membranebound cell fragment after step (b).

29. The method of claim 28, wherein cryopreserving the cell or membrane-bound cell fragment comprises reducing the temperature of the cell or membrane-bound cell fragment to a temperature of -20 °C or less.

30. The method of claim 28, wherein cryopreserving the cell or membrane-bound cell fragment comprises reducing the temperature of the cell or membrane-bound cell fragment to a temperature of about -15 °C to about -200 °C, about -30 °C to about -180 °C, about -60 °C to about -160 °C, or about -80 °C to about -140 °C.

31. The method of any one of claims 28-30, wherein introducing the cryoprotective agent into the cell or the membrane-bound cell fragment increases a percentage of viable cells or membrane-bound cell fragments after cry opreservation and thawing when compared to a percentage of viable cells or membrane-bound cell fragments which have been subject to cry opreservation and thawing without the cryoprotective agent.

32. The method of claim 31, wherein introducing the cryoprotective agent increases the percentage of viable membrane-bound cell fragments by at least about 5%, about 10%, about 15% or about 20%.

33. The method of claim 32, wherein introducing the cryoprotective agent increases the percentage of viable cells by at least about 30%, about 40%, about 50%, about 60% or about 70%.

34. The method of any one of claims 1-27, further comprising lyophilizing the cell or membrane-bound cell fragment after step (b).

35. The method of claim 30, wherein the lyophilizing comprises: i. contacting the cell or membrane-bound cell fragment with a lyophilization buffer; ii. cooling the cell or membrane-bound cell fragment to less than at least - 60 °C; and iii. drying the cell or membrane bound cell fragment.

36. The method of claim 35, wherein drying the cell or membrane bound cell fragment comprises: (1) drying the cell or membrane-bound cell fragment at about -45 °C and about 200 mTorr for about 15 hours; and (2) drying the cell or membrane-bound cell fragment at about 15 °C for about 10 hours.

37. The method of claim 35 or 36, wherein the lyophilization buffer comprises: a. glucose; b. adenine; c. NaCl; d. mannitol; e. KC1; f. dextran; g. bovine serum albumin (BSA); and / or h. sodium citrate.

38. A pharmaceutical composition comprising a cryopreserved cell or membrane-bound cell fragment produced by the method of any one of claims 1-33.

39. A pharmaceutical composition comprising a lyophilized cell or membrane-bound cell fragment produced by the method of any one of claims 1-27 or 34-37.

40. A kit, comprising the pharmaceutical composition of claim 38 or 39, and instructions for use.

41. The kit of claim 40, comprising a rehydration buffer.

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