Methods of preparing and freezing biological samples for analytical use and associated compositions

Abstract

Provided herein are methods of preparing a biological sample for analysis. In some embodiments, the methods comprise cryopreserving the cells and assessing biological features. Also provided are methods of analyzing biological samples which have been prepared according to the steps of the present technology. In some embodiments, the methods of the present technology preserve native cell structures of biological samples relative to samples prepared by other methods.

Description

METHODS OF PREPARING AND FREEZING BIOLOGICAL SAMPLES FOR ANALYTICAL USE AND ASSOCIATED COMPOSITIONSCROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 483,945, filed on February 8, 2023. The contents of this provisional application are incorporated by reference in their entirety.BACKGROUND

[0002] Existing technologies for processing patient samples to assess cellular features have many disadvantages that prevent them from being widely used or that compromise integrity of various cell structures. For example, clinical flow cytometry is typically performed on fresh peripheral blood mononuclear cells (PBMCs) processed from whole blood using Ficoll solutions and cryopreserved in the vapor phase of a liquid nitrogen freezer and later analyzed. With fresh PBMC, surface and intracellular proteins are present in their native form having no chemical modifications, permitting strong binding and staining with fluorescent epitope specific antibodies. Cryopreservation, however, has been shown to result in the shedding or loss of some surface proteins such as CD197 (CCR7), BCMA and CD69. Further, the clinical flow cytometry panels are typically limited to about 15 antibodies conjugated to stable, readily available fluorochromes, such as Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE), and Allophycocyanin (APC). Flow cytometers are standard, non-spectral instruments, where timing is also a limiting factor. Time from initiation of staining to the end of data collection may take up to about 3 days.

[0003] Similarly, gene expression analyses, such as both single cell and bulk cell RNA sequencing, not received widespread use in clinical settings or clinical trials due to cost, sample processing constraints, and complexity in data analysis. Therefore, improvements in data processing which yield more accurate cell assessments are needed.SUMMARY

[0004] The present technology comprises methods of preparing a biological sample for one or more analyses. In some embodiments, the methods comprise (i) combining the biological sample with a storage media comprising at least about 12% to at least about 20% DMSO to form a first combination at a combining temperature of about 18°C to about 25°C; (ii) incubating the first combination at an incubation temperature of about -70°C to about -90°C for at least about 16 hours to at least about 48 hours; (iii) transferring the incubated first combination from (ii) to a freezing temperature of at least about -130°C to at least about -190°C; (iv) thawing the first combination from (iii) to a processing temperature of about 36°C to about 38°C; (v) combining the thawed first combination from (iv) with a working media at the processing temperature forming a second combination; (vi)depleting neutrophils from the second combination by at least about 50% relative to a control; and (vii) performing the one or more analyses using the neutrophil depleted second combination.

[0005] In some embodiments, the methods comprise: (i) receiving a biological sample processed by the steps of: (a) combining the biological sample with a storage media comprising at least about 12% to at least about 20% DMSO to form a first combination at a combining temperature of about 18°C to about 25°C; (b) incubating the first combination at an incubation temperature of about -70°C to about -90°C for at least about 16 hours to at least about 48 hours; and (c) transferring the incubated first combination from (b) to a freezing temperature of at least about -130°C to at least about -190°C; and (ii) thawing the first combination from (c) to a processing temperature of about 36°C to about 38°C; (iii) combining the thawed first combination from (ii) with a working media at a temperature of about 36°C to about 38°C, forming a second combination; (iv) depleting neutrophils from the second combination by at least about 50% relative to a control; and (v) performing the one or more analyses using the neutrophil depleted second combination.

[0006] In some embodiments, the methods (i) receiving a biological sample processed by the steps of: (a) combining the biological sample with a storage media comprising at least about 12% to at least about 20% DMSO to form a first combination at a combining temperature of about 18°C to about 25°C; (b) incubating the first combination at an incubation temperature of about -70°C to about -90°C for at leastabout 16 hours to at least about 48 hours; (c) transferring the incubated first combination from (b) to a freezing temperature of at least about -130°C to at least about -190°C; (d) thawing the first combination from (i)(c) to a processing temperature of about 36°C to about 38°C; (e) combining the thawed first combination from (d) with a working media at the processing temperature forming a second combination; and (f) depleting neutrophils from the second combination by at least about 50% relative to a control; and (ii) performing one or more analyses using the biological sample received in (i).

[0007] In some embodiments, the methods further comprise adding a fixative to the second combination before the neutrophil depletion.

[0008] In some embodiments, the fixative is added to at least a portion of the second combination.

[0009] In some embodiments, the one or more analyses comprise a bulk cell analysis or a single cell analysis.

[0010] In some embodiments, the bulk cell analysis comprises one or more analyses selected from the group consisting of a genomic analysis, a transcriptomic analysis, an epigenomic analysis, a spatial genomics analysis, a chromatin accessibility analysis, a proteomics analysis, and a metabolomics analysis.

[0011] In some embodiments, the chromatin accessibility analysis comprises an Assay for Transposase-Accessible Chromatin (ATAC) sequencing.

[0012] In some embodiments, the single cell analysis comprises a single cell RNA- sequencing analysis.

[0013] In some embodiments, the single cell analysis comprises a Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq).

[0014] In some embodiments, the bulk cell analysis or the single cell analysis comprises a functional analysis.

[0015] In some embodiments, the functional analysis comprises providing an agent to the biological sample.

[0016] In some embodiments, the functional analysis comprises a cell differentiation assay, a cell expansion assay, am agonist screening assay, or an Activation Induced Marker (AIM) assay.

[0017] In some embodiments, the agent is a cell stimulatory agent.

[0018] In some embodiments, the cell stimulatory agent comprises one or more agents selected from the group consisting of phorbol 12-myristate 13-acetate (PMA), ionomycin, phytohemagglutinin, an anti-CD3 antibody, lipopolysaccharide, resiquimod, CD3 stimulation, or CD28 stimulation.

[0019] In some embodiments, the single cell analysis or the bulk cell analysis assesses a level of expression of one or more genes selected from the group consisting of A1 BG, ABLIM1 , AC020656.1 , AC243960.1 , ADTRP, AFF3, ALDH2, ANXA2R, APOBEC3C, APP, AQP3, ARID5B, ATF7IP2, BANK1 , BCL11 A, BCL11 B, BIRC3, BLK, CAMK4, CAPG, CARS, CASP8AP2, CBL, CCDC167, CCDC50, CCL4, CCND2, CCR7, CD14, CD27, CD36, CD6, CD68, CD79A, CD79B, CD8A, CD8B, CD96, CDKN1 C, CEBPD, CFD, CFP, CLEC10A, CLEC12A, CLIC3, CMC1 , CPVL, CSF3R, CST7, CSTA, CTSH, CXXC5, CYBB, CYTOR, DCTPP1 , DNAJB1 , DOK2, DYNLL2, DYRK2, EAF2, EBP, ERN1 , FCER1A, FCER1 G, FCER2, FCGR3A, FCN1 , FCRL1 , FGFBP2, FGL2, FHIT, FKBP1 1 , GATA3, GBP5, GIMAP7, GNG2, GPR65, GRN, GZMA, GZMB, GZMH, GZMK, HLA-DMA, HLA-DMB, HLA-DQA1 , HOPX, IFITM3, IFT57, IGHD, IGHM, IGLC2, IGSF6, IKZF3, IL2RB, IL3RA, IL4R, IL6ST, INPP4B, IRF7, IRF8, ITGAL, ITM2C, JAML, JCHAIN, JUN, KLRB1 , KLRC1 , KLRD1 , KLRF1 , KLRG1 , LEF1 , LGALS2, LGALS3, LILRA4, LINC00623, LINC00861 , LINC00926, LINC01857, LINC01871 , LINC02446, LRRC25, LY86, LYN, LYST, MAL, MAML2, MAPKAPK2, MARCHF1 , MARCKS, MATK, MEF2C, MHENCR, MNDA, MS4A1 , MS4A6A, MS4A7, MT1 X, MYBL1 , MYC, MYO1 F, MZB1 , NCF2, NCR3, NELL2, ORAI2, OXNAD1 , PAG1 , PASK, PDE3B, PDLIM1 , PECAM1 , PHACTR2, PIK3IP1 , PILRA, PITPNC1 , PLD4, PLPP5, POU2AF1 , POU2F2, PPP1 R10, PRF1 , PRKCH, PRR5, PTPN4, PTPN6, PYHIN1 , RALGPS2, RASSF1 , RCAN3, RFLNB, RHOC, RNF130, RTKN2, S100A12, S100B, SAMD3, SELENOM, SERPINA1 , SERPINF1 , SESN3, SLC2A4RG, SLC4A10, SMIM25, SP140, SPI1 , SPIB, SPON2, STMN1 , STMN3, STX7, SWAP70, SYNE1 , TAGAP, TBC1 D15, TC2N, TCF4, TCL1 A, THEM4, TMEM154, TMEM156, TMIGD2, TNFRSF13C, TNFRSF1 B, TPD52, TPM2, TPST2, TRABD2A, TRDC, TRG-AS1 , TRGC1 , TRGC2, TSHZ2, TSPAN3, TULP4, UGCG, VCAN, XCL1 , XCL2, ZAP70, and ZEB2.

[0020] In some embodiments, combining the biological sample with the storage media occurs within about 60 minutes of collection of the biological sample.

[0021] some embodiments, the storage media comprises at least about 12%DMSO.

[0022] some embodiments, the storage media comprises at least about 14%DMSO.

[0023] some embodiments, the storage media comprises at least about 15%DMSO.

[0024] some embodiments, the storage media comprises at least about 16%DMSO.

[0025] In some embodiments, the storage media comprises at least about 18%DMSO.

[0026] In some embodiments, the storage media comprises at least about 20%DMSO.

[0027] In some embodiments, the first combination is incubated in an enclosure comprising at least a polystyrene foam.

[0028] In some embodiments, the enclosure is a polystyrene foam box.

[0029] In some embodiments, the first combination is incubated for at least about16 hours at a first incubation temperature of at least about -70°C to about -80°C.

[0030] In some embodiments, the first combination is incubated for no more than about 48 hours at a second incubation temperature of at least about -80°C to about - 90°C.

[0031] In some embodiments, thawing the first combination comprises a thaw speed of about 1 °C to about 2°C per minute.

[0032] In some embodiments, the storage media is a freezing media.

[0033] In some embodiments, the storage media or the working media is a serum- free media.

[0034] In some embodiments, the thawed first combination comprises live cells.

[0035] In some embodiments, the live cells comprise at least about 25% of the total cells in the first combination.

[0036] In some embodiments, the live cells comprise at least about 50% of the total cells in the first combination.

[0037] In some embodiments, the live cells comprise at least about 75% of the total cells in the first combination.

[0038] In some embodiments, the working media is warmed to the processing temperature.

[0039] In some embodiments, depleting the neutrophils comprises detecting a cell comprising one or more neutrophil surface proteins.

[0040] In some embodiments, the cell comprising the one or more neutrophil surface proteins is depleted from the second combination.

[0041] In some embodiments, the one or more neutrophil surface proteins comprise CD15 or CD66b.

[0042] In some embodiments, the biological sample is selected from the group consisting of a urine sample, a saliva sample, a circulatory fluid sample, a synovial fluid sample, and a solid tissue sample.

[0043] In some embodiments, the circulatory fluid sample is a blood sample.

[0044] In some embodiments, the blood sample is a peripheral blood mononuclear cells (PBMC) sample.

[0045] In some embodiments, the method preserves a state of one or more features in the biological sample at the time of collection relative to a control.BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1 illustrates Uniform Manifold Approximation and Projection (UMAP) analyses for samples prepared according to the methods of the present technology relative to controls under different stimulatory conditions.

[0047] FIGS. 2A and 2B illustrate unique molecular identifiers (UMIs) (FIG. 2A) and genes (FIG. 2B) per cell for samples prepared according to the methods of thepresent technology under different stimulatory conditions, relative to non-stimulated controls.

[0048] FIGS 3A-3D illustrate pathway enrichment analysis for cells prepared according to the methods of the present technology and stimulated with phorbol 12- myristate 13-acetate (PMA) and ionomycin under four different stimulatory conditions, relative to non-stimulated controls.

[0049] FIGS 4A-4D illustrate pathway enrichment analysis for cells prepared according to the methods of the present technology and stimulated with Resiquimod (R848) under four different stimulatory conditions, relative to non-stimulated controls.

[0050] FIGS 5A-5D illustrate pathway enrichment analysis for cells prepared according to the methods of the present technology and stimulated with CD3 and CD28 cells under four different stimulatory conditions, relative to non-stimulated controls.

[0051] FIGS. 6A and 6B illustrate Venn diagrams (6A) and bar chart (6B) quantification of differentially expressed genes (DEGs) for cells prepared according to the methods of the present technology and stimulated with PAM and ionomycin under four different stimulatory conditions, relative to non-stimulated controls.

[0052] FIGS. 7A and 7B illustrate Venn diagrams (7A) and bar chart (7B) quantification of DEGs for cells prepared according to the methods of the present technology and stimulated with R848 under four different stimulatory conditions, relative to non-stimulated controls.

[0053] FIGS. 8A and 8B illustrate Venn diagrams (8A) and bar chart (8B) quantification of DEGs for cells prepared according to the methods of the present technology and stimulated with CD3 and CD28 cells under four different stimulatory conditions, relative to non-stimulated controls.

[0054] FIG. 9 illustrates steps for performing the methods of the present technology using whole blood samples. Step 1 indicates receiving or collecting the biological sample. Step 2 indicates combining the biological sample with a storage media comprising dimethyl sulfoxide (DMSO). Step 3 indicates incubating the sample and transferring the sample to a freezing temperature. Step 4 indicates thawing the sample to a processing temperature. Step 5 indicates combining the sample with a working media and optionally adding a fixative. Step 6 indicates analyzing the sampleusing a functional assay. Step 7 indicates analyzing the sample using single cell or bulk cell analysis.DETAILED DESCRIPTION

[0055] While the present technology is capable of being embodied in various forms, the description below of several embodiments is made with the understanding that the present technology is to be considered as an exemplification of the present technology and is not intended to limit the present technology to the specific embodiments illustrated. Headings are provided for convenience only and are not to be construed to limit the present technology in any manner. Embodiments illustrated under any heading may be combined with embodiments illustrated under any other heading.

[0056] The use of numerical values in the various quantitative values specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word “about.” It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about.” It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. Furthermore, the term “about,” as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1 % of the specified amount.

[0057] Also, the ranges of the present technology are intended as a continuous range, including every value between the minimum and maximum values recited, as well as any ranges that may be formed by such values. The present technologycomprises any and all (and ranges of any such ratios) that may be formed by dividing a numeric value of the present technology into any other numeric value of the present technology. Accordingly, the skilled person will appreciate that many such ratios, ranges, and ranges of ratios may be unambiguously derived from the numerical values presented herein and in all instances, such ratios, ranges, and ranges of ratios represent various embodiments of the present technology.

[0058] As used herein, a “control” when referring to a biological sample may refer to the biological sample at baseline in the native state (i.e., prior to the biological being prepared or analyzed according to the methods of the present technology), a biological sample that is subjected to a different amount or order of steps of the methods of the present technology (e.g., a sample prepared by a methods lacking one or more steps of the methods of the present technology), or a biological sample prepared by methods that are different from the methods of the present technology.Associated Methods

[0059] The present technology comprises methods of preparing biological samples for analyses. The methods preserve cellular features (e.g., cell structures, post translational modifications, cell surface proteins) in a state that correlates to the cell at the time of sample collection. In some embodiments, the methods preserve a state of one or more features in the biological sample at the time of collection relative to a control.

[0060] In some embodiments, the methods comprise steps for preparing a biological sample for freezing, freezing the sample, thawing the sample, depleting cells from the sample, and analyzing the sample. Any one of the steps of the methods may be performed at a site that is different from that of a previous step.

[0061] The samples of the methods may comprise the biological sample in its native form or the state of the biological sample at an intermediate form (e.g., a biological sample prepared in a first combination or a second combination) or the final form, prepared according to the methods of the present technology.Preparing Samples

[0062] In some embodiments, the present technology comprises methods of preparing a biological sample for one or more analyses. In some embodiments, themethods of preparing include preparing the sample for freezing (e.g., cryopreservation). The methods may comprise combining the biological sample with a storage media comprising dimethyl sulfoxide (DMSO). The combining occurs at a temperature that is warmer relative to conventional methods.

[0063] In some embodiments, the biological sample is selected from the group consisting of a urine sample, a saliva sample, a circulatory fluid sample (e.g., a blood sample, a peripheral blood mononuclear cells (PBMC) sample), a synovial fluid sample, and a solid tissue sample.

[0064] In some embodiments, the biological sample is combined with the storage media within about 60 minutes of collection of the biological sample (e.g., collecting the biological sample by blood draw, biopsy, urine collection). In some embodiments, the biological sample is combined with the storage media within about 60 minutes, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, or about 1 minute of collection of the biological sample.

[0065] In some embodiments, the biological sample is combined with the storage media within at least 60 minutes, at least 50 minutes, at least 40 minutes, at least 30 minutes, at least 20 minutes, at least 10 minutes, at least 5 minutes, or at least 1 minute of collection of the biological sample.

[0066] In some embodiments, the biological sample is combined with the storage media within at least about 60 minutes, at least about 50 minutes, at least about 40 minutes, at least about 30 minutes, at least about 20 minutes, at least about 10 minutes, at least about 5 minutes, or at least about 1 minute of collection of the biological sample.

[0067] The storage media may comprise DMSO in an amount of at least about 10% to at least about 25%. In some embodiments, the storage media comprises DMSO in an amount of at least about 12% to at least about 20%. In some embodiments, the storage media comprises DMSO in an amount of about 10%, about 1 1%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25%.

[0068] In some embodiments, the storage media comprises DMSO in an amount of at least 10%, at least 1 1 %, at least 12%, at least 13%, at least 14%, at least 15%, atleast 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21 %, at least 22%, at least 23%, at least 24%, or at least 25%.

[0069] In some embodiments, the storage media comprises DMSO in an amount of at least about 10%, at least about 1 1%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21 %, at least about 22%, at least about 23%, at least about 24%, or at least about 25%.

[0070] In some embodiments, the storage media is a freezing media (e.g., CryoStor®). The storage media may further comprise serum or is supplemented with serum. The serum may comprise natural or synthetic serums. In some embodiments, the serum is selected from the group consisting of bovine serum (e.g., fetal bovine serum, newborn calf serum, bovine serum albumin), horse serum, human serum, goat serum, rabbit serum, porcine serum, sheep serum, canine serum, or rodent serum (e.g., rat or mouse serum). In some embodiments, the storage media is serum-free.

[0071] A first combination is formed by combining the biological sample with the storage media. The first combination may be formed at a combining temperature of about 18°C to about 25°C. In some embodiments, the combining temperature is about 18°C, about 19°C, about 20°C, about 21 °C, about 22°C, about 23°C, about 24°C, or about 25°C.

[0072] In some embodiments, the combining temperature is at least 18°C, at least 19°C, at least 20°C, at least 21 °C, at least 22°C, at least 23°C, at least 24°C, or at least 25°C.In some embodiments, the combining temperature is at least about 18°C, at least about 19°C, at least about 20°C, at least about 21 °C, at least about 22°C, at least about 23°C, at least about 24°C, or at least about 25°C.Freezing Samples

[0073] The present technology further comprises methods of freezing (e.g., cryopreserving) the first combination formed by combing the biological sample with the storage media. The first combination may be prepared for freezing by incubating at an incubation temperature that is lower than the combining temperature. After incubating, the first combination may be transferred to a freezing temperature, which is lower thanthe incubation temperature. In some embodiments the first combination is incubated in an enclosure comprising at least a polystyrene foam (e.g., a polystyrene foam box).

[0074] The first combination may be incubated at an incubation temperature of at least about -65°C to at least about -100°C. In some embodiments, the incubation temperature is at least about -70°C to at least about -90°C. In some embodiments, the incubation temperature is at least about -77°C to at least about -87°C.

[0075] In some embodiments, the incubation temperature is about -70°C, about - 72°C, about -74°C, about -76°C, about -78°C, about -80°C, about -82°C, about -84°C, about -86°C, about -88°C, or about -90°C.

[0076] In some embodiments, the incubation temperature is at least -70°C, at least -72°C, at least -74°C, at least -76°C, at least -78°C, at least -80°C, at least -82°C, at least -84°C, at least -86°C, at least -88°C, or at least -90°C.

[0077] In some embodiments, the incubation temperature is at least about -70°C, at least about -72°C, at least about -74°C, at least about -76°C, at least about -78°C, at least about -80°C, at least about -82°C, at least about -84°C, at least about -86°C, at least about -88°C, or at least about -90°C.

[0078] In some embodiments, the first combination is incubated at the incubation temperature for at least about 10 hours to at least about 72 hours. In some embodiments, the first combination is incubated at the incubation temperature for at least about 16 hours to at least about 48 hours. In some embodiments, the first combination is incubated at the incubation temperature for about 16, about 18, about 20, about 22, about 24, about 26, about 28, about 30, about 32, about 34, about 36, about 38, about 40, about 42, about 44, about 46, or about 48 hours.

[0079] In some embodiments, the first combination is incubated at the incubation temperature for at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, at least 32, at least 34, at least 36, at least 38, at least 40, at least 42, at least 44, at least 46, or at least 48 hours.

[0080] In some embodiments, the first combination is incubated at the incubation temperature for at least about 16, at least about 18, at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about32, at least about 34, at least about 36, at least about 38, at least about 40, at least about 42, at least about 44, at least about 46, or at least about 48 hours.

[0081] After incubating at the incubation temperature, the first combination is frozen (e.g., cryopreserved) at a freezing temperature. The first combination may be frozen until further use (e.g., shipment or analysis), and may be frozen for an extended period of time, for example, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, or longer.

[0082] In some embodiments, the freezing temperature is at least about -100°C to at least about -200°C. In some embodiments, the freezing temperature is at least about -130°C to at least about -190°C. In some embodiments, the freezing temperature is about -130°C, about -140°C, about -150°C, about -160°C, about -170°C, about -180°C, or about -190°C.

[0083] In some embodiments, the freezing temperature is at least -130°C, at least -140°C, at least -150°C, at least -160°C, at least -170°C, at least -180°C, or at least - 190°C.

[0084] In some embodiments, the freezing temperature is at least about -130°C, at least about -140°C, at least about -150°C, at least about -160°C, at least about - 170°C, at least about -180°C, or at least about -190°C.Thawing Samples

[0085] The present technology comprises steps of thawing samples for further processing and / or analysis. In some embodiments, the samples are prepared and / or frozen in a first site, and the thawing steps are performed in a second site. In other embodiments, the samples are prepared, frozen, and / or thawed at the same site. In some embodiments, the thawing steps comprise receiving a sample that was prepared and / or frozen by the methods of the present technology.

[0086] Thawing the samples comprises bringing the frozen first combination to a processing temperature. The first combination may be thawed to the processing temperature at a thaw speed of about 1 °C to about 2°C per minute. The processing temperature may be about 35°C to about 40°C. In some embodiments, the processing temperature is about 36°C to about 38°C. In some embodiments, the processing temperature is about 36°C, about 37°C, or about 38°C.

[0087] In some embodiments, the processing temperature is at least 36°C, at least 37°C, or at least 38°C.

[0088] In some embodiments, the processing temperature is at least about 36°C, at least about 37°C, or at least about 38°C.

[0089] The thawed first combination may comprise live cells. In some embodiments, the live cells comprise at least about 10%, at least about 20%, 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or at least about 100% of the total cells in the first combination.

[0090] When the first combination is brought to the processing temperature, the first combination may be combined with a working media at the processing temperature, forming a second combination. The second combination may be subjected to additional processing steps or may be analyzed at this point. In some embodiments, the second combination only comprises the live cells from the thawed first combination.

[0091] In some embodiments, the working media is warmed to the processing temperature before combining with the first combination. The working media may further comprise serum or is supplemented with serum. The serum may comprise natural or synthetic serums. In some embodiments, the serum is selected from the group consisting of bovine serum (e.g., fetal bovine serum, newborn calf serum, bovine serum albumin), horse serum, human serum, goat serum, rabbit serum, porcine serum, sheep serum, canine serum, or rodent serum (e.g., rat or mouse serum). In some embodiments, the working media is serum-free.

[0092] A fixative may be added to the second combination at any step (e.g., after freezing or after thawing). In some embodiments, a fixative is added to at least a portion of the second combination. Nonlimiting examples of fixatives include formaldehydes (e.g., paraformaldehyde), glutaraldehyde, methanol, acetone, and osmium tetroxide.

[0093] In some embodiments, a lysis solution (e.g., a red blood cell lysis buffer) or a DNAse solution is added to the first combination after thawing to the processing temperature or to the second combination.

[0094] In some embodiments, the sample is pelleted (e.g., by centrifugation) or resuspended before adding the working media, the fixative, the lysis solution, and / or the DNAse solution.Immune Cell Depletion

[0095] The methods of the present technology comprise steps of depleting cells from samples. In some embodiments, the cells are immune cells, such as white blood cells. Nonlimiting examples of white blood cells include neutrophils, eosinophils, basophils, monocytes, and lymphocytes. In some embodiments, the methods comprise depleting neutrophils from a sample. Neutrophils may be depleted before or after freezing samples at the freezing temperature. In some embodiments, the neutrophils are depleted after thawing samples. In some embodiments, neutrophils are depleted from the biological sample before and / or during preparing the biological sample for freezing. In some embodiments, the neutrophils are depleted from the first combination. In some embodiments, the neutrophils are depleted from the second combination (e.g., the second combination comprising a fixative). In some embodiments, the neutrophils are depleted from a sample (e.g., the biological sample, the first combination, the second combination) comprising at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% live cells relative to the total cells in the sample.

[0096] Neutrophil depletion may comprise detecting a cell comprising one or more neutrophil cell markers (e.g., neutrophil-specific genes or neutrophil-specific cell surface proteins) and depleting the cells comprising the one or more neutrophil surface markers. In some embodiments, the one or more neutrophil cell markers comprise CD15 or CD66b. In some embodiments, neutrophil depletion comprises selecting for cells expressing markers (e.g., genes or surface proteins) that are not expressed by neutrophils and depleting the cells which do not express the markers. In some embodiments, neutrophils are depleted using a density gradient (e.g., density gradient centrifugation).

[0097] In some embodiments, the neutrophils are depleted from the sample (e.g., the biological sample after collection, the biological sample during processing, the first combination before freezing, the first combination after thawing, the second combination comprising working media) by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to a control. In some embodiments, theneutrophils are depleted from the sample by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% relative to a control. In some embodiments, the neutrophils are depleted from the sample by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% relative to a control. In some embodiments, the neutrophils are depleted from the sample in an amount necessary to perform one or more analyses.Analyzing Samples

[0098] The samples of the present technology (e.g., the biological sample after collection, the biological sample during processing, the first combination before freezing, the first combination after thawing, the second combination comprising working media, the neutrophil depleted sample) may be analyzed to assess molecular features. In some embodiments, the methods of the present technology comprise preparing, freezing, and thawing samples before performing one or more analyses. In some embodiments, the present technology comprises receiving a sample that has been prepared and frozen according to the methods of the present technology and thawing the sample and performing one or more analyses. In some embodiments, the present technology comprises receiving a sample that has been prepared, frozen, and / or thawed according to the methods of the present technology and performing one or more analyses. In some embodiments, the samples are prepared, frozen, and / or thawed in a site that is different from the analysis site.

[0099] The analyses of the present technology may comprise bulk cell or single cell analyses. The bulk cell and / or the single cell analyses may assess molecular features including, but not limited to, gene expression, protein levels, post translational modifications, epigenetic markings, chromatin states, cell and / or organelle localization, cell and / or organelle morphology, cell proliferation, cell viability, cell-cell interactions, cell cycle progression, cell motility, and cell migration. In some embodiments, the analyses determine the identity, condition, or state of a cell in the biological sample.

[0100] In some embodiments, the bulk or single cell analyses are used (i) to determine the molecular characteristics of a biological sample taken during the course of a clinical trial, (ii) for in vitro assays (e.g. functional assays such as drug screening, (iii) for cell proliferation & differentiation studies, for cell line generation, and genetic manipulation, (iv) for in vivo assays & animal models to include xenograft assays, (v)for making a diagnostic or prognostic decision in a subject, (vi) for identifying molecules that play essential roles in disease pathogenesis and can be modulated by small molecules, RNAi molecules, proteins, antibodies, etc., for therapeutic purposes, (vii) for high throughput screening.

[0101] In some embodiments, the bulk cell or single cell analyses assess a level of expression of one or more genes selected from the group consisting of A1 BG, ABLIM1 , AC020656.1 , AC243960.1 , ADTRP, AFF3, ALDH2, ANXA2R, APOBEC3C, APP, AQP3, ARID5B, ATF7IP2, BANK1 , BCL1 1 A, BCL1 1 B, BIRC3, BLK, CAMK4, CAPG, CARS, CASP8AP2, CBL, CCDC167, CCDC50, CCL4, CCND2, CCR7, CD14, CD27, CD36, CD6, CD68, CD79A, CD79B, CD8A, CD8B, CD96, CDKN1 C, CEBPD, CFD, CFP, CLEC10A, CLEC12A, CLIC3, CMC1 , CPVL, CSF3R, CST7, CSTA, CTSH, CXXC5, CYBB, CYTOR, DCTPP1 , DNAJB1 , DOK2, DYNLL2, DYRK2, EAF2, EBP, ERN1 , FCER1 A, FCER1 G, FCER2, FCGR3A, FCN1 , FCRL1 , FGFBP2, FGL2, FHIT, FKBP1 1 , GATA3, GBP5, GIMAP7, GNG2, GPR65, GRN, GZMA, GZMB, GZMH, GZMK, HLA-DMA, HLA-DMB, HLA-DQA1 , HOPX, IFITM3, IFT57, IGHD, IGHM, IGLC2, IGSF6, IKZF3, IL2RB, IL3RA, IL4R, IL6ST, INPP4B, IRF7, IRF8, ITGAL, ITM2C, JAML, JCHAIN, JUN, KLRB1 , KLRC1 , KLRD1 , KLRF1 , KLRG1 , LEF1 , LGALS2, LGALS3, LILRA4, LINC00623, LINC00861 , LINC00926, LINC01857, LINC01871 , LINC02446, LRRC25, LY86, LYN, LYST, MAL, MAML2, MAPKAPK2, MARCHF1 , MARCKS, MATK, MEF2C, MHENCR, MNDA, MS4A1 , MS4A6A, MS4A7, MT1X, MYBL1 , MYC, MYO1 F, MZB1 , NCF2, NCR3, NELL2, ORAI2, OXNAD1 , PAG1 , PASK, PDE3B, PDLIM1 , PECAM1 , PHACTR2, PIK3IP1 , PILRA, PITPNC1 , PLD4, PLPP5, POU2AF1 , POU2F2, PPP1 R10, PRF1 , PRKCH, PRR5, PTPN4, PTPN6, PYHIN1 , RALGPS2, RASSF1 , RCAN3, RFLNB, RHOC, RNF130, RTKN2, S100A12, SWOB, SAMD3, SELENOM, SERPINA1 , SERPINF1 , SESN3, SLC2A4RG, SLC4A10, SMIM25, SP140, SPI1 , SPIB, SPON2, STMN1 , STMN3, STX7, SWAP70, SYNE1 , TAGAP, TBC1 D15, TC2N, TCF4, TCL1 A, THEM4, TMEM154, TMEM156, TMIGD2, TNFRSF13C, TNFRSF1 B, TPD52, TPM2, TPST2, TRABD2A, TRDC, TRG-AS1 , TRGC1 , TRGC2, TSHZ2, TSPAN3, TULP4, UGCG, VCAN, XCL1 , XCL2, ZAP70, and ZEB2. In some embodiments, the bulk cell or single cell analyses assess a level of expression of one or more genes disclosed in International Application No PCT / US22 / 81977, which is hereby incorporated by reference in its entirety. In some embodiments, the level of expression of the one or more genes is used to identify PBMC types; monitor immune health anddiagnosing disease; resolve reproducibility issues suffered by sequencing methods (e.g., single cell sequencing methods); enhance data quality relative to a larger gene panel.

[0102] In some embodiments, the bulk cell analysis or the single cell analysis comprises one or more analyses selected from the group consisting of a genomic analysis, a transcriptom ic analysis, an epigenomic analysis, a spatial genomics analysis, a chromatin accessibility analysis, a proteomics analysis, and a metabolomics analysis.

[0103] In some embodiments, the genomics analysis comprises one or more methods selected from the group consisting of Sanger sequencing, next-generation sequencing, nanopore sequencing, shotgun sequencing, pyrosequencing, and singlemolecule real-time sequencing.

[0104] In some embodiments, the transcriptomic analysis comprises one or more methods selected from the group consisting of poly(A) selection sequencing, total RNA sequencing, strand-specific RNA sequencing, ribosome profiling sequencing, small RNA sequencing, long read RNA sequencing, capture-based RNA sequencing, and chimeric RNA sequencing.

[0105] In some embodiments, the epigenomic analysis comprises one or more methods selected from the group consisting of DNA methylation profiling, histone modification profiling, and RNA methylation profiling.

[0106] In some embodiments, the spatial genomics analysis comprises one or more methods selected from the group consisting of Hi-throughput chromosome conformation capture (Hi-C) sequencing, Capture Hi-C sequencing, Chromosome Conformation Capture (3C) sequencing, Circular Chromosome Conformation Capture (4C) sequencing, Chromosome Conformation Capture Carbon Copy (5C) sequencing, Hi-C with Chromatin Immunoprecipitation (HiChIP) sequencing, Capture-C sequencing, Targeted Chromosome Conformation Capture (T2C) sequencing, and Promoter Capture Hi-C sequencing.

[0107] In some embodiments, the chromatin analysis comprises one or more methods selected from the group consisting of Assay for Transposase-Accessible Chromatin (ATAC) sequencing, DNase sequencing, MNase sequencing,Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) sequencing, Cleavage Under Targets and Release Using Nuclease (CUT&RUN), and Chromatin Immunoprecipitation (ChIP) sequencing, Cleavage Under Targets and Tag mentation (CUT&Tag) sequencing.

[0108] In some embodiments, the proteomic analysis comprises one or more methods selected from the group consisting of mass spectrometry, tandem mass spectrometry, label-free quantification, isotope labeling, a protein-protein interaction analysis, and structural proteomics.

[0109] In some embodiments, the single cell analysis comprises a single cell RNA- sequencing analysis or a Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq). In some embodiments, the single cell analysis comprises methods outlined in Swanson, E., Lord, et aL, (2021 ). Simultaneous trimodal single-cell measurement of transcripts, epitopes, and chromatin accessibility using TEA-seq. eLife, 10, e63632, the entirety of which is incorporated herein by reference.

[0110] In some embodiments, the single cell analysis or the bulk cell analysis comprises methods or analyses that are sensitive to cell handling, including those disclosed in Savage, A. K., et al. (2021 ). Multimodal analysis for human ex vivo studies shows extensive molecular changes from delays in blood processing. iScience, 24(5), 102404, the entirety of which is herein incorporated by reference.

[0111] The bulk cell analysis or the single cell analysis may comprise a functional analysis. The functional analysis may comprise providing an agent to the sample (e.g., the biological sample after collection, the biological sample during processing, the first combination before freezing, the first combination after thawing, the second combination comprising working media, the neutrophil depleted sample) and assessing one or more phenotypes. Nonlimiting examples of functional analyses include a cell differentiation assay, a cell expansion assay, am agonist screening assay, or an Activation Induced Marker (AIM) assay.

[0112] In some embodiments the agent is a cell stimulatory agent. Nonlimiting examples of cell stimulatory agents include phorbol 12-myristate 13-acetate (PMA), ionomycin, phytohemagglutinin, an anti-CD3 antibody, lipopolysaccharide, resiquimod, CD3 stimulation, or CD28 stimulation. In some embodiments, the functional analysescomprise providing one or more agents to the sample. In some embodiments, the functional analyses comprise providing two or more agents to the sample.EXAMPLESExample 1 : Preparing and Cryopreservinq Biological Samples

[0113] Whole blood samples were prepared and frozen by cryopreservation according to the methods of the present technology. 5 mL of 100% DMSO (anhydrous) was added to 100 mL of a media comprising 10% DMSO (CryoStor CS10, Sigma Aldrich) to create a storage media. 2 mL aliquots of the whole blood storage media were transferred to barcode-labeled cryovials. Barcoded cryovials were stored at 2 - 8°C until use or shipment.

[0114] At the time of whole blood collection, the cryovials prefilled with 2 mL whole blood freezing media to were warmed to about 25°C for at least about 30 minutes. Whole blood was collected from a donor into a Sodium Heparin tube. Within 60 minutes of blood collection, the tube of about 25°C whole blood was inverted to mix, and 2 mL aliquots of whole blood was transferred to each pre-filled 5 mL cryovial and mixed, resulting in a DMSO concentration of about 7.5%. The filled cryovials were placed in a about 25°C foam freeze box. Empty slots remaining in the freeze box with were filled with empty 5 mL cryovials. The foam freeze box was then placed on dry ice with the base fully submerged and stored for at least 6 hours. Samples were maintained on dry ice or transferred to a -77°C to -87°C freezer for up to 2 days. Samples were then transferred to liquid nitrogen storage after 2 days until use or shipment.Example 2: Thawing and Processing Cryopreserved Biological Samples

[0115] Samples prepared and frozen according to example 1 were thawed and prepared. To thaw samples, 30 mL of warmed serum-free media (AIM V medium, Gibco) was transferred to a conical tube and placed in a 37°C water bath at least 30 minutes. 80 mL of cold serum-free media (4°C), 10 mL of cold 1 x RBC lysis buffer (BioLegend) (4°C), 100 pL of 0.2 mg / mL DNase Buffer (25°C), and 200 pL of cold Isolation Buffer (Mojosort, BioLegend) (4°C) per was prepared per sample.

[0116] Samples were removed from liquid nitrogen storage and were stored at - 80°C or on dry ice until ready for thaw. Samples were thawed by placing the sampletubes in a foam tube holder and placing the holder into the 37°C water bath. Tubes were removed after 2 minutes and placed on ice.

[0117] Contents of each sample tube were transferred to a single 50mL conical polypropylene tube containing 30mL pre-warmed serum-free media by pouring directly into the tube. 1 ml_ of the serum-free media from the destination tube was added back into to the starting cryovial.

[0118] Samples were centrifuged for 10 mins at 400g and 4°C. Supernatant was aspirated from each tube and pellets were resuspended in cold serum-free media. Resuspended cells were centrifuged for 5 mins at 400g and 4°C and transferred to ice until use.

[0119] Supernatants from samples were aspirated and cold 1 X RBC lysis buffer was added to each sample tube. Samples were incubated on ice for 15 minutes, protected form light. After incubation, 20 ml_ cold serum-free media was added to each tube and pelleted via centrifugation. Pellets were resuspended in 100pL DNase buffer and incubated at about 25°C for 15 minutes. 30 mL cold serum free media was again added and samples were pelleted via centrifugation. Supernatants were resuspended in phosphate buffered saline and optionally plated in a well plate. Samples were incubated on ice until subsequent use or shipment.Example 3: Neutrophil Depletion of Biological Samples

[0120] Neutrophils were depleted from the samples prepared and thawed according to Example 2. To deplete neutrophils, a 1 X isolation buffer (Mojosort Isolation Buffer, BioLegend) was prepared at about 4°C. Antibody cocktails were prepared by adding equal volumes of CD15 and CD66b phycoerythrin (PE)-conjugated antibodies to a tube and kept on ice until use.

[0121] Samples were plated in an input well plate, pelleted by centrifugation, and resuspended in isolation buffer. Antibody cocktails were added to each sample an incubated for about 20 minutes. CD15 and CD66b proteins on cell surfaces in the samples were bound by anti-phycoerythrin (PE) beads. After the 20-minute incubation, dead cell beads and dead cell monomers were mixed for 5 minutes. Well volumes were brought to 200 pL with isolation buffer and centrifuged to pellet cells at 4°C.

[0122] Anti-PE beads and bead-monomers were aspirated distributed to a column and mixed with the sample. Beads were pelleted, resuspended in isolation buffer, and transferred to a plate magnet at about 25°C to allow bead pelleting. Cells were then resuspended in isolation buffer.

[0123] A portion of the Anti PE-bead monomer mix was aspirated and dispensed into an input plate sample well. Bead tubes were mixed and incubated for 15 minutes at about 4°C. After incubation, additional chilled isolation buffer was added. The input plate was then incubated at about 25°C for 5 minutes and beads rings were pelleted on a magnet. 150uL of supernatant was added to a cleanup well plate, and cells were resuspended off the magnet using isolation buffer.

[0124] The intermediate plate to the magnet at about 25°C for 5 minutes to allow bead ring pelleting on the magnet. Additional supernatant was aspirated and transferred to the cleanup well plate. The cleanup well plate was moved to the magnet to allow remaining beads to pellet. Output plates were centrifuged to pellet cells and plate magnets were removed. Cells were stained using acridine orange (AO) and propidium iodide (PI) (AOPI) dye to assess cell viability. Cell counts were quantified using the Cellaca MX software.Example 4: Cryopreserved Biological Samples Retain Function

[0125] Whole blood samples A-C prepared and cryopreserved by the steps according to Examples 1 -3 and an industry standard leukopak control sample D were stimulated with conditions CD3_CD28 PMA-lonomycin and R848 or a control no stimulation. Single cell RNA sequencing was performed and data represented by Uniform Manifold Approximation and Projection (UMAP) analysis compared clustering across the samples prepared by the steps of the present technology (CS15) relative to ficoll-based non-cryopreserved PBMC samples under each condition (FIG. 1 ). CS15 cells demonstrated similar clustering patterns to that of the ficoll-based non- cryopreserved PBMCs.

[0126] Unique molecular identifiers (UMIs) (FIG. 2A) and genes (FIG. 2B) per cell were quantified for CS15 samples and controls under stimulatory conditions A-D. Controls were samples comprising collected leukocytes that were live and had not beenfrozen. No significant differences were observed between CD15 samples and leukopak samples under each condition.

[0127] Pathway enrichment analysis using single cell RNA sequencing (scRNA- seq) data was conducted CS15 cells under stimulatory conditions relative to nonstimulatory conditions for conditions A-D (FIGS. 3-5). Analysis was conducted for PMA / ionomycin stimulation (FIGS. 3A-3D), R848 stimulation (FIGS. 4A-4D), and CD3 / CD28 stimulation (FIGS. 5A-5D). PMA / lonomycin stimulation resulted in differentially expressed genes (DEGs) under each of stimulatory conditions A-D (FIG. 6A), with 67% of all DEGs common among each condition (FIG. 6B). Under CD3 / CD28 cell stimulation, condition A had the most unique DEGs among conditions A-D (14%) (FIG. 7A), with 43% of all DEGs common among each condition (FIG. 7B). R848 stimulation resulted in 52% of all DEGs shared among conditions A-D (FIGS. 8A and 8B). Whole blood for this analysis was collected from a first site (e.g., a clinical site) and transferred to a second site for additional processing and analysis (FIG. 9). This illustrated that whole blood prepared and frozen according to the methods of the present technology retain the ability to perform functional assays across all major immune cell types.Additional Embodiments

[0128] Various embodiments of the present technology are set forth below in paragraphs

[0129] to

[0172] :

[0129] 1. A method of preparing a biological sample for one or more analyses, the method comprising:(vii) combining the biological sample with a storage media comprising at least about 12% to at least about 20% DMSO to form a first combination at a combining temperature of about 18°C to about 25°C;(ii) incubating the first combination at an incubation temperature of about - 70°C to about -90°C for at least about 16 hours to at least about 48 hours;(iii) transferring the incubated first combination from (ii) to a freezing temperature of at least about -130°C to at least about -190°C;(iv) thawing the first combination from (iii) to a processing temperature of about 36°C to about 38°C;(v) combining the thawed first combination from (iv) with a working media at the processing temperature forming a second combination;(vi) depleting neutrophils from the second combination by at least about 50% relative to a control; and(vii) performing the one or more analyses using the neutrophil depleted second combination.

[0130] 2. A method of preparing a biological sample for one or more analyses, the method comprising:(i) receiving a biological sample processed by the steps of:(a) combining the biological sample with a storage media comprising at least about 12% to at least about 20% DMSO to form a first combination at a combining temperature of about 18°C to about 25°C;(b) incubating the first combination at an incubation temperature of about -70°C to about -90°C for at least about 16 hours to at least about 48 hours; andI transferring the incubated first combination from (b) to a freezing temperature of at least about -130°C to at least about -190°C; and(ii) thawing the first combination frl(c) to a processing temperature of about 36°C to about 38°C;(iii) combining the thawed first combination from (ii) with a working media at a temperature of about 36°C to about 38°C, forming a second combination;(iv) depleting neutrophils from the second combination by at least about 50% relative to a control; and(v) performing the one or more analyses using the neutrophil depleted second combination.

[0131] 3. A method of analyzing a biological sample, the method comprising:(i) receiving a biological sample processed by the steps of:(a) combining the biological sample with a storage media comprising at least about 12% to at least about 20% DMSO to form a first combination at a combining temperature of about 18°C to about 25°C;(b) incubating the first combination at an incubation temperature of about -70°C to about -90°C for at least about 16 hours to at least about 48 his;(c) transferring the incubated first combination from (b) to a freezing temperature of at least about -130°C to at least about -190°C;(d) thawing the first combination from (i)(c) to a processing temperature of about 36°C to about 38°C;(e) combining the thawed first combination from (d) with a working media at the processing temperature forming a second combination; and(f) depleting neutrophils from the second combination by at least about 50% relative to a control; and(ii) performing one or more analyses using the biological sample received in(i)-

[0132] 4. A method of preparing a blood sample for one or more analyses, the method comprising:(vii) (i) combining the blood sample with a storage media comprising at least about 12% to at least about 20% DMSO to form a first combination at a combining temperature of about 18°C to about 25°C;(ii) incubating the first combination at an incubation temperature of about - 70°C to about -90°C for at least about 16 hours to at least about 48 hours;(iii) transferring the incubated first combination from (ii) to a freezing temperature of at least about -130°C to at least about -190°C;(iv) thawing the first combination from (iii) to a processing temperature of about 36°C to about 38°C;(v) combining the thawed first combination from (iv) with a working media at the processing temperature forming a second combination;(vi) depleting neutrophils from the second combination by at least about 50% relative to a control; and(vii) performing the one or more analyses using the neutrophil depleted second combination.

[0133] 5. The method of any one of embodiments 1 -4, further comprising adding a fixative to the second combination before the neutrophil depletion.

[0134] 6. The method of embodiment 5, wherein the fixative is added to at least a portion of the second combination.

[0135] 7. The method of any one of embodiments 1 -6, wherein the one or more analyses comprise a bulk cell analysis or a single cell analysis.

[0136] 8. The method of embodiment 7, the bulk cell analysis comprises one or more analyses selected from the group consisting of a genomic analysis, a transcriptomic analysis, an epigenomic analysis, a spatial genomics analysis, a chromatin accessibility analysis, a proteomics analysis, and a metabolomics analysis.

[0137] 9. The method of embodiment 8, wherein the chromatin accessibility analysis comprises an Assay for Transposase-Accessible Chromatin (ATAC) sequencing.

[0138] 10. The method of embodiment 7, wherein the single cell analysis comprises a single cell RNA-sequencing analysis.

[0139] 11. The method of embodiment 7, wherein the single cell analysis comprises a Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE- seq).

[0140] 12. The method of embodiment 7, wherein the bulk cell analysis or the single cell analysis comprises a functional analysis.

[0141] 13. The method of embodiment 12, wherein the functional analysis comprises providing an agent to the biological sample.

[0142] 14. The method of embodiment 12 or 13, wherein the functional analysis comprises a cell differentiation assay, a cell expansion assay, am agonist screening assay, or an Activation Induced Marker (AIM) assay.

[0143] 15. The method of embodiment 13, wherein the agent is a cell stimulatory agent.

[0144] 16. The method of embodiment 15, wherein the cell stimulatory agent comprises one or more agents selected from the group consisting of phorbol 12- myristate 13-acetate (PMA), ionomycin, phytohemagglutinin, an anti-CD3 antibody, lipopolysaccharide, resiquimod, CD3 stimulation, or CD28 stimulation.

[0145] 17. The method of embodiment 7, wherein the single cell analysis or the bulk cell analysis assesses a level of expression of one or more genes selected from the group consisting of A1 BG, ABLIM1 , AC020656.1 , AC243960.1 , ADTRP, AFF3, ALDH2, ANXA2R, APOBEC3C, APP, AQP3, ARID5B, ATF7IP2, BANK1 , BCL1 1A, BCL1 1 B, BIRC3, BLK, CAMK4, CAPG, CARS, CASP8AP2, CBL, CCDC167, CCDC50, CCL4, CCND2, CCR7, CD14, CD27, CD36, CD6, CD68, CD79A, CD79B, CD8A, CD8B, CD96, CDKN1 C, CEBPD, CFD, CFP, CLEC10A, CLEC12A, CLIC3, CMC1 , CPVL, CSF3R, CST7, CSTA, CTSH, CXXC5, CYBB, CYTOR, DCTPP1 , DNAJB1 , DOK2, DYNLL2, DYRK2, EAF2, EBP, ERN1 , FCER1A, FCER1 G, FCER2, FCGR3A, FCN1 , FCRL1 , FGFBP2, FGL2, FHIT, FKBP11 , GATA3, GBP5, GIMAP7, GNG2, GPR65, GRN, GZMA, GZMB, GZMH, GZMK, HLA-DMA, HLA-DMB, HLA-DQA1 , HOPX, IFITM3, IFT57, IGHD, IGHM, IGLC2, IGSF6, IKZF3, IL2RB, IL3RA, IL4R, IL6ST, INPP4B, IRF7, IRF8, ITGAL, ITM2C, JAML, JCHAIN, JUN, KLRB1 , KLRC1 , KLRD1 , KLRF1 , KLRG1 , LEF1 , LGALS2, LGALS3, LILRA4, LINC00623, LINC00861 , LINC00926, LINC01857, LINC01871 , LINC02446, LRRC25, LY86, LYN, LYST, MAL, MAML2, MAPKAPK2, MARCHF1 , MARCKS, MATK, MEF2C, MHENCR, MNDA, MS4A1 , MS4A6A, MS4A7, MT1X, MYBL1 , MYC, MYO1 F, MZB1 , NCF2, NCR3, NELL2, ORAI2, OXNAD1 , PAG1 , PASK, PDE3B, PDLIM1 , PECAM1 , PHACTR2, PIK3IP1 , PILRA, PITPNC1 , PLD4, PLPP5, POU2AF1 , POU2F2, PPP1 R10, PRF1 , PRKCH, PRR5, PTPN4, PTPN6, PYHIN1 , RALGPS2, RASSF1 , RCAN3, RFLNB, RHOC, RNF130, RTKN2, S100A12, SWOB, SAMD3, SELENOM, SERPINA1 , SERPINF1 , SESN3, SLC2A4RG, SLC4A10, SMIM25, SP140, SPI1 , SPIB, SPON2, STMN1 , STMN3, STX7, SWAP70, SYNE1 , TAGAP, TBC1 D15, TC2N, TCF4, TCL1A, THEM4, TMEM154, TMEM156, TMIGD2, TNFRSF13C, TNFRSF1 B, TPD52, TPM2, TPST2, TRABD2A, TRDC, TRG-AS1 , TRGC1 , TRGC2, TSHZ2, TSPAN3, TULP4, UGCG, VCAN, XCL1 , XCL2, ZAP70, and ZEB2.

[0146] 18. The method of any one of embodiments 1-17, wherein combining the biological sample with the storage media occurs within about 60 minutes of collection of the biological sample.

[0147] 19. The method of any one of embodiments 1 -18, wherein the storage media comprises at least about 12% DMSO.

[0148] 20. The method of any one of embodiments 1 -18, wherein the storage media comprises at least about 14% DMSO.

[0149] 21. The method of any one of embodiments 1 -18, wherein the storage media comprises at least about 15% DMSO.

[0150] 22. The method of any one of embodiments 1 -18, wherein the storage media comprises at least about 16% DMSO.

[0151] 23. The method of any one of embodiments 1 -18, wherein the storage media comprises at least about 18% DMSO.

[0152] 24. The method of any one of embodiments 1 -18, wherein the storage media comprises at least about 20% DMSO.

[0153] 25. The method of any one of embodiments 1-18, wherein the first combination is incubated in an enclosure comprising at least a polystyrene foam.

[0154] 26. The method of embodiment 25, wherein the enclosure is a polystyrene foam box.

[0155] 27. The method of any one of embodiments 1-26, wherein the first combination is incubated for at least about 16 hours at a first incubation temperature of at least about -70°C to about -80°C.

[0156] 28. The method of embodiment 27, wherein the first combination is incubated for no more than about 48 hours at a second incubation temperature of at least about -80°C to about -90°C.

[0157] 29. The method of any one of embodiments 1 -28, wherein thawing the first combination comprises a thaw speed of about 1 °C to about 2°C per minute.

[0158] 30. The method of any one of embodiments 1 -29, wherein the storage media is a freezing media.

[0159] 31 . The method of any one of embodiments 1 -30, wherein the storage media or the working media is a serum-free media.

[0160] 32. The method of any one of embodiments 1 -30, wherein the thawed first combination comprises live cells.

[0161] 33. The method of embodiment 32, wherein the live cells comprise at least about 25% of the total cells in the first combination.

[0162] 34. The method of embodiment 32, wherein the live cells comprise at least about 50% of the total cells in the first combination.

[0163] 35. The method of embodiment 32, wherein the live cells comprise at least about 75% of the total cells in the first combination.

[0164] 36. The method of any one of embodiments 1 -35, wherein the working media is warmed to the processing temperature.

[0165] 37. The method of any one of embodiments 1 -36, wherein depleting the neutrophils comprises detecting a cell comprising one or more neutrophil surface proteins.

[0166] 38. The method of embodiment 37, wherein the cell comprising the one or more neutrophil surface proteins is depleted from the second combination.

[0167] 39. The method of embodiment 38, wherein the one or more neutrophil surface proteins comprise CD15 or CD66b.

[0168] 40. The method of any one of embodiments 1 -3, wherein the biological sample is selected from the group consisting of a urine sample, a saliva sample, a circulatory fluid sample, a synovial fluid sample, and a solid tissue sample.

[0169] 41 . The method of embodiment 40, wherein the circulatory fluid sample is a blood sample.

[0170] 42. The method of embodiment 4 or 41 , wherein the blood sample is a peripheral blood mononuclear cells (PBMC) sample.

[0171] 43. The method of any one of embodiments 1 -42, wherein the method preserves a state of one or more features in the biological sample at the time of collection relative to a control.

[0172] 44. The method of embodiment 43, wherein the control is a biological sample subjected to a method lacking one or more steps of embodiment 1 or 2.

Claims

CLAIMSWe claim:1 . A method of preparing a biological sample for one or more analyses, the method comprising:(i) combining the biological sample with a storage media comprising at least about 12% to at least about 20% DMSO to form a first combination at a combining temperature of about 18°C to about 25°C;(ii) incubating the first combination at an incubation temperature of about - 70°C to about -90°C for at least about 16 hours to at least about 48 hours;(iii) transferring the incubated first combination from (ii) to a freezing temperature of at least about -130°C to at least about -190°C;(iv) thawing the first combination from (iii) to a processing temperature of about 36°C to about 38°C;(v) combining the thawed first combination from (iv) with a working media at the processing temperature forming a second combination;(vi) depleting neutrophils from the second combination by at least about 50% relative to a control; and(vii) performing the one or more analyses using the neutrophil depleted second combination.

2. A method of preparing a biological sample for one or more analyses, the method comprising:(i) receiving a biological sample processed by the steps of:(a) combining the biological sample with a storage media comprising at least about 12% to at least about 20% DMSO to form a first combination at a combining temperature of about 18°C to about 25°C;(b) incubating the first combination at an incubation temperature of about-70°C to about -90°C for at least about 16 hours to at least about 48 hours; and(c) transferring the incubated first combination from (b) to a freezing temperature of at least about -130°C to at least about -190°C; and(ii) thawing the first combination from (c) to a processing temperature of about 36°C to about 38°C;(iii) combining the thawed first combination from (ii) with a working media at a temperature of about 36°C to about 38°C, forming a second combination;(iv) depleting neutrophils from the second combination by at least about 50% relative to a control; and(v) performing the one or more analyses using the neutrophil depleted second combination.

3. A method of analyzing a biological sample, the method comprising:(i) receiving a biological sample processed by the steps of:(a) combining the biological sample with a storage media comprising at least about 12% to at least about 20% DMSO to form a first combination at a combining temperature of about 18°C to about 25°C;(b) incubating the first combination at an incubation temperature of about-70°C to about -90°C for at least about 16 hours to at least about 48 hours;(c) transferring the incubated first combination from (b) to a freezing temperature of at least about -130°C to at least about -190°C;(d) thawing the first combination from (i)(c) to a processing temperature of about 36°C to about 38°C;(e) combining the thawed first combination from (d) with a working media at the processing temperature forming a second combination; and(f) depleting neutrophils from the second combination by at least about50% relative to a control; and(ii) performing one or more analyses using the biological sample received in (i).

4. A method of preparing a blood sample for one or more analyses, the method comprising:(i) combining the blood sample with a storage media comprising at least about 12% to at least about 20% DMSO to form a first combination at a combining temperature of about 18°C to about 25°C;(ii) incubating the first combination at an incubation temperature of about - 70°C to about -90°C for at least about 16 hours to at least about 48 hours;(iii) transferring the incubated first combination from (ii) to a freezing temperature of at least about -130°C to at least about -190°C;(iv) thawing the first combination from (iii) to a processing temperature of about 36°C to about 38°C;(v) combining the thawed first combination from (iv) with a working media at the processing temperature forming a second combination;(vi) depleting neutrophils from the second combination by at least about 50% relative to a control; and(vii) performing the one or more analyses using the neutrophil depleted second combination.

5. The method of any one of claims 1 -4, further comprising adding a fixative to the second combination before the neutrophil depletion.

6. The method of claim 5, wherein the fixative is added to at least a portion of the second combination.

7. The method of any one of claims 1 -6, wherein the one or more analyses comprise a bulk cell analysis or a single cell analysis.

8. The method of claim 7, the bulk cell analysis comprises one or more analyses selected from the group consisting of a genomic analysis, a transcriptomic analysis, an epigenomic analysis, a spatial genomics analysis, a chromatin accessibility analysis, a proteomics analysis, and a metabolomics analysis.

9. The method of claim 8, wherein the chromatin accessibility analysis comprises an Assay for Transposase-Accessible Chromatin (ATAC) sequencing.

10. The method of claim 7, wherein the single cell analysis comprises a single cell RNA-sequencing analysis.11 . The method of claim 7 , wherein the single cell analysis comprises a Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq).

12. The method of claim 7, wherein the bulk cell analysis or the single cell analysis comprises a functional analysis.

13. The method of claim 12, wherein the functional analysis comprises providing an agent to the biological sample.

14. The method of claim 12 or 13, wherein the functional analysis comprises a cell differentiation assay, a cell expansion assay, am agonist screening assay, or an Activation Induced Marker (AIM) assay.

15. The method of claim 13, wherein the agent is a cell stimulatory agent.

16. The method of claim 15, wherein the cell stimulatory agent comprises one or more agents selected from the group consisting of phorbol 12-myristate 13-acetate (PMA), ionomycin, phytohemagglutinin, an anti-CD3 antibody, lipopolysaccharide, resiquimod, CD3 stimulation, or CD28 stimulation.

17. The method of claim 7, wherein the single cell analysis or the bulk cell analysis assesses a level of expression of one or more genes selected from the group consisting of A1 BG, ABLIM1 , AC020656.1 , AC243960.1 , ADTRP, AFF3, ALDH2, ANXA2R, APOBEC3C, APP, AQP3, ARID5B, ATF7IP2, BANK1 , BCL11A, BCL11 B, BIRC3, BLK, CAMK4, CAPG, CARS, CASP8AP2, CBL, CCDC167, CCDC50, CCL4, CCND2, CCR7, CD14, CD27, CD36, CD6, CD68, CD79A, CD79B, CD8A, CD8B, CD96, CDKN1 C, CEBPD, CFD, CFP, CLEC10A, CLEC12A, CLIC3, CMC1 , CPVL,CSF3R, CST7, CSTA, CTSH, CXXC5, CYBB, CYTOR, DCTPP1 , DNAJB1, DOK2, DYNLL2, DYRK2, EAF2, EBP, ERN1, FCER1A, FCER1G, FCER2, FCGR3A, FCN1, FCRL1, FGFBP2, FGL2, FHIT, FKBP11, GATA3, GBP5, GIMAP7, GNG2, GPR65, GRN, GZMA, GZMB, GZMH, GZMK, HLA-DMA, HLA-DMB, HLA-DQA1, HOPX, IFITM3, IFT57, IGHD, IGHM, IGLC2, IGSF6, IKZF3, IL2RB, IL3RA, IL4R, IL6ST, INPP4B, IRF7, IRF8, ITGAL, ITM2C, JAML, JCHAIN, JUN, KLRB1, KLRC1, KLRD1, KLRF1, KLRG1, LEF1, LGALS2, LGALS3, LILRA4, LINC00623, LINC00861, LINC00926, LINC01857, LINC01871, LINC02446, LRRC25, LY86, LYN, LYST, MAL, MAML2, MAPKAPK2, MARCHF1, MARCKS, MATK, MEF2C, MHENCR, MNDA, MS4A1, MS4A6A, MS4A7, MT1X, MYBL1, MYC, MY01 F, MZB1, NCF2, NCR3, NELL2, ORAI2, OXNAD1, PAG1, PASK, PDE3B, PDLIM1, PECAM1, PHACTR2, PIK3IP1, PILRA, PITPNC1, PLD4, PLPP5, POU2AF1, POU2F2, PPP1R10, PRF1, PRKCH, PRR5, PTPN4, PTPN6, PYHIN1, RALGPS2, RASSF1, RCAN3, RFLNB, RHOC, RNF130, RTKN2, S100A12, SWOB, SAMD3, SELENOM, SERPINA1, SERPINF1, SESN3, SLC2A4RG, SLC4AW, SMIM25, SP140, SPI1, SPIB, SPON2, STMN1, STMN3, STX7, SWAP70, SYNE1, TAGAP, TBC1D15, TC2N, TCF4, TCL1A, THEM4, TMEM154, TMEM156, TMIGD2, TNFRSF13C, TNFRSF1B, TPD52, TPM2, TPST2, TRABD2A, TRDC, TRG-AS1, TRGC1, TRGC2, TSHZ2, TSPAN3, TULP4, UGCG, VCAN, XCL1 , XCL2, ZAP70, and ZEB2.

18. The method of any one of claims 1-17, wherein combining the biological sample with the storage media occurs within about 60 minutes of collection of the biological sample.

19. The method of any one of claims 1-18, wherein the storage media comprises at least about 12% DMSO.

20. The method of any one of claims 1-18, wherein the storage media comprises at least about 14% DMSO.

21. The method of any one of claims 1-18, wherein the storage media comprises at least about 15% DMSO.

22. The method of any one of claims 1 -18, wherein the storage media comprises at least about 16% DMSO.

23. The method of any one of claims 1 -18, wherein the storage media comprises at least about 18% DMSO.

24. The method of any one of claims 1 -18, wherein the storage media comprises at least about 20% DMSO.

25. The method of any one of claims 1 -18, wherein the first combination is incubated in an enclosure comprising at least a polystyrene foam.

26. The method of claim 25, wherein the enclosure is a polystyrene foam box.

27. The method of any one of claims 1 -26, wherein the first combination is incubated for at least about 16 hours at a first incubation temperature of at least about -70°C to about -80°C.

28. The method of claim 27, wherein the first combination is incubated for no more than about 48 hours at a second incubation temperature of at least about -80°C to about -90°C.

29. The method of any one of claims 1-28, wherein thawing the first combination comprises a thaw speed of about 1 °C to about 2°C per minute.

30. The method of any one of claims 1 -29, wherein the storage media is a freezing media.31 . The method of any one of claims 1 -30, wherein the storage media or the working media is a serum-free media.

32. The method of any one of claims 1-30, wherein the thawed first combination comprises live cells.

33. The method of claim 32, wherein the live cells comprise at least about 25% of the total cells in the first combination.

34. The method of claim 32, wherein the live cells comprise at least about 50% of the total cells in the first combination.

35. The method of claim 32, wherein the live cells comprise at least about 75% of the total cells in the first combination.

36. The method of any one of claims 1 -35, wherein the working media is warmed to the processing temperature.

37. The method of any one of claims 1 -36, wherein depleting the neutrophils comprises detecting a cell comprising one or more neutrophil surface proteins.

38. The method of claim 37, wherein the cell comprising the one or more neutrophil surface proteins is depleted from the second combination.

39. The method of claim 38, wherein the one or more neutrophil surface proteins comprise CD15 or CD66b.

40. The method of any one of claims 1 -3, wherein the biological sample is selected from the group consisting of a urine sample, a saliva sample, a circulatory fluid sample, a synovial fluid sample, and a solid tissue sample.

41. The method of claim 40, wherein the circulatory fluid sample is a blood sample.

42. The method of claim 4 or 41 , wherein the blood sample is a peripheral blood mononuclear cells (PBMC) sample.

43. The method of any one of claims 1-42, wherein the method preserves a state of one or more features in the biological sample at the time of collection relative to a control.

44. The method of claim 43, wherein the control is a biological sample subjected to a method lacking one or more steps of claim 1 or 2.

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