Methods and assays performed with cell populations

Abstract

The present disclosure relates to methods for enriching a first cell population positive for a target moiety and / or a second cell population positive for a target moiety from a sample, wherein the level of the target moiety in the first cell population is relatively lower than the level of the target moiety in the second cell population. The methods of the present disclosure can also be applied to assays for determining different cell populations positive for a target moiety in a sample, as well as assays for optimizing enrichment conditions. Finally, the present disclosure relates to kits of parts useful for carrying out the methods and assays.

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 979,025, filed on February 20, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the isolation of cells, and more specifically, to the preferential enrichment of cell populations in samples characterized by target regions at different levels. Background Technology

[0003] Multicellular organisms contain a large number of cells and the products of these cells. Cells in multicellular organisms can be classified on a variety of different bases. For example, cells can be distinguished based on their structure and / or function. Alternatively, cells can be classified according to their origin, developmental stage, or tissue residency. Cell classification can also be facilitated by gene expression markers or the expression of gene expression, such as by the localization of peptides or proteins on the cell surface.

[0004] Given the complexity of multicellular organisms and their vast array of cell types, cell isolation techniques have greatly aided in the study of these cells. Early models for isolating cell types were based on differential adhesion to substrates, preferential survival / expansion under specific culture conditions, size grading, or differential sedimentation rates (e.g., in density gradient media). More recent models utilize the presence / absence of target portions that may be present on the cell surface, and the recognition of such target portions in conjunction with mate bodies.

[0005] STEMCELL Technologies and other companies have commercialized immunomagnetic cell separation reagents that can be used for either positive or negative cell separation. In positive cell separation, cells of interest are separated based on the presence of the target region of interest. In negative cell separation, cells of interest are separated based on the presence of the target region of interest in non-interested cells. The main difference between the two methods is that negative cell separation produces “untouched cells,” which can have special uses in downstream applications. In fact, sequential separation using either positive or negative methods, or both, is often used to generate populations of cells of interest.

[0006] However, these methods may fail when the cell population of interest and the cells of non-interest are positive for the same target region. Therefore, improved methods are needed to separate and / or isolate cells based on different levels of the target region. Summary of the Invention

[0007] This disclosure relates to a method for separating target-positive cell populations in a sample based on the presence level of the target portion, and a determination for identifying different target-positive cell populations in a sample based on the presence level of the target portion.

[0008] In one key aspect of this disclosure, a method is provided for enriching (or separating) a first cell population and / or a second cell population that are positive for a target moiety from a sample, comprising labeling the first and second cell populations with particles to form a cell:particle complex; contacting the cell:particle complex with an enrichment agent to substantially remove the particle labeling from the first population; and separating the first population from the sample, wherein the level of the target moiety in the first cell population is relatively lower than the level of the target moiety in the second cell population.

[0009] In one embodiment, the method may further include contacting the residual cell:particle complex in the sample with a separation reagent to substantially separate the second population from the particles.

[0010] In one embodiment, the method may further include separating the second population from the sample.

[0011] In one embodiment, the target portion is a cell surface marker. In one embodiment, the marker is human CD271, human CD25, human CD49d, mouse CD138, human CD8, or human CD56.

[0012] In one embodiment, the particles are coated with a polymer. In another embodiment, the particles respond to a magnetic field.

[0013] In one embodiment, the method may further include separating the cell:particle complex from the sample after step a) and before step c).

[0014] In one embodiment, the method may further include providing at least a saturated amount of particles relative to the target portion level.

[0015] In one embodiment, the labeling of a first or second cell population with particles is mediated by an antibody or antibody fragment. In one embodiment, the antibody or antibody fragment comprises a particle-specific member and a target-part-specific member. In one embodiment, the particle-specific member is directly or indirectly linked to the target-part-specific member. In one embodiment, the particle-specific member and the target-part-specific member form a bispecific complex.

[0016] In one embodiment, the enrichment reagent is formulated differently from the separation reagent. In one embodiment, both the enrichment reagent and the separation reagent comprise a polymer. In one embodiment, the concentration of the polymer in the enrichment reagent is relatively low compared to the separation reagent. In one embodiment, the polymer is PEG (polyethylene glycol), PEG-based, or PEG-like. In one embodiment, the polymer is dextran, dextran-based, or dextran-like.

[0017] In one embodiment, the method may further include enriching the cell:labeled complex from the sample after step a) and before step b).

[0018] In another key aspect of this disclosure, an assay is provided for identifying different cell populations that are positive for a target moiety in a sample, comprising labeling the target moiety with particles to form a cell:particle complex; and obtaining readings of the cell:particle complex by flow cytometry, wherein the level of the target moiety in the first cell population is (relatively) lower than the level of the target moiety in the second cell population.

[0019] In one embodiment, the readings of the cell:particle complex of the first population are different from the readings of the cell:particle complex of the second population. In one embodiment, the readings of both the cell:particle complex of the first population and the cell:particle complex of the second population are different from the readings of uncomplexed target-positive cells. In one embodiment, the readings are a lateral scattering distribution.

[0020] In one embodiment, the target portion is a cell surface marker. In one embodiment, the marker is human CD271, human CD25, human CD49d, mouse CD138, human CD8, or human CD56.

[0021] In one embodiment, the particles respond to a magnetic field.

[0022] In one embodiment, the determination may further include providing at least a saturated amount of particles relative to the target portion level of the first and second populations.

[0023] In one embodiment, the labeling of a first or second cell population with particles is mediated by an antibody or antibody fragment. In one embodiment, the antibody or antibody fragment comprises a particle-specific member and a target-part-specific member. In one embodiment, the particle-specific member is directly or indirectly linked to the target-part-specific member. In one embodiment, the particle-specific member and the target-part-specific member form a bispecific complex.

[0024] In one embodiment, the determination may further include gating based on the lateral scattering distribution on the target portion.

[0025] In one embodiment, the assay may further include separating the cell:particle complex from the sample before obtaining the reading.

[0026] In one embodiment, the assay may further include contacting the cell:particle complex with an enrichment reagent and reacquiring the reading by flow cytometry to assess any shift in the reading. Therefore, in another key aspect of this disclosure, an assay is provided for measuring the dose-responsiveness of the cell:particle complex to the enrichment reagent.

[0027] In another key aspect of this disclosure, a kit is provided for enriching a first cell population and / or a second cell population that is positive for a target moiety from a sample, comprising a tube containing polymer-coated particles; a tube containing an antibody composition comprising particle-specific members linked to target moiety-specific members; a tube containing an enrichment reagent; and optionally a tube containing a separation reagent.

[0028] In one embodiment, the enrichment reagent is PEG-containing or dextran-containing.

[0029] In one embodiment, the separating agent is PEG-containing or dextran-containing.

[0030] In one embodiment, the concentration of PEG or dextran in the enrichment reagent is relatively lower than that in the separation reagent.

[0031] Other features and advantages of the invention will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific embodiments indicate preferred embodiments of the invention, they are given by way of illustration only, and various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art based on this detailed description. Attached Figure Description

[0032] To better understand the various embodiments described herein, and to more clearly illustrate how these various embodiments can be implemented, reference will be made to the accompanying drawings by way of example, which illustrate at least one exemplary embodiment and are now described therein. The drawings are not intended to limit the scope of the teachings described herein.

[0033] Figure 1 The results of neural crest cell differentiation from various human ES and human iPS cell lines are shown. In mTeSR TM 1 or TeSR TM -E8 TM Various ES and iPS cell lines were maintained in the middle, and then STEMdiff was used. TM Neural crest differentiation kit (STEMCELL Technologies) was used for differentiation. A) Human embryonic stem cells (“ES”) and induced pluripotent stem cells (“iPS”) were effectively differentiated into SOX10. +Neural crest cells (85.5 ± 1.6%; mean ± SEM; n = 9). PAX6 in cultures of B) and A) + The level of neuroectodermal cells varied in a cell line-dependent manner (5.6 ± 0.7%; mean ± SEM; n = 9). Figures represent the positive percentage of total DAPI in the tiled image. Dots indicate results from a single experiment.

[0034] Figure 2 Displayed in mTeSR TM Undifferentiated H9 cells maintained in STEMCELL Technologies and using STEMdiff TM Flow cytometry image of CD271 levels in H9 cells differentiated into neural crest cells using the neural crest differentiation kit. On day 6, data were analyzed using ACCUTASE. TM (STEMCELL Technologies) breaks down cells into single-cell suspensions. The cells are then processed using DAPI. - Gating of live cells. Although undifferentiated H9 cells and H9 cells differentiated into neural crest cells showed relatively consistent expression of the surface antigen CD57, undifferentiated H9 cells were characterized by lower surface CD271 levels (A) compared to surface CD271 levels (B) in differentiated neural crest cells.

[0035] Figure 3 Showing the use of STEMdiff TM Flow cytometry image of CD49d surface level in H9 or B004 cells differentiated using a neural crest differentiation kit. Cells were treated with DAPI. - Gating was performed on live cells. In the H9 cells tested, CD49d... 高 (A) and CD271 高 (B) expression was 90% and 87%, respectively. In the B004 cells tested, CD49d... 高 (C) and CD271 高 The expression rates for (D) were 34% and 33%, respectively.

[0036] Figure 4 The results showed that the single surface antigen CD271 can distinguish PAX6. + Nerve cells and SOX10 + Neural crest cells. Assay performed at STEMdiff. TM Surface CD271 levels (A) and intracellular SOX10 and PAX6 levels (B) in F016 cells differentiated using a neural crest differentiation kit. Measurements were taken using STEMdiff. TMSurface CD271 levels (C) and intracellular SOX10 and PAX6 levels (D) in R038 cells differentiated using a neural crest differentiation kit. Measurements were taken using STEMdiff. TM Surface CD271 levels (E) and intracellular SOX10 and PAX6 levels (F) of H1 cells differentiated using a neural crest differentiation kit. Prior to fixation and permeabilization, GloCell was used. TM Fixable Viability Dye Violet 450 (STEMCELL Technologies) labeled cells. This was achieved by excluding GloCell. TM Fixable Viability Dye 450 signal was used to gate live cells. Baseline fluorescence signal was determined using a fluorescent dye and an isotype-matched control antibody. Each flow cytometry plot was gated to visualize CD271. 低 Cells (gray spots) or CD271 高 Neural crest cells (black dots). SOX10 expression was correlated with CD271 expression in all three cell lines tested. 高 Cell overlap, while PAX6 expression is associated with CD271. 低 Cell overlap.

[0037] Figure 5 The differential expression of CD271 in flow cytometry was shown. STEMdiff was used. TM Neural crest differentiation kits were used to differentiate neural crest cells from 1C and H9 cells. After 6 days of culture, cells were collected, pooled, and labeled with a PE-conjugated anti-human CD271 antibody. The images show preferential isolation of CD271 from the pooled cell population by flow cytometry (A), after isolation based on CD271 antigen expression (B), and by treatment with an enrichment reagent (B). 高 Assays were performed after cell division (C). This population consisted of active singlet CD271 cells. 高 Cells perform gating.

[0038] Figure 6 The particles used to label cells increase the side-scattered (SSC) signal detected by flow cytometry. (A) H9, 1C, and M001 cells in STEMdiff TM Differentiation in the neural crest differentiation kit produces CD271. + Cells are separated by positive selection (e.g.) Figure 5 (As shown in B). Enriched CD271 + Cells in STEMdiff TM The cells were further cultured for 6 days in a neural crest differentiation kit, and then used to isolate CD271. +The cells are incubated with an antibody composition or PE-conjugated antibody for the particles. Each dot in A) represents the starting population before positive selection (start) or different conditions tested to optimize the cell isolation procedure (Experiments 1, 2, and 3), where the same fill color represents a head-to-head experiment. In B) through D), DAPI is used... - Live-cell-gated flow cytometry plots show merged populations of H9 and 1C cells without particle labeling ((B) and (D)), or CD271 isolated from the merged populations using antibody compositions and particles. + Cells ((C) and (E)). CD271 expression was assessed, showing CD271... 低 (Gray dot) or CD271 高 (Black dots). Unlabeled cells show CD271. 低 and CD271 高 SSC between cells + No difference in signal (D). When cells were labeled with granules, the signal was different from that of CD271. 低 Compared to cells, the relatively large SSC + Signals and CD271 高 Cell-associated (E). (F) CD271 from a merged population of H9 and 1C cells. 高 and CD271 低 Cells showed varying separation efficiencies depending on the concentration of the enrichment reagent (data for n=1 neural crest cell cultures).

[0039] Figure 7 The effects of different concentrations of enrichment reagents on CD271 were shown. 高 The effect of neural crest cell purity and recovery rate. Differentiation of 1C or H9 cells, such as... Figure 5 As shown. (A) Separate CD271 + Cells were incubated with different concentrations of PEG-containing enrichment reagents (“first enrichment reagents”) and CD271 was added. 高 The purity and recovery rate of neural crest cells were compared with those incubated in a standard PBS-based washing solution (“0%”). Different CD271 cells with n=6 are shown. 高 Results of neural crest cell isolation. Mean purity or recovery rate is indicated by a "+" sign in the box plot. Paired two-tailed t-tests were performed on logit-transformed data (p≥0.05ns, p<0.05*, p<0.01**, p<0.001***). (B) Isolated CD271 + Cells were incubated with different concentrations of dextran-containing enrichment reagents (“second enrichment reagents”), and CD271 was... 高The purity and recovery rate of neural crest cells were compared with those incubated in a standard PBS-based washing solution (“0%”). CD271 is shown. 高 Neural crest cell isolation results, where n = 5 (0%), n = 4 (0.05%), or n = 1 (0.01% and 0.5%). Mean purity or recovery is indicated by a "+" sign in box plots. Paired two-tailed t-tests were performed on logit-transformed data (p ≥ 0.05 ns, p < 0.01**). (C) A rapid and accurate assay was developed to determine the responsiveness of positively selected target moiety positive cells to enrichment reagents of different concentrations / formulations, wherein the positively selected target moiety positive cells are stratified in at least two populations based on the level of the target moiety. The geometric mean of the lateral scattering signal of the particle-labeled cells was determined by flow cytometry analysis. A dose-response curve was generated by plotting log[inhibitor] versus response using a variable slope fitting (four parameters). The vertical dashed line on the dose-response curve represents the calculated IC50. 50 value.

[0040] Figure 8 The enrichment reagent was shown to be effective on CD 271. 高 Added at different times during neural crest cell isolation. (A) In CD271 + During immunomagnetic positive selection of cells, the order of addition of antibody composition (“C”), particles (“P”), and enrichment reagent (“E”) was varied, and CD271 was evaluated. 高 % purity and % recovery rate of neural crest cells. Results for different neural crest cell cultures with n=4 are shown. (B) on CD271 + During the immunomagnetic positive selection of cells, the timing of the enrichment reagent addition was varied (whether before incubating cells with the antibody composition, after incubating cells with the antibody composition and particles (“after the first fill”), or after magnetic separation (“after the first pour”)), and CD271 was evaluated. 高 % purity and % recovery of neural crest cells. Results are shown from different neural crest cell cultures of n=19 (enrichment reagent added before antibody composition), n=6 (enrichment reagent added after the first fill), or n=10 (enrichment reagent added after the first pour). Mean purity or recovery is indicated by a "+" sign in the box plot. Paired two-tailed t-tests were performed on logit-transformed data (p≥0.05ns, p<0.01**, p<0.001***).

[0041] Figure 9 The differential expression of CD271 in flow cytometry was shown. STEMdiff was used. TMNeural crest differentiation kits were used to differentiate 1C or H9 cells. After 6 days of culture, cells were collected, pooled, and labeled with PE-conjugated anti-human CD271 antibody. CD271 levels in the pooled cell population were analyzed by flow cytometry. 低 Cell population (A) and CD271 cells separated by decanting after incubation with enrichment reagent by positively selected CD271 cells. 低 Cells (B). This population is dominated by active singlet CD271. 低 Cell gating. (C) Separated and differentiated CD271 + After cell division, the cells were incubated with different concentrations of PEG-containing enrichment reagents, and the isolated CD271 cells were... 低 Cell purity and recovery were compared with control using standard PBS-based washing reagent (“0%”). The negative efflux fractions of CD271 positive selection experiments performed with n=3 different neural crest cell cultures are shown. Paired two-tailed t-tests were performed on logit-transformed data (p ≥ 0.05 ns, p < 0.05*, p < 0.01**). (D) The same procedure as shown in (C) was performed using dextran-coated particles. In differentiated CD271... + During cell isolation, cells were incubated with different concentrations of dextran-containing enrichment reagents, and the isolated CD271 cells were... 低 Cell purity and recovery were compared with those of a control using standard PBS-based washing reagent (“0%”). The negative efflux fraction is shown in a CD271 positive selection assay performed with n=1 neural crest cell culture.

[0042] Figure 10 The enriched CD271 was displayed. 高 Neural crest cells are functional. (A) Differentiated and enriched CD271 高 The cells expanded during culture, but CD271... 低 Nerve cells did not proliferate significantly under the same culture conditions. H9 cells were cultured using STEMdiff. TM Neural crest differentiation was performed using a differentiation kit. After 6 days of culture, cells were collected, pooled, and isolated using immunomagnetic selection of CD271 cells, followed by incubation in an enrichment reagent. The enriched CD271 cells were then... 高 Groups laid on STEMdiff TM In neural crest differentiation medium, and cultured for another 7 days. Live CD271 高 Cells from 3.81 × 10 5 The number of cells expanded to 6.43 ± 0.97 × 10⁻⁶. 5 Cell count (mean ± SEM; 4 replicates per well). Only negative CD271 cells were observed under the same conditions. 低 Limited amplification of fractions, from 1.91 × 104 Up to 2.89±1.18×10 4 (a) 4 live cells (mean ± SEM; 4 replicates per well). (B) CD271 cells for further culture. 高 Cell seeding density can be varied. The white arrow indicates PAX6. + Cells, as observed in non-enriched initial culture wells. Enriched CD271. 高 The isolated population of cells formed SOX10 + The confluence layer of cells indicates the establishment of a minimum PAX6 concentration. + (C) Enriched cultures of neural crest cells containing contaminants. (C) Re-spreading, enriched CD271. 高 Neural crest cells can differentiate into peripheral neurons. Peripheral neuron differentiation was induced using a seeding density of B) by passage of neural crest cells on day 6 under the conditions described in Lee G et al. (2010) Natprotocol 5(4):688–701. White arrows point to cell bodies expressing Brn3A, a transcription factor found in the nuclei of developing peripheral neurons. The presence of axonal connections between peripheral neuron cell bodies was assessed by peripheral protein expression, a type III intermediate filament protein expressed in neurons of the peripheral nervous system.

[0043] Figure 11 CD25 was displayed 高 Cells can preferentially accumulate from leukocyte isolates. CD25 cells in leukocyte isolates were analyzed by flow cytometry (A), and CD25 cells isolated were positively selected by immunomagnetism. + Cells (B) and CD25 enrichment from population (B) using enrichment reagents. 高 Cells (C). In each of (A) to (C), the population is dominated by active singlet CD45. + CD25 高 Cell gating. (D) Enriched CD25 by different concentrations of enrichment reagents. + Cells were delabeled. The geometric mean of the lateral scattering signal of cells labeled with polymer-coated particles was determined by flow cytometry analysis. A dose-response curve was generated by plotting log[inhibitor] versus response using a variable slope fitting (four parameters). (E) CD25 cells isolated by immunomagnetic positive selection were... + Cells were incubated with different concentrations of PEG-containing enrichment reagents or controls, and CD25 was... 高Cell purity and recovery were compared with control using standard PBS-based washing reagent (“0%”). Results are shown from leukocyte isolates from n=3 donors, with the same fill color representing head-to-head experiments. Paired two-tailed t-tests were performed on logit-transformed data (p ≥ 0.05 ns, p < 0.05*, p < 0.01**). (F) Enriched CD25 in (E) 高 Cells were further incubated with an anti-CD127 removal agent. The delabeled FOXP3 cells were then... + CD25 高 The purity and recovery rate of regulatory T cells were compared with those of the control using standard PBS-based washing reagent (“0%”). Results are shown from leukocyte isolates from n=4 donors. Mean purity or recovery rate is indicated by a “+” sign in the box plot. Paired two-tailed t-tests were performed on logit-transformed data (p ≥ 0.05 ns, p < 0.05*, p < 0.01**).

[0044] Figure 12 CD56 was displayed. 高 Cells can preferentially accumulate from leukocyte-isolated samples. CD56 cells in leukocyte-isolated samples were analyzed by flow cytometry (A), and CD56 cells isolated were positively selected by immunomagnetism. + Cells (B) and CD56 enriched from population (B) using enrichment reagents. 高 Cells (C). In each of (A) to (C), the population is dominated by active singlet CD45. + CD56 高 Cell gating. (D) Enriched CD56 cells were processed using enrichment reagents of different concentrations. + Cells were delabeled. The geometric mean of the lateral scattering signal of cells labeled with polymer-coated particles was determined by flow cytometry analysis. A dose-response curve was generated by plotting log[inhibitor] versus response using a variable slope fitting (four parameters). (E) CD56 cells isolated by immunomagnetic positive selection were... + Cells were incubated with different concentrations of PEG-containing enrichment reagents or controls, and CD56 was... 高 Cell purity and recovery were compared to control with standard PBS-based washing reagent (“0%”). Results are shown from leukocyte isolates from n=4 donors. Mean purity or recovery is indicated by a “+” sign in the box plot. Paired two-tailed t-tests were performed on logit-transformed data (p ≥ 0.05 ns, p < 0.05*, p < 0.01**).

[0045] Figure 13 CD8 was displayed 高CD8 cells can be preferentially enriched from leukocyte isolates. Flow cytometry analysis was performed on CD8 cells (A) in leukocyte isolates, and immunomagnetic positive selection was used to isolate CD8 cells. + Cells (B) and CD8 enrichment from population (B) using enrichment reagents. 高 Cells (C). In each of (A) to (C), the population is dominated by active singlet CD45. + CD8 高 Cell gating. (D) Enriched CD8+ cells were processed using enrichment reagents at different concentrations. + Cells were delabeled. The geometric mean of the lateral scattering signal of cells labeled with polymer-coated particles was determined by flow cytometry analysis. A dose-response curve was generated by plotting log[inhibitor] versus response using a variable slope fitting (four parameters). (E) CD8 cells isolated by immunomagnetic positive selection were... + Cells were incubated with different concentrations of PEG-containing enrichment reagents or controls, and CD8+ were... 高 Cell purity and recovery were compared with a control using standard PBS-based washing reagent (“0%”). Results are shown from a leukocyte isolation donor of n=1.

[0046] Figure 14 CD138 was displayed. 高 CD138 cells were preferentially enriched from juvenile mouse spleen cell samples. Flow cytometry analysis was performed on CD138 cells in mouse spleen cell samples (A), and CD138 cells were positively selected using immunomagnetism. + Cells (B) and CD138 enriched from population (B) using enrichment reagents. 高 Cells. In each of (A) to (C), the population consists of active singlet CD45 cells. + CD267 (TACI) + CD138 高 Gating of plasma cells / blasts. (D) Enrichment of CD138 by different concentrations of enrichment reagents. + Cells were delabeled. The geometric mean of the lateral scattering signal of cells labeled with polymer-coated particles was determined by flow cytometry analysis. A dose-response curve was generated by plotting log[inhibitor] versus response using a variable slope fitting (four parameters). (E) CD138 cells isolated by immunomagnetic positive selection were... + Cells were incubated with different concentrations of PEG-containing enrichment reagents or controls, and CD138 was... 高Cell purity and recovery were compared with control using routine PBS-based washing reagent (“0%”). Results are shown for different mouse spleen cell samples of n=6. Mean purity or recovery is indicated by a “+” sign in the box plot. Paired two-tailed t-tests were performed on logit-transformed data (p ≥ 0.05 ns, p < 0.05*, p < 0.01**, p < 0.001***). (F) CD138 cells isolated by immunomagnetic positive selection. + Cells were incubated with different concentrations of dextran-containing enrichment reagents or controls, and CD138 was... 高 Cell purity and recovery were compared with those of a control using standard PBS-based washing reagent (“0%”). Results are shown for different mouse spleen cell samples of n=2 (0% and 0.04%) and n=1 (0.08% and 0.16%).

[0047] Figure 15 CD138 was displayed. 高 Cells were preferentially enriched from bone marrow samples of naive C57BL / 6 mice. Analysis of mouse bone marrow samples (A) by flow cytometry and positive selection of CD138 cells by immunomagnetic assay were performed. + Cells (B) and CD138 enriched from population (B) using enrichment reagents. 高 Cells (C). In each of (A) to (C), the population is dominated by active singlet CD45. + CD267 (TACI) + CD138 高 Gating of plasma cells / problasts. (D) CD138 isolated by immunomagnetic positive selection. + Cells were incubated with different concentrations of PEG-containing enrichment reagents or controls, and CD138 was... 高 Cell purity and recovery were compared with control using routine PBS-based washing reagent (“0%”). Results are shown for different mouse bone marrow cell samples of n=6. Mean purity or recovery is indicated by a “+” sign in the box plot. Paired two-tailed t-tests were performed on logit-transformed data (p ≥ 0.05 ns, p < 0.05*, p < 0.01**, p < 0.001***). (E) CD138 cells isolated by immunomagnetic positive selection. + Cells were incubated with different concentrations of dextran-containing enrichment reagents or controls, and CD138 was... 高 Cell purity and recovery were compared with those of a control using standard PBS-based washing reagent (“0%”). Results are shown for different mouse bone marrow cell samples of n=2 (0% and 0.04%) and n=1 (0.08% and 0.16%). Detailed Implementation

[0048] This disclosure relates to methods, assays, and kits for different target-positive cell populations in a sample. More specifically, the different target-positive cell populations include a first target-positive cell population and a second target-positive cell population. In one embodiment, the target level of the first target-positive cell population is (relatively) lower than the target level of the second target-positive cell population.

[0049] As used herein, the term "target motif" refers to a cell-associated motif whose presence, absence, or quantity (i.e., level) can aid in the identification or classification of a specific type of cell or cell population. In one implementation, the target motif is a cell surface marker. In some cases, a target motif can uniquely identify a specific cell population (i.e., cell type). For example, CD45 is uniquely associated with normal human hematopoietic cells. In some cases, a target motif cannot uniquely identify a specific cell population (i.e., cell type). For example, CD8 is associated with at least normal human NK cells and normal human T cells. In such cases, it may be necessary to further determine the presence or absence of different target motifs to differentiate cell types. For example, CD8 is associated with at least normal human NK cells and normal T cells, but further querying CD56 can differentiate these populations. In some cases, the level of a target motif can also be used to differentiate between two or more target motif-positive cell populations. For example, human CD271 expression is associated with cells of the neural crest cell lineage (CD271...). 高 ) and cells from neuroectodermal and non-neuroectodermal cell lineages (CD271) 低 This is related to the following: Similarly, different human cell populations can be stratified based on the levels of CD25, CD49d, CD8, or CD56. In mice, the level of CD138 can be queried to stratify different target-positive cell populations in a sample.

[0050] As used herein, the term "sample" refers to a cell preparation, such as a cell suspension. Cell preparations can be of any species, at any developmental stage, of any tissue type, or in any disease state. A cell sample will include a first cell population that is positive for the target portion, a second cell population that is also positive for the target portion, and may include a cell population that is negative for the target portion. In some embodiments, the cells are vertebrate cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. Cell preparations can be obtained by processing organs or tissues, for example, by conventional methods in the life sciences. Alternatively, cell preparations can be obtained from solutions such as blood, which may be pretreated to remove certain contaminants, such as red blood cells or plasma components. Cell preparations can also be obtained from in vitro or ex vivo cultures of cells. In vitro or ex vivo cultures of cells can be maintenance or expansion cultures, or they can be differentiation cultures. In some cases, cell preparations can be non-adherent (e.g., suspension) cell cultures, in which case such cell preparations can be readily used for the methods or assays disclosed herein, or the cells may undergo one or more pretreatment steps, such as removal of contaminants, before preparation for use. In some cases, the cell preparation may be adherent cells, in which case it may be necessary to release such cells from the cell culture medium using conventional techniques before preparing them for use in the methods or assays disclosed herein.

[0051] As used herein, the term "particle" refers to an object that can be used to label, identify, or bind cells (e.g., target moiety-positive cells). In some embodiments, the properties of the particle can be utilized in downstream detection or separation assays. Thus, the properties of the particle can determine one or more separation methods (e.g., responsive to a magnetic field or based on density (e.g., specific gravity / relative density)). Non-limiting examples of downstream assays may include separating or imaging cells that have been labeled / identified by the particle. In some embodiments, the particle may be magnetic, paramagnetic, or superparamagnetic, and thus can be employed in magnetic field-utilizing workflows to facilitate the separation of particle-labeled cells. In some cases, the particle may be functionalized to facilitate downstream assays, such as separation or detection assays. For example, the particle is typically conjugated with an anti-target moiety antibody or antibody fragment. Alternatively, the particle may be functionalized with different types of coatings that can be bound by antibodies or antibody fragments or antibody compositions that link the particle to target moiety-positive cells. For example, the particle may be functionalized with avidin or streptavidin, and such a particle may be complexed with biotin or a biotinylated entity (e.g., a biotinylated anti-target moiety antibody or antibody fragment). Alternatively, other coatings can functionalize the particles, and in one specific embodiment, the coating can be a polymer. Examples of polymer coatings include PEG, PEG-based, or PEG-like coatings. Alternatively, examples of polymer coatings include dextran, dextran-based, or dextran-like coatings.

[0052] method

[0053] In one aspect of this disclosure, the method includes steps of enriching a population of target-part positive cells based on the target part level of target-part positive cells in a sample. More specifically, the target-part positive cells in the sample include at least a first population of target-part positive cells and a second population of target-part positive cells, wherein the target part level of the first population is relatively (or on average) lower than the target part level of the second population.

[0054] This method can begin by providing a cell sample. The cell sample can come from any source, but in a preferred embodiment, the sample is a single-cell suspension of cells. In some embodiments, the cell sample may comprise or consist of relatively small clusters or clumps of cells, or the cell sample may comprise or consist of relatively large aggregates of cells.

[0055] In many cases, cell samples can be pretreated before being provided to break down tissues or organs into their components, to clarify solutions containing cells with certain contaminants, or to separate cells from the matrix.

[0056] In implementations that begin with a tissue or organ, methods for dissociating the tissue or organ into single cells are known. For example, the tissue or organ may be enzymatically digested, mechanically disrupted, or chemically decomposed. In some cases, processing a tissue or organ may involve any combination of enzymatic digestion, mechanical disruption, and chemical decomposition to produce a suitable cell suspension.

[0057] In embodiments that begin with a cell-containing solution, this solution can be clarified to remove contaminants, etc. Methods for clarifying such solutions are known and include filtration or chemical treatment. For example, if starting cells are to be obtained from a blood sample, it may be necessary to remove red blood cells and / or other types of cells of no interest. In one embodiment, the blood sample can be clarified by density gradient centrifugation. In one embodiment, agglutination of red blood cells can clarify the blood sample, for example by performing a rosette process. TM STEMCELL Technologies' approach. In one embodiment, a blood sample can be clarified by lysing red blood cells with a lysis buffer. In another embodiment, a blood sample can be clarified by altering the osmotic pressure within the red blood cells. If the solution containing cells is a bodily fluid other than blood, such as urine, it is important to concentrate the cells while also clarifying the solution of non-cellular components.

[0058] In some implementations, the starting cells may have already been cultured previously. If grown in suspension (i.e., under non-adherent conditions), the cells are readily available for use in the methods disclosed herein. However, cells grown in suspension are typically immersed in cell culture medium, precipitated, and resuspended in a suitable physiological buffer such as phosphate-buffered saline or EasySep. TM The buffer solution (STEMCELL Technologies) may be suitable. Furthermore, cell suspensions immersed in cell culture medium may not be at optimal density, which can be easily adjusted by centrifugation and resuspension (e.g., in a suitable buffer).

[0059] In some embodiments, the starting cells may have been previously adhered to a substrate for culture, whether in tissue culture dishes or flasks, or on microcarriers. In such embodiments, it is necessary to detach the cells from the substrate, which, as those skilled in the art will know, can be accomplished by mechanical, enzymatic, or chemical means. Regardless of whether the cells are detached from the substrate by mechanical, enzymatic, or chemical methods, or by any combination of these methods, it is also necessary to regulate the density of the detached cells before beginning to implement the methods disclosed herein. When detaching cells, care should be taken to minimize or avoid excessive exposure to the reagents to limit the digestion of the target portion (and / or other portions) on the cell surface.

[0060] The methods disclosed herein cover a wide range of cell densities. In one embodiment, the cell density in the sample should be approximately 1 × 10⁻⁶. 4 cells / mL and 1×10 10 Between 100 cells / mL, or around 1×1000 cells / mL 5 cells / mL and 1×10 9 Between 100 cells / mL, or around 5 × 10⁻⁶ cells / mL. 5 cells / mL and 5×10 8 Between 5 cells / mL. In one embodiment, the cell density in the sample should be approximately 5 × 10⁻⁶ cells / mL. 7 ±0.5×10 7 Cells / mL.

[0061] Once the cell sample is adjusted to the appropriate density and / or suspended in the appropriate buffer, the target positive cells in the sample are labeled with particles to form a cell:particle complex.

[0062] In one embodiment, the target moiety is a cell surface marker. Many cell surface markers are known and are therefore included in this disclosure. The target moiety should be bindable to a binding member (e.g., an antibody or antibody fragment). In one embodiment, the cell population of interest in the sample is characterized by unique or distinguishable levels of the target moiety. For example, the target moiety level in a first cell population (target moiety positive) is relatively lower than the target moiety level in a second cell population (target moiety also positive).

[0063] In one implementation, the target portion is human CD271, wherein CD271 高 Differentiating neural crest cells, CD271 低 Distinguish between neuroectodermal cells and non-neuroectodermal cells.

[0064] In one implementation, the target portion is human CD49d, wherein CD49d 高 Differentiating neural crest cells, CD49d 低 Distinguish between neuroectodermal cells and non-neuroectodermal cells.

[0065] In one implementation, the target portion is human CD25, wherein CD25 高 To distinguish between regulatory T cells and activated T cells, CD25 低 Differentiate from non-activated T cells.

[0066] In one implementation, the target portion is human CD8, wherein CD8 高 Differentiating T cells, CD8 低 Differentiate the subpopulations of natural killer cells.

[0067] In one implementation, the target portion is human CD56, wherein CD56高 Differentiating the cytokine-producing subsets of natural killer cells, CD56 低 Differentiate the cytotoxic subgroups of natural killer cells.

[0068] In one implementation, the target moiety is mouse CD138, wherein CD138 高 To distinguish terminally differentiated plasmablasts and plasma cells, CD138 低 Differentiate pre-B cells.

[0069] Skilled readers will recognize that the methods disclosed herein are not limited to the target regions specified above, but can be generalized to any system in which different cell populations in a sample can be distinguished based on the respective levels of the target regions.

[0070] The labeling of a first cell population positive for the target region and a second cell population positive for the target region with particles (to form a cell:particle complex) can be accomplished in a variety of ways. In one embodiment, the connection between the particles and the first and second cell populations is mediated by an antibody or antibody fragment, respectively. In one embodiment, the antibody or antibody fragment comprises particle-specific members and target-specific members.

[0071] The antibodies or antibody fragments disclosed herein can correspond to any structure capable of binding to the target moiety. In one embodiment, the antibody can correspond to any isotype, including IgA, IgD, IgE, IgG, and IgM. In one embodiment, the antibody fragment is F(ab), F(ab')2, scFv fragment, or any adaptation thereof. In some embodiments, the antibody or antibody fragment binds to the target moiety with high specificity. Therefore, it is preferred that the antibody or antibody fragment is monoclonal.

[0072] In one embodiment, the labeling of the target moiety with particles is mediated by a single antibody or fragment thereof. In such an embodiment, one arm of the single antibody or fragment thereof binds to the particle (i.e., the particle-specific member), and the other arm binds to the target moiety (i.e., the target moiety-specific member).

[0073] In one embodiment, the particle labeling of the target moiety is mediated by more than one antibody or fragment thereof. In one embodiment, the particle-specific member is directly or indirectly linked to the target moiety-specific member. In one embodiment, the particle-specific member and the target moiety-specific member form a bispecific complex.

[0074] In one embodiment, the bispecific complex includes a particle-specific member that binds directly to a target-specific member. In such an embodiment, a region of the particle-specific member can bind directly to a region of the target-specific member. For example, the Fc region of the particle-specific member can be directly conjugated to the Fc region of the target-specific member. In the case of an antibody fragment, the linker portion of the particle-specific member can be directly conjugated to the linker portion of the target-specific member.

[0075] In one embodiment, the bispecific complex includes a particle-specific member indirectly linked to a target-part-specific member. In such an embodiment, the indirect linking of the particle-specific member and the target-part-specific member can be achieved in any manner. For example, the particle-specific member and the target-part-specific member can each be bound to a common element. For example, the common element can be a polymer capable of conjugating an antibody or antibody fragment. Alternatively, the common element can be a particle or bead. Alternatively, the common element can be a complementary pair of entities, such as biotin and avidin / streptavidin, wherein each entity is conjugated to one of the particle-specific member or the target-part-specific member. In one embodiment, the particle-specific member and the target-part-specific member can be linked in an immune complex, wherein they are linked by one or more antibodies or antibody fragments. In one embodiment, the bispecific complex comprises two antibodies or their F(ab')2 fragments—the particle-specific member and the target-part-specific member—linked in any manner described herein.

[0076] The method may further include providing at least a saturated amount of particles relative to the target fraction level (i.e., the sum of the target fractions in a first target fraction-positive cell population and a second target fraction-positive cell population). Therefore, a sufficient amount of particles can enhance the recovery of target fraction-positive cells from the sample.

[0077] In one embodiment, the particles are coated with a polymer. In such an embodiment, the polymer may be PEG, PEG-based, or PEG-like. Alternatively, the polymer may be dextran, dextran-based, or dextran-like. Regardless of the nature of the polymer coating the particles, the particle-specific members should be specific to the polymer (of the particles) rather than to the particles themselves.

[0078] The particles may have properties that facilitate the separation of the cell:particle complex from other cells in the sample, said other cells having a non-positive target portion or a target portion that has been delabeled (as described below). In one embodiment, the particles respond to a magnetic field. In such an embodiment, the cell:particle complex formed in the sample (linked by an antibody or antibody fragment) can be exposed to a magnetic field, as commonly done in immunomagnetic separation, to separate the cell:particle complex from other cells in the sample that have a non-positive target portion (or a target portion that has been delabeled). In one embodiment, the particles respond to the density of the solution in which the cells are suspended. For example, the density of the particles may be lower than the density of the solution, in which case the cell:particle complex will float in the solution. Alternatively, the density of the particles may be higher than the density of the solution and the density of cells not contained in the cell:particle complex, in which case the cell:particle complex will sink in the solution. Thus, the cell:particle complex can be separated from other cells in the sample that have a non-positive target portion (or a target portion that has been delabeled). The particles considered in this disclosure are available from various suppliers and / or manufacturers, including STEMCELL Technologies. Therefore, an early or preliminary step of the method disclosed herein may include separating the cell:particle complex from the sample after conjugating the particle and the target-positive cell (by antibody or antibody fragment).

[0079] To enrich (and ultimately separate) target-part-positive cell populations characterized by different target-part levels, the resulting cell:particle complex (with or without a separation step) is contacted with an enrichment reagent. The cell:particle complex should be incubated with the enrichment reagent for a sufficient duration to substantially delabel the first target-part-positive cell population. As used herein, “substantially delabel” means (by the enrichment reagent) delabeling or debinding particles from the target parts of most (if not all) of the first cell population. Although the first cell population is characterized by relatively lower target-part levels than the second cell population, a range of target-part levels may be present between cells within the first population. Therefore, even if not all, a significant proportion of the first cell population will be delabeled, or they may retain only subthreshold levels of particles, rendering them unresponsive to separation means (as described above). In one embodiment, after incubation of the cell:particle complex in the enrichment reagent, approximately 100%, or approximately 95%, or approximately 90%, or approximately 80%, or approximately 70%, or approximately 60%, or approximately 50% of the target parts of the first cell population may be delabeled. In summary, the enrichment reagent serves to make the first cell population more easily labeled than the second cell population, thus making the first cell population less sensitive (i.e., unresponsive) to the separation method compared to the second cell population (retained in the cell:particle complex). This allows the first cell population to be separated or enriched from the second cell population, which was previously impossible in immunomagnetic separation of cells.

[0080] In one implementation, the enrichment agent separates / enriches the first population from the second population, thereby allowing the second population to remain near the magnetic field after incubation of the cell:granule complex in the presence of the enrichment agent, while the first population does not respond to the magnetic field.

[0081] The enrichment reagent should be mild and non-toxic to cells (e.g., it should not directly cause cell death). For example, the enrichment reagent should have physiological (relative to isolated animal cells, more specifically mammalian cells) pH, osmolarity, osmolality, temperature, etc. Furthermore, the enrichment reagent may contain salts and other minerals commonly encountered by cells, but only at or near physiological levels.

[0082] In one embodiment, the enrichment reagent may include a buffer suitable for soaking mammalian cells, such as phosphate-buffered saline, HEPES, MOPS, or Hank's balanced salt solution. In one embodiment, the enrichment reagent may further contain fetal bovine serum (FBS) and / or bovine serum albumin (BSA) and / or serum-derived or recombinant albumin from any species. When the enrichment reagent contains FBS, the concentration of FBS in the buffer-based enrichment reagent may be about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 1%, about 0.5%, or lower. When the enrichment reagent contains BSA or albumin from another species, the concentration of albumin in the buffer-based enrichment reagent may be about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, or about 0.1%, or lower.

[0083] In one embodiment, the enrichment reagent may include a chelating agent, such as EDTA, suitable for use in soaking cells. When the enrichment reagent contains a chelating agent, the concentration of the chelating agent in the buffer-based enrichment reagent may be about 10 μM, about 5 μM to 10 μM, about 1 μM to 5 μM, about 0.5 μM to 1 μM, about 0.1 μM to 0.5 μM, about 50 mM to 100 mM, about 10 mM to 50 mM, about 5 mM to 10 mM, about 1 mM to 5 mM, or lower.

[0084] In one embodiment, the enrichment agent comprises one or more polymers, and said polymers should be cell-mild and non-toxic at the concentrations used. In one embodiment, the polymer is PEG, PEG-based, or PEG-like. In one embodiment, the polymer is dextran, dextran-based, or dextran-like. The polymers included in the enrichment agent should be identical or structurally equivalent to the polymer used to coat the particles, at least at the monomer level.

[0085] In one embodiment, the concentration of one or more polymers in the enrichment reagent should be non-toxic to cells. Therefore, the polymer should be included in the enrichment reagent at a relatively low concentration (compared to the separation reagent, as described below). In one embodiment, the polymer may be included in the enrichment reagent at the minimum concentration required to achieve separation of a first target-part positive cell population from a retained cell:particle complex containing a second target-part positive cell population. In one embodiment, the concentration of the polymer in the enrichment reagent reaches IC50. 50This can be determined by the determinations disclosed below. In one embodiment, the concentration of the polymer in the enrichment reagent is less than 10% (w / v), less than 5% (w / v), less than 1% (w / v), less than 0.5% (w / v), less than 0.25% (w / v), less than 0.125% (w / v); less than 0.0625% (w / v); less than 0.03125% (w / v), less than 0.015625% (w / v); or less than 0.01% (w / v).

[0086] In one embodiment, the components of the enrichment reagent may be pre-formulated as stock solutions, which can be appropriately diluted by the end user to achieve separation of a first target-positive cell population from a second target-positive cell population. In another embodiment, the components of the enrichment reagent may be formulated separately, for example, as a basal solution (e.g., a buffer solution which may or may not contain FBS and / or BSA and / or serum-derived or recombinant albumin and / or chelating agents from any species) and a concentrated solution of the active ingredient. In such embodiments, the active ingredient solution is diluted in the basal solution to an appropriate concentration, which can be determined using the assays disclosed below or by a simple titration experiment.

[0087] Depending on the cell type (and the associated target moiety), it may be necessary to optimize the dose-responsiveness of the enrichment reagent concentration to improve separation / enrichment efficiency and thus enrich the first cell population that is positive for the target moiety from the second cell population that is positive for the target moiety. For example, a higher concentration of enrichment reagent may be required when both the first and second cell populations in the sample exhibit high levels (albeit different) of the target moiety. On the other hand, a lower concentration of enrichment reagent may be sufficient when at least the first cell population and possibly the second cell population in the sample exhibit low (and different) levels of the target moiety.

[0088] After contacting the cell:particle complex with an enrichment reagent, a first cell population can be separated from the sample. Separation of the first cell population is possible because, after substantially removing the particle label, a significant proportion of these cells no longer respond to the separation method (depending on the particle quality). However, a second cell population persists within the cell:particle complex and is therefore still susceptible to particle influence. For clarification purposes only, and not intended as a limitation, in embodiments where the particles respond to a magnetic field, the substantially delabeled first cell population can be separated in the negative fraction, while the second cell population (within the cell:particle complex) is retained in the presence of the magnetic field.

[0089] In some applications, separating the cell:particle complex from the sample may be important before separating the substantially delabeled first cell population. The initial separation step can be performed after the cell:particle complex has formed, preferably before it comes into contact with the enriching reagent. Separation can be performed as described above, for example by utilizing the mass of the particles (whether buoyant, dense, or responsive to a magnetic field). Initial separation may be particularly important when a substantially pure first cell population is the desired output of the disclosed method, because otherwise, the substantially delabeled first cell population separated from the sample will be included in cells that are not positive for the target region. However, if a second cell population is the desired output of the disclosed method, such initial separation may not be necessary.

[0090] In one specific implementation, the cell:particle complex can be formed in a tube using a magnetic field-responsive particle and antibody complex, including target-specific members and particle-specific members, with the antibody complex not directly conjugated to the particles. Optional separation steps may include positioning the tube near the magnetic field and removing cells from the sample that are not positive for the target region while retaining the cell:particle complex. The cell:particle complex can be resuspended in a buffer such as an enrichment reagent, with the tube near or away from the magnetic field. Alternatively, the unseparated cell:particle complex can be similarly contacted with an enrichment reagent. However, the first cell population will be substantially delabeled and can similarly be removed from the retained (or residual) cell:particle complex containing the second cell population. Alternatively, the aforementioned methods can be performed using a column instead of a tube. Variations in these methods, depending on the type of container used—whether tube, dish, flask, column, bag, or other type of container—will be apparent to those skilled in the art.

[0091] The enrichment reagent can be added at any time before separating the first population of cells that are positive for the target fraction. More specifically, the enrichment reagent can be added before or after the formation of the cell:particle complex. Still more specifically, the enrichment reagent can be added before or after the addition of the particle or bispecific complex to the sample.

[0092] After isolating a primary delabeled cell population that is positive for the target region, this cell population is separated / enriched from a secondary cell population that is positive for the target region. Similarly, the secondary cell population that is positive for the target region has been isolated from the primary cell population. On the one hand, if the downstream application only requires the primary cell population, the cell:particle complex remaining in the container (as well as the free particles after the primary population label has been removed) can be discarded. On the other hand, if the secondary cell population in the residual cell:particle complex is required by the downstream application, it can also be isolated. Naturally, in some cases, it may be desirable to use the primary and secondary cell populations separately in the downstream application.

[0093] After isolating a substantially delabeled first cell population that is positive for the target region from the sample, the residual cell:particle complex (containing the second cell population) can be further processed. In one embodiment, the residual cell:particle complex can be separated by removing it from the influence of a separation means (e.g., a magnetic field or others). In one embodiment, it may be necessary to substantially separate the second cell population by contacting the residual cell:particle complex with a separation reagent. The phrase “substantially separated” as used herein has essentially the same meaning as the phrase “substantially delabeled,” except for necessary modifications to specifically apply to the separation reagent and the second cell population that is positive for the target region.

[0094] The separation reagent should be mild and non-toxic to cells (e.g., it should not directly cause cell death). For example, the separation reagent should have physiological (relative to isolated animal cells, more specifically mammalian cells) pH, co-osmotic concentration, co-osmotic gravity, temperature, density, etc. Furthermore, the separation reagent may contain salts and other minerals commonly encountered by cells.

[0095] In one embodiment, the separation reagent may include a buffer suitable for soaking mammalian cells, such as phosphate-buffered saline, HEPES, MOPS, or Hank's balanced salt solution. In one embodiment, the separation reagent may further contain fetal bovine serum (FBS) and / or bovine serum albumin (BSA) and / or serum-derived or recombinant albumin from any species. When the separation reagent contains FBS, the concentration of FBS in the buffer-based enrichment reagent may be about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 1%, about 0.5%, or lower. When the separation reagent contains BSA or albumin from any species, the concentration of albumin in the buffer-based enrichment reagent may be about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, or about 0.1%, or lower.

[0096] In one embodiment, the separation reagent may include a chelating agent, such as EDTA, suitable for use in soaking cells. When the separation reagent contains a chelating agent, the concentration of the chelating agent in the buffer-based separation reagent may be about 10 μM, about 5 μM to 10 μM, about 1 μM to 5 μM, about 0.5 μM to 1 μM, about 0.1 μM to 0.5 μM, about 50 mM to 100 mM, about 10 mM to 50 mM, about 5 mM to 10 mM, about 1 mM to 5 mM, or lower.

[0097] In one embodiment, the separation agent may include one or more polymers, and said polymers should be cell-mild and non-toxic at the concentrations used. In one embodiment, the polymer is PEG, PEG-based, or PEG-like. In one embodiment, the polymer is dextran, dextran-based, or dextran-like. The polymers included in the separation agent should be identical or structurally equivalent to, at least at the monomer level, the polymer used to coat the particles.

[0098] In one embodiment, the concentration of one or more polymers in the separation reagent should be non-toxic to cells. Therefore, the polymer should be included in the separation reagent at a relatively low concentration. In one embodiment, the polymer may be included in the separation reagent at the minimum concentration required to achieve separation of a second cell population that is positive for both the particle and the target portion. In one embodiment, the concentration of the polymer in the separation reagent reaches IC50. 50 This can be determined by the determinations disclosed below. In one embodiment, the concentration of the polymer in the separating reagent is less than 10% (w / v), less than 5% (w / v), less than 1% (w / v), less than 0.5% (w / v), less than 0.25% (w / v), less than 0.125% (w / v); less than 0.0625% (w / v); less than 0.03125% (w / v), less than 0.015625% (w / v); or less than 0.01% (w / v).

[0099] In one embodiment, the components of the separation reagent may be pre-formulated as stock solutions, which can be appropriately diluted by the end user to achieve the separation of a second population of target-positive cells from the particles. In another embodiment, the components of the separation reagent may be formulated separately, for example, as a base solution (e.g., a buffer solution which may or may not contain FBS and / or BSA and / or serum-derived or recombinant albumin and / or chelating agents from any species) and a concentrated solution of the active ingredient. In such an embodiment, the end user may add an appropriate volume of the active ingredient solution to an appropriate volume of the base solution to obtain a properly formulated separation reagent, which can be determined using the assays disclosed below or by a simple titration experiment.

[0100] Depending on the cell type (and the associated target moiety), it may be necessary to measure the dose-responsiveness of different concentrations of the separation agent in order to optimize the separation of the marker from a second cell population that is positive for the target moiety. For example, a higher concentration of the separation agent may be required when the second cell population presents high levels of the target moiety. Because the separation agent is mild and non-toxic to cells, there are few restrictions on the concentration of its components. However, since excessively high concentrations of the separation agent can be unnecessary and / or lead to unintended consequences, relatively low concentrations of the separation agent may be sufficient for the purpose of separating most (if not all) of the particles from the second cell population (within the residual cell:particle complex), both for the cells and in downstream applications. Specifically, a lower concentration of the separation agent may be sufficient when the second cell population presents low levels of the target moiety.

[0101] Therefore, the enrichment reagent preferentially achieves substantially delabeling of particles from a first cell population that is positive for the target region, while the separation reagent achieves substantially separation of particles from a second cell population that is positive for the target region (within the residual cell:particle complex). Although the enrichment reagents function differently from the separation reagents, each reagent may contain many or substantially all of the same components. For example, each reagent may be formulated from a common base solution. In one embodiment, the base solution may be water. In one embodiment, the base solution may be a buffer, such as phosphate-buffered saline, HEPES, MOPS, or Hank's balanced salt solution. In one embodiment, each reagent may further contain a chelating agent, such as EDTA.

[0102] However, assuming the enrichment reagent preferentially enriches a first cell population in the sample that is positive for the target portion, and the sample also contains a second cell population that is positive for the target portion, the respective formulations of the enrichment reagent and the separation reagent may differ in one or more respects. In one embodiment, the enrichment reagent is formulated differently from the separation reagent in at least one respect. For example, the enrichment reagent and the separation reagent may contain different concentrations of the same active ingredient. In one embodiment, the active ingredient is a polymer; therefore, in one embodiment, the concentration of the polymer in the enrichment reagent is relatively low compared to the separation reagent. In one embodiment, the polymer is PEG, PEG-based, or PEG-like. In one embodiment, the polymer is dextran, dextran-based, or dextran-like.

[0103] Based on the foregoing, the method of this disclosure includes those steps of separating / enriching different target-part positive cell populations in a sample based on the target part level in different target-part positive cell populations; the target part level of the first cell population is relatively (or on average) lower than the target part level of the second cell population.

[0104] The separation method adapted to this invention may require separating, isolating, or enriching different cell populations characterized by different levels of first and second target regions. For example, a first enrichment reagent can be used to enrich a first cell population positive for the first target region, and a second enrichment reagent can be used to enrich a first cell population positive for the second target region. Subsequently, a first separation reagent can be used to enrich a second cell population positive for the first target region, and a second separation reagent can be used to enrich a second cell population positive for the second target region.

[0105] In the aforementioned adaptation, the first enrichment reagent and the second enrichment reagent will respectively achieve specific enrichment of the first cell population positive for the first target portion and the second cell population positive for the second target portion. Therefore, the first enrichment reagent and the second enrichment reagent are different in at least one aspect, for example, with respect to the properties of the active ingredients contained therein. Similarly, the first separation reagent and the second separation reagent will respectively achieve specific enrichment of the second cell population positive for the first target portion and the second cell population positive for the second target portion. Therefore, the first separation reagent and the second separation reagent are different in at least one aspect, for example, with respect to the properties of the active ingredients contained therein.

[0106] The methods disclosed herein may further include culturing one, some, or all of the isolated cell populations under suitable culture conditions. This method may include seeding the isolated cell populations at effective cell densities and under effective culture conditions to expand such isolated cell populations.

[0107] In one implementation, the isolated CD271 高 Cells can be cultured under conditions suitable for neural crest cells, and isolated CD271 低 The cells can be discarded or cultured under conditions suitable for neuroectodermal cells.

[0108] If the cell is CD271 高 If the cells are sufficient, the effective cell density can be greater than 1×10⁻⁶. 5 cells / cm 2 If the cell is CD271 低 The effective cell density can be greater than or less than 1×10⁻⁶ cells. 5 cells / cm 2 .

[0109] Measurement

[0110] In one aspect, the assays disclosed herein include those steps for identifying different cell populations that are positive for the target portion in a sample.

[0111] As described above, the assay can begin with the provision of a cell sample. The cell sample can be from any source, but in a preferred embodiment, the sample is a single-cell suspension. In some embodiments, the cell sample may comprise or consist of relatively small clusters or clumps of cells, or the cell sample may comprise or consist of relatively large aggregates of cells.

[0112] In many cases, cell samples can be pretreated before being provided to break down tissues or organs into their components, to clarify solutions containing cells with certain contaminants, or to separate cells from the matrix.

[0113] In implementations that begin with a tissue or organ, methods for dissociating the tissue or organ into single cells are known. For example, the tissue or organ may be enzymatically digested, mechanically disrupted, or chemically decomposed. In some cases, processing a tissue or organ may involve any combination of enzymatic digestion, mechanical disruption, and chemical decomposition to produce a suitable cell suspension.

[0114] In embodiments that begin with a cell-containing solution, this solution can be clarified to remove contaminants, etc. Methods for clarifying such solutions are known and include filtration or chemical treatment. For example, if starting cells are to be obtained from a blood sample, it may be necessary to remove red blood cells and / or other types of cells of no interest. In one embodiment, the blood sample can be clarified by density gradient centrifugation. In one embodiment, agglutination of red blood cells can clarify the blood sample, for example by performing a rosette process. TM STEMCELL Technologies' approach. In one embodiment, a blood sample can be clarified by lysing red blood cells with a lysis buffer. In another embodiment, a blood sample can be clarified by altering the osmotic pressure within the red blood cells. If the solution containing cells is a bodily fluid other than blood, such as urine, it is important to concentrate the cells while also clarifying the solution of non-cellular components.

[0115] In some implementations, the starting cells may have already been cultured previously. If grown in suspension (i.e., under non-adherent conditions), the cells are readily available for use in the methods disclosed herein. However, cells grown in suspension are typically immersed in cell culture medium, precipitated, and resuspended in a suitable physiological buffer such as phosphate-buffered saline or EasySep. TM The buffer solution (STEMCELL Technologies) may be suitable. Furthermore, cell suspensions immersed in cell culture medium may not be at optimal density, which can be easily adjusted by centrifugation and resuspension (e.g., in a suitable buffer).

[0116] In some embodiments, the starting cells may have been previously adhered to a substrate for culture, whether in tissue culture dishes or flasks, or on microcarriers. In such embodiments, it is necessary to detach the cells from the substrate, which, as those skilled in the art will know, can be accomplished by mechanical, enzymatic, or chemical means. Regardless of whether the cells are detached from the substrate by mechanical, enzymatic, or chemical methods, or by any combination of these methods, it is also necessary to regulate the density of the detached cells before beginning to implement the methods disclosed herein. When detaching cells, care should be taken to minimize or avoid excessive exposure to the reagents, as target portions (and / or other portions) present on the cell surface may be digested.

[0117] The methods disclosed herein cover a wide range of cell densities. In one embodiment, the cell density in the sample should be approximately 1 × 10⁻⁶. 4 cells / mL and 1×10 10 Between 100 cells / mL, or around 1×1000 cells / mL 5 cells / mL and 1×10 9 Between 100 cells / mL, or around 5 × 10⁻⁶ cells / mL. 5 cells / mL and 5×10 8 Between 5 cells / mL. In one embodiment, the cell density in the sample should be approximately 5 × 10⁻⁶ cells / mL. 7 ±0.5×10 7 Cells / mL.

[0118] Once the cell sample is suspended in a suitable buffer and adjusted to the appropriate density, the target positive cells in the sample are labeled with particles to form a cell:particle complex.

[0119] In one embodiment, the target moiety is a cell surface marker. Many cell surface markers are known and are therefore included in this disclosure, provided that the target moiety can be bound by a binding member such as an antibody or antibody fragment. Importantly, for the assays disclosed herein, the cell population in the sample is characterized by unique or distinguishable levels of the target moiety. For example, the target moiety level in a first cell population (target moiety positive) is relatively lower than the target moiety level in a second cell population (target moiety also positive).

[0120] In one implementation, the target portion is human CD271, wherein CD271 高 Differentiating neural crest cells, CD271 低 Distinguish between neuroectodermal cells and non-neuroectodermal cells.

[0121] In one implementation, the target portion is human CD49d, wherein CD49d 高 Differentiating neural crest cells, CD49d 低Distinguish between neuroectodermal cells and non-neuroectodermal cells.

[0122] In one implementation, the target portion is human CD25, wherein CD25 高 To distinguish between regulatory T cells and activated T cells, CD25 低 Differentiate from non-activated T cells.

[0123] In one implementation, the target portion is human CD8, wherein CD8 高 Differentiating T cells, CD8 低 Differentiate the subpopulations of natural killer cells.

[0124] In one implementation, the target portion is human CD56, wherein CD56 高 Differentiating the cytokine-producing subsets of natural killer cells, CD56 低 Differentiate the cytotoxic subgroups of natural killer cells.

[0125] In one implementation, the target moiety is mouse CD138, wherein CD138 高 To distinguish terminally differentiated plasmablasts and plasma cells, CD138 低 Differentiate pre-B cells.

[0126] Experienced readers will recognize that the methods disclosed in this article are not limited to the target regions specified above, but can be generalized to any system in which different cell populations can be distinguished at the target region level.

[0127] Labeling a first cell population and a second cell population that are positive for the target region with particles (to form a cell:particle complex) can be accomplished in a variety of ways. In one embodiment, the labeling, binding, or linkage between the particles and the first and second cell populations is mediated by an antibody or antibody fragment, respectively. In one embodiment, the antibody or antibody fragment comprises particle-specific members and target-specific members.

[0128] The antibodies or antibody fragments disclosed herein can correspond to any structure capable of binding to the target moiety. In one embodiment, the antibody can correspond to any isotype, including IgA, IgD, IgE, IgG, and IgM. In one embodiment, the antibody fragment is F(ab), F(ab')2, scFv fragment, or any adaptation thereof. In some embodiments, the antibody or antibody fragment binds to the target moiety with high specificity. Therefore, it is preferred that the antibody or antibody fragment is monoclonal.

[0129] In one embodiment, the labeling of the target moiety with particles is mediated by a single antibody or fragment thereof. In such an embodiment, one arm of the single antibody or fragment thereof binds to the particle (i.e., the particle-specific member), and the other arm binds to the target moiety (i.e., the target moiety-specific member).

[0130] In one embodiment, the particle labeling of the target moiety is mediated by more than one antibody or fragment thereof. In one embodiment, the particle-specific member is directly or indirectly linked to the target moiety-specific member. In one embodiment, the particle-specific member and the target moiety-specific member form a bispecific complex.

[0131] In one embodiment, the bispecific complex includes a particle-specific member that binds directly to a target-specific member. In such an embodiment, a region of the particle-specific member can bind directly to a region of the target-specific member. For example, the Fc region of the particle-specific member can be directly conjugated to the Fc region of the target-specific member. In the case of an antibody fragment, the linker portion of the particle-specific member can be directly conjugated to the linker portion of the target-specific member.

[0132] In one embodiment, the bispecific complex includes a particle-specific member indirectly linked to a target-part-specific member. In such embodiments, the indirect linking of the particle-specific member and the target-part-specific member can be achieved in any manner. For example, the particle-specific member and the target-part-specific member can each bind to a common element. For example, the common element can be a polymer capable of conjugating an antibody or antibody fragment. Alternatively, the common element can be a particle or a bead. Alternatively, the common element can be a complementary pair of entities, such as biotin and avidin / streptavidin or two oligonucleotides, wherein each entity is conjugated to one of the particle-specific member or the target-part-specific member. In one embodiment, the particle-specific member and the target-part-specific member can be linked in an immune complex, wherein they are linked by one or more antibodies or antibody fragments. In one embodiment, the bispecific complex comprises two antibodies or their F(ab')2 fragments—the particle-specific member and the target-part-specific member—linked in any manner described herein.

[0133] The assay may further include providing at least a saturated amount of particles relative to the target fraction level (i.e., the sum of the target fractions in a first target fraction-positive cell population and a second target fraction-positive cell population). Therefore, a sufficient amount of particles can be used to label all target fraction-positive cells in the sample, which can improve recovery.

[0134] In one embodiment, the particles are coated with a polymer. In such an embodiment, the polymer may be PEG, PEG-based, or PEG-like. Alternatively, the polymer may be dextran, dextran-based, or dextran-like. Regardless of the nature of the polymer coating the particles, the particle-specific members should be specific to the polymer (of the particles) rather than to the particles themselves.

[0135] The particles may have properties that facilitate the separation of the cell:particle complex from other cells in the sample, where the target portion of the other cells is not positive. In one embodiment, the particles respond to a magnetic field. In such an embodiment, exposing the cell:particle complex formed in the sample (linked by an antibody or antibody fragment) to a magnetic field, as is typically done in immunomagnetic separation, allows the cell:particle complex to be separated from other cells in the sample where the target portion is not positive. In one embodiment, the particles respond to the density of the solution in which the cells are suspended. For example, the density of the particles may be lower than the density of the solution, in which case the cell:particle complex will float in the solution. Alternatively, the density of the particles may be higher than the density of the solution and the density of cells not contained in the cell:particle complex, in which case the cell:particle complex will sink in the solution. Thus, the cell:particle complex can be separated from other cells in the sample where the target portion is not positive. The aforementioned particles are available from various suppliers and / or manufacturers, including STEMCELL Technologies. Therefore, an early or initial step in the assay disclosed herein may further include separating the cell:particle complex from the sample after linking the particles and target-part positive cells (by an antibody or antibody fragment).

[0136] Regardless of other properties of the particles, when labeled with particles (e.g., via antibodies or antibody fragments, as described above), the particles are sufficient to alter the readings (e.g., side-scatter readings) of target-part positive cells. In one embodiment, the flow cytometry readings of the cell:particle complex are sensitive enough to distinguish between a cell:particle complex containing a first cell population that is target-part positive and a cell:particle complex containing a second cell population that is target-part positive. In such an embodiment, the target level of the first target-part positive cell population is relatively lower than the target level of the second target-part positive cell population.

[0137] Therefore, once the cell:particle complex is formed, the assay includes obtaining readings of the cell:particle complex by flow cytometry. Assuming that the target fraction level in a first cell population that is positive for the target fraction is relatively lower than the target fraction level in a second cell population that is positive for the target fraction, the readings of the cell:particle complex containing the first cell population will differ from the readings of the second cell population.

[0138] Furthermore, the readings for the cell-granule complex containing the first cell population that is positive for the target portion and the cell-granule complex containing the second cell population that is positive for the target portion differ from the readings for the uncomplexed target-positive cells.

[0139] Furthermore, in some embodiments, such as when the readings are lateral scattering distributions of the cell:particle complex, the readings will show different cell populations corresponding to their respective target fraction levels. Therefore, the assay may further include (after obtaining the readings by flow cytometry) contacting the cell:particle complex with an enrichment reagent (as described above) and re-obtaining the readings by flow cytometry to assess the shift in the readings. A shift in the readings (e.g., lateral scattering distribution) may preferentially occur in the first cell population that is positive for the target fraction, indicating that this population has been substantially delabeled. Similarly, the assay may also include contacting the cell:particle complex with different concentrations of an enrichment reagent and assessing the shift in the readings corresponding to each concentration of the enrichment reagent by flow cytometry. Therefore, such an assay can be used to assess the dose-responsiveness of the cell:particle complex (particularly to a specific cell population labeled with particles) to an enrichment reagent (as described above herein).

[0140] In one embodiment, the measurement may further include gating with readings on the target portion, such as the lateral scattering distribution.

[0141] In one embodiment, as described above, the assay may further include enriching cells in the sample that are positive for the target portion and are therefore contained in the cell:particle complex before acquiring the reading. In one embodiment, the enrichment of the cell:particle complex in the sample may be performed by immunomagnetic cell separation, as described above.

[0142] Reagent test kit

[0143] In another aspect of this disclosure, kits are provided for carrying out the methods and assays disclosed herein. Specifically, the kits of this disclosure can be used to enrich a first target-part positive cell population and / or a second target-part positive cell population from a sample.

[0144] In one embodiment, the kit includes a tube containing the particles of the present invention. In one embodiment, the particles contained in the kit are polymer-coated. In one embodiment, the particles are coated with PEG or a PEG derivative. In one embodiment, the particles are coated with dextran or a dextran derivative.

[0145] In one embodiment, the kit further includes a tube containing the antibody complex of the present invention. In one embodiment, the antibody complex is a bispecific complex. In one embodiment, the bispecific complex includes a particle-specific member linked to a target-specific member.

[0146] In one embodiment, the kit further includes a tube containing the enrichment reagent of the present invention. In one embodiment, the enrichment reagent contains PEG or a PEG derivative. In one embodiment, the enrichment reagent contains dextran or a dextran derivative.

[0147] In one embodiment, the kit further includes a tube containing a separation reagent. In one embodiment, the separation reagent contains PEG or a PEG derivative. In one embodiment, the separation reagent contains dextran or a dextran derivative.

[0148] In one embodiment, the concentration of PEG (or PEG derivative) or dextran (or dextran derivative) in the enrichment reagent is relatively lower than the concentration in the separation reagent.

[0149] In one embodiment, the kit also includes instructions on how to perform the methods and assays disclosed herein.

[0150] The following non-limiting embodiments are illustrative of the present invention.

[0151] Example

[0152] Example 1: Differentiation of CD271 presenting cells using different PSC lineages

[0153] Neural crest cells can spontaneously arise in neural and other differentiation protocols, but in other applications, it may be necessary to differentiate the starting cells into neural crest cells. The efficiency of PSC differentiation into neural crest cells may depend on the cell line.

[0154] According to the manufacturer's plan, in STEMdiff TM Differentiation of various PSC lineages was performed in the presence of the neural crest differentiation kit (STEMCELL Technologies Part ID#08610). Figure 1 Showing the use of STEMdiff TM The neural crest differentiation kit (STEMCELL Technologies) differentiates H1 ES cells, H7 ES cells, H9 ES cells, WLS-1C iPS cells, STiPS-M001 iPS cells, STiPS-B004 iPS cells, and STiPS-R038 iPS cells into CD271 cells. 高 The efficiency of neural crest cells.

[0155] Following the differentiation protocol, room temperature PBS (without Ca) was used. 2+ and Mg 2+ Differentiated cells were washed with DMEM / F12 or preheated (37°C) 0.25% w / v trypsin-EDTA or Accutase at 37°C. TMIncubate for 5–7 minutes. Both are available from STEMCELL Technologies. If using a solution containing trypsin, it may be necessary to inactivate it, for example, by 1 mL of 0.5% w / v soybean trypsin inhibitor ACF (STEMCELL Technologies) preheated (37°C). Cells can be removed using a serum pipette before centrifugation at 300×g for 5 minutes (low braking setting) and resuspending in a suitable buffer, and similar samples can be pooled.

[0156] Example 2: Flow cytometry

[0157] Cells harvested according to Example 1 are typically resuspended in an appropriate volume of serum-free solution, such as RoboSep Buffer 2 (STEMCELL Technologies) or any other phosphate-buffered saline solution. In this example, a composition of Dulbecco's phosphate-buffered saline containing 0.5% w / v bovine serum albumin and 2 mM EDTA was used to resuspend the cells to obtain 2.5 × 10⁻⁶ cells. 7 The cell concentration is [total cells / mL]. Optionally, the harvested cells can be passed through a 70µm cell filter before or after centrifuging the sample to remove larger cells.

[0158] Up to 1×10 on the Beckman Coulter CytoFLEX instrument 6 All cells stained with antibodies conjugated with fluorescent dyes were subjected to flow cytometry, and data analysis was performed using FCS Express (version 5). Cells were typically stained in 96-well round plates.

[0159] For intracellular staining (typically using antibodies conjugated with anti-SOX10 and / or anti-PAX6 fluorescent dyes), cells are first fixed with 200 μL of cold 4% paraformaldehyde and incubated at 2–8°C for approximately 15 minutes. After fixation, cells are centrifuged at 700 × g for 3 minutes, resuspended in 250 μL of room temperature PBS + 0.1% Tween 20, and incubated at room temperature for approximately 15 minutes. After permeabilization, cells are centrifuged at 700 x g for 3 minutes, resuspended in 50 μL of RoboSep buffer 2 (STEMCELL Technologies), and stained with 50 μL of a 2X staining mixture of one or more antibodies. The sample is incubated in the dark for 15 minutes, then washed twice with RoboSep buffer 2 (STEMCELL Technologies). After the final wash, cells are resuspended in 100 μL of RoboSep buffer 2 (STEMCELL Technologies).

[0160] Example 3: Markers of neural crest cells

[0161] Neural crest cells have historically been defined based on the expression of intracellular markers such as SOX10, PAX7, and TFAP2. Because the fluorescently conjugated antibodies used to detect these intracellular markers are relatively large, cell fixation and permeabilization are required, preventing cell viability. Neural crest cells can also be defined based on the expression of surface antigens such as CD57, CD271, and CD49d. Using surface-expressed antigens is advantageous for analyzing neural crest cells and other cells because they do not undergo harsh intracellular staining procedures and remain viable. Neural crest cells share the surface expression of some cellular markers with ectoderm-associated cell species. One such example is neuroectoderm cells, defined by the intracellular expression of the PAX6 marker, which can also be identified by the surface expression of the CD271 marker. However, the surface expression levels of these shared antigens can differ, for example, CD271. When evaluated by flow cytometry, the expression levels of SOX10, PAX7, and TFAP2 can vary. + Compared to neural crest cells, PAX6 + Neuroectodermal cells express up to 10 times less of the CD271 marker on their cell surface.

[0162] Undifferentiated PSCs express relatively low levels of the neural crest marker CD271, which was assessed by flow cytometry of H9 cells using a PE-conjugated anti-CD271 antibody (as described in Example 2). Figure 2 A). After culturing cells as described in Example 1, the increased CD271 targeted two different populations of CD271 in the positive cells of the target region. 高 and CD271 低 Layering ( Figure 2 B).

[0163] Similar to CD271, neural crest cells differentiated from H9 or B004 cells, as described in Example 1, are also stratified into two distinct CD49d-presenting cell populations. 高 and CD49d 低 ( Figure 3 ).

[0164] Intracellular staining (as described in Example 1) was performed on differentiated PSCs (as described in Example 2) to correlate surface marker expression with intracellular markers of neural crest cells. SOX10 is a known marker of neural crest cells (Liu and Cheung (2016), Developmental Biology, 419, 199–216), while PAX6 is a known marker of neuroectodermal cells (Liu and Cheung (2016)). CD271 cells differentiated from STiPS-F016 (iPS), R038, and H1 cells were also stained.+ Cells were stained with fluorescently conjugated anti-SOX10 and anti-PAX6 antibodies and analyzed by flow cytometry (as described in Example 2). Figure 4 ).

[0165] Example 4: Preparation of a bispecific complex

[0166] A bispecific complex is prepared by incubating a target-specific member, a particle-specific member, and a adaptor member in PBS. The concentration of individual antibodies or antibody fragments can vary; theoretically, approximately equimolar concentrations of the target-specific member and the particle-specific member will produce the highest proportion of the bispecific complex. As stated above, the disclosed subject matter is independent of the type of adaptor member, although in this case, the adaptor member is an antibody specific to both the target-specific member and the particle-specific member. After incubation, the resulting bispecific complex of antibody (or antibody fragment) can be used for downstream assays, such as cell separation protocols.

[0167] When CD271 + When cells are intended to be targeted by a bispecific complex, an anti-CD271 antibody and an antibody or antibody fragment targeting the polymer of the coated particles can be included in the initial incubation. When CD56... + When cells are intended to be targeted by a bispecific complex, anti-CD56 antibody and antibodies or antibody fragments targeting the polymer of the coated particles can be included in the initial incubation. When CD25... + When cells are intended to be targeted by a bispecific complex, anti-CD25 antibody and antibodies or antibody fragments targeting the polymer of the coated particles can be included in the initial incubation. When CD127... + When cells are intended to be targeted by a bispecific antibody complex, an anti-CD127 antibody and an antibody or antibody fragment targeting the polymer of the coated particles may be included in the initial incubation. When CD8... + When cells are intended to be targeted by a bispecific antibody complex, anti-CD8 antibody and antibody or antibody fragments targeting the polymer of the coated particles can be included in the initial incubation. When mouse CD138... + When cells are intended to be targeted by a bispecific antibody complex, an anti-CD138 antibody and an antibody or antibody fragment targeting the polymer of the coated particles may be included in the initial incubation.

[0168] After the bispecific complexes are formed, they are incubated with a cell sample, preferably a single-cell suspension, which can be obtained using conventional techniques. Following incubation of the bispecific complexes and cells, particles are added to the sample. In this study, the particles are EasySep Releasable RapidSpheres (STEMCELL Technologies, #50201) or EasySep Dextran RapidSpheres (STEMCELL Technologies, #50100). Thus, cells and particles are linked by the bispecific complexes, which can be separated based on the properties of the particles. In this study, the particles are at least responsive to a magnetic field, enabling immunomagnetic separation of cells of interest (in the positive selection protocol).

[0169] Example 5: Enrichment of neural crest cells

[0170] As described in Example 1, neural crest cells were differentiated from 1C and H9 cells. After 6 days of culture, cells were harvested from the culture plate as described in Example 2 and aggregated into a single-cell suspension. Flow cytometry was performed on the initiating differentiated cell population using a PE-conjugated anti-human CD271 antibody as described in Example 2. The initiating differentiated cell population comprised less than 25% CD271. 高 Neural crest cells ( Figure 5 A). Using the antibody complex described in Example 4, CD271 was separated by immunomagnetic separation. + Cells and CD271 - After cell separation, CD271 高 The proportion of neural crest cells increases to approximately 50%. Figure 5 B). In the positive selection of CD271 + After incubation with 0.008% PEG enrichment reagent, CD271 高 The proportion of neural crest cells can be further increased to approximately 90%. Figure 5 C).

[0171] Example 6: Components used for cell enrichment are retained on the cell surface

[0172] In separating cells such as CD271 + After the cells (as described in Example 5) are cultured, they can then be seeded in a suitable culture medium. In CD271 + In the case of neural crest cells, they are re-layered in STEMdiff. TMCells were cultured for 6 days using a neural crest differentiation kit (STEMCELL Technologies). Subsequently, cells were harvested as described in Example 1 and stained with a fluorescently conjugated anti-isotype antibody (targeting a bispecific complex) and a fluorescently conjugated anti-particle antibody. The stained cells were analyzed by flow cytometry as described in Example 2, and the antibody composition and CD271 were detected on a small fraction of the cells. + Cellular granules ( Figure 6 A). As previously mentioned, neural crest cells differentiated from pluripotent stem cells (“PSCs”) are stratified into CD271. 高 and CD271 低 group( Figure 6 B, unlabeled cells; and Figure 6 C, cells labeled with particles via antibody complexes). However, it was also shown that, compared with unlabeled cells ( Figure 6 In contrast, the lateral scattering distribution of particles-labeled cells can also distinguish different CD271 cells. + Cell population ( Figure 6 E).

[0173] Due to the enrichment of CD271 + Cells can retain particles on their surface that facilitate their separation; therefore, in some applications, it may be necessary to actively remove these components. Separation of CD271 according to Example 5. + After cell collection, CD271 was tested using different concentrations of enrichment reagent (STEMCELL Technologies, #17900). 高 Or CD271 低 Release efficiency from particles ( Figure 6 F). Of all tested concentrations, CD271 低 Its release efficiency is higher than that of CD271 高 However, at high concentrations of the enrichment reagent (e.g., ≥0.1%), almost all CD271... + The cells were separated from the particles. Therefore, relatively low concentrations of enrichment reagents, such as between approximately 0.001% and 0.1%, were used as a starting point for optimizing the experiments.

[0174] Example 7: Effects of different enrichment reagent concentrations on label-delabeled CD271 高 Cellular effects

[0175] Further optimization of the enrichment reagent concentration showed that after adding a low concentration of the PEG-containing enrichment reagent (“first enrichment reagent”), CD271 高 Cells can obtain CD271 + CD271 further accumulates in cells. It is found in differentiated PSC samples. 高 The average purity of the cells was approximately 30% (not shown) Figure 5 A). As described in Example 5, CD271 was isolated. + After cell initiation, the initial cell purity can be increased to an average of approximately 70%. Figure 7 A) Adding 0.004%, 0.008%, or 0.016% of the first enrichment reagent during the enrichment protocol resulted in CD271. 高 Increased purity after cell enrichment indicates CD271 低 The number of contaminated cells decreased. However, using relatively high concentrations of enrichment reagents on CD271... 高 Cell enrichment followed by recycling is harmful. Figure 7 A).

[0176] Further optimization of the enrichment reagent concentration showed that, after adding a low concentration of the dextran-containing enrichment reagent (“second enrichment reagent”), CD271 高 Cells can obtain CD271 + CD271 further accumulates in cells. It is found in differentiated PSC samples. 高 The average purity of the cells was approximately 30% (not shown) Figure 5 A). As described in Example 5, CD271 was isolated. + After cell inoculation, the initial cell purity can be increased to an average of approximately 80%. Figure 7 B). Adding 0.01%, 0.05%, or 0.5% of a second enrichment reagent during the enrichment process resulted in CD271. 高 Increased purity after cell enrichment indicates CD271 低 The number of contaminated cells decreased. However, using relatively high concentrations of enrichment reagents on CD271... 高 Cell enrichment followed by recycling is harmful. Figure 7 B).

[0177] A rapid and accurate flow cytometry assay was developed to screen for optimal concentrations of enrichment reagents. Briefly, as described in Example 4, a cell population was first incubated with a bispecific complex that is specific to both the target moiety and the particles of the cells of interest to allow binding of target moiety-positive cells. Subsequently, such a complex was incubated with particles exhibiting high side-scattering to form a cell:particle complex. High side-scattering particles were defined as those producing approximately 2.5 x 10⁻⁶ cells / particles on a Beckman Coulter CytoFLEX instrument when collected in the absence of cells and antibodies. 4 The geometric mean fluorescence intensity. Therefore, the lateral scattering of the cell:granule complex can produce 1×10⁻⁶ fluorescence intensity. 5Or a higher geometric mean fluorescence intensity signal, depending on the original scattering distribution of the unlabeled population. Excess bispecific complex and particles were washed away, and then cells were stained with fluorescently conjugated antibodies. After a second wash to remove excess fluorescently conjugated antibodies, the cell:particle complex was incubated with various concentrations of enrichment reagents. Due to the high lateral scattering of the cell:particle complex, it is possible to assess the release efficiency of a given concentration of enrichment reagent by flow cytometry through the shift in lateral scattering. The geometric mean of the lateral scattering signal of the particle-labeled cells was determined by flow cytometry analysis. Figure 7 C). Dose-response curves were generated using two enrichment reagent formulations: a first enrichment reagent containing PEG and a second enrichment reagent containing dextran. The dose-response curves were generated by plotting the log[inhibitor] versus the response using a variable slope fitting (four parameters).

[0178] The reduction in lateral scattering is associated with delabeling of the cell population (i.e., removal of granules from it). The average 2.5-fold decrease in SSC signal from the maximum lateral scattering signal represents adequate delabeling and a reduced ability of cells to migrate toward the magnetic field during immunomagnetic separation, allowing such cells to be poured out of the negative fraction.

[0179] Example 8: The enrichment reagent can be added at any time during cell enrichment.

[0180] The timing of adding the enrichment reagent in the enrichment protocol was investigated. In the cell isolation protocol described in Example 4, the order in which the antibody composition (“C”), particles (“P”), and enrichment reagent (“E”) were added to the cell sample was varied. The order of addition did not appear to affect CD271. 高 Average % purity or average % recovery of cells ( Figure 8 A).

[0181] Furthermore, whether before adding the antibody composition, after incubating the sample with the antibody composition and particles (“after the first fill”), or after the first magnetic separation (“after the first pour”), the enriched CD271 高 The average percentage purity of the cells did not appear to have changed. Figure 8 B). However, when the enrichment reagent is added after the first filling or after the first pouring, CD271 高 The average % recovery rate of cells appears to increase ( Figure 8 B).

[0182] Example 9: Enrichment reagents can be used to extract CD271 高 CD271 isolated from neural crest cells 低 cell

[0183] As described in Example 1, neural crest cells were differentiated from 1C and H9 cells. After 6 days of culture, cells were harvested from the culture plate as described in Example 2 and aggregated into a single-cell suspension. Flow cytometry was performed on the initially differentiated cell population using a PE-conjugated anti-human CD271 antibody as described in Example 2. The initially differentiated cell population comprised approximately 70% CD271 cells. 低 cell( Figure 9 A). In CD271 as described in Example 5 + Following the initial cell isolation (using an anti-CD271 bispecific complex as described in Example 4), cells were subsequently incubated in a PEG-containing enrichment reagent to inhibit CD271. 低 The proportion of cells in the negative efflux fraction increased to approximately 95%. Figure 9 B). With increasing concentration of the first enrichment reagent, CD271 was observed to... 低 The average purity of the cells increased slightly, while the average recovery rate increased significantly. Figure 9 C). Perform essentially the same procedures as described in (B) and (C), but use EasySep Dextran RapidSpheres (STEMCELL Technologies) for immunomagnetic separation. Separate differentiated CD271... + After cell collection, they were incubated with different concentrations of dextran-containing enrichment reagents to enhance CD271. 低 Cell purity and recovery rate compared with control washing reagent (EasySep) TM The results obtained were compared with those obtained using the washing reagents typically used in the protocol (“0%”) (D).

[0184] Example 10: Isolated CD271 高 Neural crest cells are functional

[0185] Enriched from cell populations and reacted with CD271 in enrichment reagent. 低 CD271 isolated from cells 高 Neural crest cells (using an anti-CD271 bispecific complex as produced in Example 4) can be detected in STEMdiff. TM Further culture and / or maintenance in a neural crest differentiation kit (STEMCELL Technologies). Enriched CD271 will be... 高 Cells were replated (day 0) and incubated in culture medium for 7 days. On day 7, the cell culture was harvested, counted, and analyzed by flow cytometry as described in Example 2 to quantify CD271. 高 and CD271 低 The number of cells. Figure 10 A shows that CD271 高 Cells showed strong expansion during 7 days of culture, while CD271...低 The cells showed low or no amplification.

[0186] In addition, CD271 was enriched from the cell population and reacted with the enrichment reagent. 低 CD271 isolated from cells 高 Neural crest cells (using the anti-CD271 bispecific complex produced as in Example 4) can be further cultured at a cell density optimal for establishing neural crest cells. In this example, at 2 × 10⁻⁶ cells / year... 5 cells / cm 2 Up to 4×10 5 cells / cm 2 The optimal seeding density for observing neural crest cell populations at a live cell density ( Figure 10 B). In the same embodiment, along with the inoculation of CD271 高 Cell purity was increased through a publicly available enrichment procedure, and contaminating PAX6 could be observed. + The reduction in cell population is indicated by a white arrow. Figure 10 B).

[0187] In addition, CD271 was enriched from the cell population and reacted with the enrichment reagent. 低 CD271 isolated from cells 高 Neural crest cells (using an anti-CD271 bispecific complex as produced in Example 4) could be further cultured under conditions that promoted differentiation into peripheral neurons. In this example, the differentiation protocol of Lee G et al. (2010) Nat Protoc 5(4):688–701 was used. CD271 cells were cultured under conditions that promoted differentiation into peripheral neurons expressing peripheral proteins and Brn3A. 高 Neural crest cells ( Figure 10 C).

[0188] Example 11: CD25 高 Cells can be preferentially enriched from leukocyte isolated samples.

[0189] First, the leukocyte separation sample was treated by lysing the red blood cells using an ammonium chloride solution (STEMCELL Technologies Part ID#07850) according to the product information sheet (STEMCELL Technologies Part ID#70500). EasySep was then used. TM Wash the sample twice with buffer (STEMCELL Part ID#20144), add the sample to 50 mL, and centrifuge at 150 x g for 10 minutes (without brake setting). Remove the supernatant with a pipette and transfer to a CD25 culture medium. 高 Prior to the enrichment process (essentially as described in Example 5), the cell pellet was resuspended in EasySep. TM In buffer solution.

[0190] Detection was performed using a PE-conjugated anti-human CD25 antibody, and flow cytometry was used on the treated leukocyte isolate sample as described in Example 2. The initial cell population comprised approximately 2% CD25. 高 cell( Figure 11 A). Using the anti-CD25 bispecific complex as described in Example 4 to target CD25. + After the first cell separation, CD25 高 The proportion of cells increased to approximately 57% ( Figure 11 B). After incubating isolated cells with an enrichment reagent containing PEG, CD25 高 The proportion of cells can be further increased to approximately 82%. Figure 11 C).

[0191] such as dashed lines (IC) 50 As shown, the appropriate concentration of the enrichment reagent can be determined by the determination described in Example 7. Figure 11 D).

[0192] As the concentration of the enrichment reagent increases, the average CD25 高 Increased cell purity meant that only higher concentrations of enrichment reagents affected the average CD25. 高 Cell recovery rate is harmful ( Figure 11 E).

[0193] CD25 was enriched using an enrichment reagent. 高 Cells and CD25 低 After cell separation, CD25 高 Cells were incubated with an anti-CD127 bispecific complex formed essentially as described in Example 4. This CD127 removal procedure is similar to EasySep. TM Human CD4 + CD127 低 CD25 + The procedure described in the Regulatory T Cell Isolation Kit (STEMCELL Technologies Part ID#18063) was followed. CD25 was enriched using the first enrichment reagent as described in Example 7. 高 After cells, from CD25 高 Remove CD127 from the population + Cells lead to average FOXP3+CD25 高 Increased purity of regulatory T cells ( Figure 11 F). In CD127 + After cell removal, it was used to enrich CD25. 高 The concentration of the first enrichment reagent gradually increased with the average FOXP3+CD25 concentration. 高Regulatory T cell recovery has a progressively increasing, more detrimental effect. Figure 11 F).

[0194] Example 12: CD56 高 Cells can be preferentially enriched from leukocyte isolated samples.

[0195] First, the leukocyte isolate sample (STEMCELL Part ID#70500) was treated by lysing red blood cells with ammonium chloride solution (STEMCELL Part ID#07850) according to the product information sheet. EasySep was then used. TM Wash the sample twice with buffer (STEMCELL Part ID#20144), add the sample to 50 mL, and centrifuge at 150 x g for 10 minutes (without brake). Remove the supernatant with a pipette, taking care not to disturb the cell pellet. Perform CD56... 高 Prior to the enrichment process (essentially as described in Example 5), the sample was suspended in EasySep. TM In buffer solution.

[0196] Detection was performed using a PE-conjugated anti-human CD25 antibody, and flow cytometry was used on the treated leukocyte isolates as described in Example 2. The initial cell population consisted of approximately 0.5% CD56. 高 cell( Figure 12 A). Anti-CD56 bispecific complex (as described in Example 4) was used in immunomagnetic separation to target CD56. + After cell separation, CD56 高 The proportion of cells increased to approximately 8% ( Figure 12 B). After incubating the separated sample with an enrichment reagent containing PEG, CD56 高 The proportion of cells can be further increased to approximately 50%. Figure 12 C).

[0197] such as dashed lines (IC) 50 As shown, the appropriate concentration of the first enrichment reagent can be determined by the determination described in Example 7. Figure 12 D).

[0198] As the concentration of the enrichment reagent increases, the average CD56 高 Increased cell purity, but increasingly higher concentrations of enrichment reagents have a greater impact on average CD56. 高 Cell recovery rate has an increasingly unfavorable impact. Figure 12 E).

[0199] Example 13: CD8 高 Cells can be preferentially enriched from leukocyte isolated samples.

[0200] First, the leukocyte isolate sample (STEMCELL Part ID#70500) was treated by lysing red blood cells with ammonium chloride solution (STEMCELL Part ID#07850) according to the product information sheet. EasySep was then used. TM Wash the sample twice with buffer (STEMCELL Part ID#20144), add the sample to 50 mL, and centrifuge at 150 x g for 10 minutes (without brake setting). Remove the supernatant with a pipette and transfer to CD8. 高 Prior to the enrichment process (essentially as described in Example 5), the cell pellet was resuspended in EasySep. TM In buffer solution.

[0201] Detection was performed using a PE-conjugated anti-human CD8 antibody, and flow cytometry was used on the treated leukocyte isolate sample as described in Example 2. The initial cell population comprised approximately 8% CD8. 高 cell( Figure 13 A). Anti-CD8 bispecific complex (as described in Example 4) was used in immunomagnetic separation to target CD8. + After cell separation, CD8 高 The proportion of cells increased to approximately 84% ( Figure 13 B). After incubating the separated samples with an enrichment reagent containing PEG, CD8 高 The proportion of cells can be further increased to approximately 92%. Figure 13 C).

[0202] such as dashed lines (IC) 50 As shown, the appropriate concentration of the first enrichment reagent can be determined by the determination described in Example 7. Figure 13 D).

[0203] As the concentration of the enrichment reagent increases, the average CD8+... 高 Cell purity generally increases, but increasingly higher concentrations of enrichment reagents have a greater impact on the average CD8+ cell count. 高 Cell recovery rate typically has an increasingly unfavorable impact. Figure 13 E).

[0204] Example 14: CD138 高 Cells can be preferentially enriched from spleen cell samples of juvenile mice.

[0205] Spleens were removed from juvenile C57BL / 6 mice and mechanically separated into 50 mL tubes using a 70 μm nylon mesh cell filter. Cells were then stored in EasySep at 2–8 °C. TM Preparing the lysed spleen suspension to 50 mL before centrifugation at 300 x g for 10 minutes (low braking setting) using buffer (STEMCELL Part ID#20144), remove the supernatant and resuspend the cell pellet in EasySep.TM In buffer solution. In CD138 高 Prior to the enrichment process (essentially as described in Example 5), the single-cell suspension was stored on ice.

[0206] Flow cytometry was performed on the isolated spleen cell population as described in Example 2, using APC-conjugated anti-mouse CD267 (TACI) antibody and PE-conjugated anti-mouse CD138 antibody. The starting cell population contained less than 1% CD138. 高 cell( Figure 14 A). Separation of CD138 using an anti-CD138 bispecific complex (as described in Example 4) in immunomagnetic separation. + After cells, CD138 高 The proportion of cells increased to approximately 65% ​​( Figure 14 B). After incubating the isolated cells in a PEG-containing enrichment reagent, CD138 高 The proportion of cells can be further increased to approximately 76%. Figure 14 C).

[0207] such as dashed lines (IC) 50 As shown in Example 7, the appropriate concentrations of the first PEG-containing enrichment reagent and the second dextran-containing enrichment reagent can be determined by the determination method described in Example 7. Figure 14 D).

[0208] Firstly, the enrichment reagent increased the average CD138. 高 Cell purity, but higher concentrations of enrichment reagents are not conducive to the average CD138. 高 Cell recovery rate ( Figure 14 E).

[0209] The second enrichment reagent increased the average CD138. 高 Cell purity, and the enrichment reagent with gradually increasing concentrations, appeared to affect the average CD138. 高 Cell recovery rate was not affected. Figure 14 F).

[0210] Example 15: CD138 高 Cells can be preferentially enriched from bone marrow samples of juvenile mice.

[0211] Femurs and tibias were removed from juvenile C57BL / 6 mice, mechanically separated using a mortar and pestle, and then passed through a 70 μm nylon mesh cell filter into 50 mL tubes. The cells were then stored in EasySep at 2–8 °C. TM Preparing the bone marrow cell suspension to 50 mL with buffer (STEMCELL PartID#20144) before centrifugation at 300 x g for 10 minutes (low braking setting), remove the supernatant and resuspend the cell pellet in EasySep. TMIn buffer solution. In CD138 高 Prior to the enrichment process (essentially as described in Example 5), the single-cell suspension was stored on ice.

[0212] Flow cytometry was performed on the isolated bone marrow cell population using APC-conjugated anti-mouse CD267 (TACI) antibody and PE-conjugated anti-mouse CD138 antibody, as described in Example 2. The starting cell population contained less than 1% CD138. 高 cell( Figure 15 A). Separation of CD138 using an anti-CD138 bispecific complex (as described in Example 4) in immunomagnetic separation. + After cells, CD138 高 The proportion of cells increased to approximately 30%. Figure 15 B). After incubation with a PEG-containing enrichment reagent to isolate cells, CD138 高 The proportion of cells can be further increased to approximately 75%. Figure 15 C).

[0213] The first enrichment reagent containing PEG increased the average CD138. 高 Cell purity, but only the highest concentration of enrichment reagent is not conducive to the average CD138. 高 Cell recovery rate ( Figure 15 D).

[0214] The second enrichment reagent containing dextran increased the average CD138. 高 Cell purity, and the enrichment reagent with gradually increasing concentrations, appeared to affect the average CD138. 高 Cell recovery rate was not affected. Figure 15 E).

[0215] While this disclosure has been described with reference to examples which are now considered preferred, it should be understood that this disclosure is not limited to the disclosed examples. Rather, this disclosure is intended to cover various modifications and equivalents included within the spirit and scope of the appended claims.

[0216] All publications, patents and patent applications are incorporated herein by reference in their entirety, to the same extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated herein by reference in its entirety.

Claims

1. A method for enriching a first cell population that is positive for a target region from a sample comprising a first cell population and a second cell population that are positive for a target region, the method comprising: a) Labeling a first and second population with polymer-containing particles to form a cell:particle complex, wherein labeling of the first and second cell populations with particles is mediated by an antibody or antibody fragment, and wherein the antibody or antibody fragment comprises a particle-specific member and a target-specific member; b) Contacting the cell:particle complex with an enrichment agent to substantially and preferentially remove the particle markers from the first cell population, while the second cell population remains substantially within the cell:particle complex, wherein the enrichment agent comprises the polymer; and c) Separate the first population from the sample, such that the first population is separated from the second population. The target portion in the first cell population is the same as the target portion in the second cell population, and the level of the target portion in the first cell population is lower than the level of the target portion in the second cell population. The concentration of the polymer in the enrichment reagent does not exceed 0.1% w / v.

2. The method of claim 1, further comprising: d) Contact the residual cell:particle complex in the sample with a separation reagent to substantially separate the second population from the particles, wherein the separation reagent contains the polymer and the concentration of the polymer in the enrichment reagent is relatively low compared to the separation reagent.

3. The method of claim 2, further comprising: e) Separate the second population from the sample.

4. The method of claim 1, wherein the target portion is a cell surface marker.

5. The method of claim 4, wherein the cell surface marker is human CD271, human CD25, human CD49d, mouse CD138, human CD8, or human CD56.

6. The method of claim 1, wherein the particles respond to a magnetic field.

7. The method of claim 1, further comprising separating the cell:granule complex from the sample after step a) and before step c).

8. The method of any one of claims 1 to 7, further comprising providing at least a saturated amount of particles relative to the target portion of the first group and the second group.

9. The method of claim 1, wherein the particle-specific member is directly or indirectly connected to the target-specific member.

10. The method of claim 1, wherein the particle-specific member and the target-specific member form a bispecific complex.

11. The method of claim 1 or 2, wherein the polymer is PEG (polyethylene glycol), a PEG-based polymer, or a PEG-like polymer.

12. The method of claim 1 or 2, wherein the polymer is dextran, a dextran-based polymer, or a dextran-like polymer.

13. An assay for identifying different cell populations positive for a target region in a sample, the assay comprising: a) Labeling the target portion with particles containing a polymer to form a cell:particle complex, wherein labeling of the target portion of a first cell population and a second cell population with particles is mediated by an antibody or antibody fragment, and wherein the antibody or antibody fragment contains particle-specific members and target-specific members. b) Obtain cell:granule complex readings by flow cytometry, wherein the cell:granule complex readings for the first cell population differ from those for the second cell population. c) Contacting the cell:particle complex with an enrichment reagent, wherein the enrichment reagent comprises the polymer, and wherein the concentration of the polymer in the enrichment reagent does not exceed 0.1% w / v; and d) Reacquire readings by flow cytometry to assess the shift in readings. The target portion in the first cell population is the same as the target portion in the second cell population, and the level of the target portion in the first cell population is lower than the level of the target portion in the second cell population.

14. The assay as described in claim 13, wherein the readings of both the first group of cells:granule complex and the second group of cells:granule complex are different from the readings of the uncomplexed target portion positive cells.

15. The measurement as claimed in claim 13, wherein the reading is a lateral scattering distribution.

16. The assay as described in any one of claims 13 to 15, wherein the target portion is a cell surface marker.

17. The assay as described in claim 16, wherein the cell surface marker is human CD271, human CD25, human CD49d, mouse CD138, human CD8, or human CD56.

18. The determination as claimed in claim 13, wherein the particles respond to a magnetic field.

19. The determination as claimed in claim 13 further includes providing at least a saturated amount of particles relative to the level of the target portion of the first population and the second population.

20. The determination as described in claim 13, wherein the particle-specific member is directly or indirectly connected to the target-specific member.

21. The determination as described in claim 13, wherein the particle-specific member and the target-specific member form a bispecific complex.

22. The determination as claimed in claim 15 further includes gating based on the lateral scattering distribution on the target portion.

23. The assay of claim 13 further includes separating the cell:granule complex from the sample before acquiring the reading.

24. The assay as described in claim 13, used to measure the dose-responsiveness of the cell:particle complex to the enrichment reagent.

25. A kit for enriching a first cell population and / or a second cell population that are positive for a target moiety from a sample, said kit comprising: a) A tube containing polymer-coated particles; b) A tube containing an antibody composition, wherein the antibody composition comprises a particle-specific member linked to a target-specific member; c) Tubes containing enrichment reagents; and d) Tubes containing separating reagents, The target portion in the first cell population is the same as the target portion in the second cell population, and the level of the target portion in the first cell population is lower than the level of the target portion in the second cell population; and The concentration of PEG or dextran in the enrichment reagent is relatively lower than that in the separation reagent, and the concentration of PEG or dextran in the enrichment reagent does not exceed 0.1% w / v.

Images