Method for removing undifferentiated cells

Abstract

The present invention relates to methods and devices for size-based isolation of undifferentiated cells from a population of cells using curvilinear microfluidic channels. The curvilinear microfluidic channel may be a spiral microfluidic channel. The differentiated cells are spinal progenitor cells (SCPCs), neural progenitor cells (NPCs), and / or cells differentiated therefrom. The method may include flowing an input cell population through first and second curvilinear microfluidic channels connected in sequence and in fluid communication, where the first channel is coupled to an inlet and the second channel is coupled to one or more outlets, where the first curvilinear microfluidic channel has a cross-sectional area smaller than a cross-sectional area of the second curvilinear microfluidic channel.

Description

Technical Field

[0001] The present invention generally relates to methods for cell separation. Specifically, the present invention relates to methods and devices for separating undifferentiated cells from a cell population based on size using curvilinear microfluidic channels. Background Art

[0002] Pluripotent stem cells, such as embryonic stem cells (ESCs), have broad application prospects in cell therapy because they can differentiate into many different lineages according to clinical needs. The ability to convert a patient's somatic cells into induced pluripotent stem cells (iPSCs) to generate different cell types not only solves the problem of immune rejection in cell therapy, but also circumvents the ethical issues associated with the use of ESCs. However, the presence of residual iPSCs in differentiated cells poses a clinical risk because these cells may form teratomas and other forms of tumors after transplantation. The potential tumorigenicity of residual iPSCs depends on the number of such cells in the final cell population and the nature of the differentiated cell lineage, and therefore varies depending on the cell manufacturing protocol and clinical application.

[0003] Methods for removing residual iPSCs or ESCs have been reported. These methods include the use of cytotoxic antibodies and small molecule drugs targeting stem cells, fluorescence-activated cell sorting (FACS) using fluorescently spiked antibodies that recognize stem cell surface markers, and magnetic-activated cell sorting (MACS) using magnetically coupled antibodies. In these methods, the added small molecules or fluorescent or magnetic markers may exist as contaminants and subsequently interfere with cell proliferation and behavior. In addition, cell viability may be reduced due to the high in-line pressure used during the sorting process. These methods also rely heavily on specific surface markers, whose expression may vary depending on a variety of factors such as the culture medium used and the differentiation protocol. In addition, these methods are inefficient and costly to implement when performing cell manufacturing on an industrial scale. Therefore, there is a need for a label-free, efficient and scalable method for separating undifferentiated cells from cell mixtures during cell manufacturing.

[0004] It would be desirable in the art to overcome or ameliorate at least one of the above problems, or at least provide a useful alternative. Summary of the Invention

[0005] Disclosed herein is a method for removing undifferentiated cells from a population of differentiated cells and undifferentiated cells, wherein the method comprises: a) providing an input population of differentiated cells and undifferentiated cells to an inlet of a microfluidic device; b) flowing the input cell population through at least one curvilinear microfluidic channel coupled to the inlet, wherein the curvilinear microfluidic channel is configured to separate undifferentiated cells from the input cell population based on size; and c) collecting a first output cell population through one or more outlets of the curvilinear microfluidic channel coupled to the microfluidic device, wherein the first output cell population has a lower proportion of undifferentiated cells than the input cell population.

[0006] Also disclosed herein is a method for purifying differentiated cells from differentiated cells and undifferentiated cell populations, wherein the method comprises: a) providing differentiated cell and undifferentiated cell input populations to an inlet of a microfluidic device; b) flowing the cell input population through at least one curved microfluidic channel connected to the inlet, wherein the curved microfluidic channel is configured to separate differentiated cells from the cell input population based on size; and c) collecting a first cell output population through one or more outlets of the curved microfluidic channel connected to the microfluidic device, wherein the first cell output population has a higher proportion of differentiated cells than the cell input population.

[0007] The present invention also provides a device for removing undifferentiated cells from a population of differentiated cells and undifferentiated cells, wherein the device comprises: a) an inlet for receiving an input population of differentiated and undifferentiated cells; b) at least one curved microfluidic channel connected to the inlet, wherein the curved microfluidic channel is configured to separate undifferentiated cells from the cell input population based on size; and c) one or more outlets connected to the curved microfluidic channel, which are used to collect a cell output population, wherein the proportion of undifferentiated cells in the cell output population is lower than that in the cell input population.

[0008] Disclosed herein is a device for purifying differentiated cells from differentiated and undifferentiated cell populations, wherein the device comprises: a) an inlet for receiving an input population of differentiated and undifferentiated cells; b) at least one curved microfluidic channel connected to the inlet, wherein the curved microfluidic channel is configured to separate differentiated cells from the input cell population based on size; and c) one or more outlets connected to the curved microfluidic channel for collecting a cell output population, wherein the proportion of undifferentiated cells in the cell output population is higher than that in the cell input population. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Embodiments of the invention are described below by way of non-limiting examples with reference to the accompanying drawings, in which:

[0010] Figure 1Schematic diagram of the spiral sorting device. The spiral sorting device contains two spiral channels: (1) directly connected to the inlet, used to concentrate cells on the inner wall (IW); (2) directly leading to the outlet, used for cell size separation, where larger cells are close to the inner wall (IW) and smaller cells are close to the outer wall (OW).

[0011] Figure 2 Figure 3: Average size of unsorted and sorted populations containing spinal cord progenitor cells (SCPCs) differentiated from induced pluripotent stem cells (iPSCs). (A and B) The average cell size of the "small cell" and "large cell" output populations differed significantly, and both populations also showed differences in average cell size compared to the unsorted population. (C) Cell viability of SCPCs in different size groups before and after cell transplantation. No significant differences were observed between the groups.

[0012] Figure 3 : In vivo cell viability of SCPC injected into the rat spinal cord: SCPC (unsorted, sorted-small, sorted-large, or mixed) were injected into the T9-T10 level of the rat spinal cord. Four weeks after implantation, higher cell viability was observed in the unsorted and sorted-small groups compared to the sorted-large or mixed groups. The cell viability of the sorted-large cells was almost negligible. The mixed group (prepared by mixing sorted-small and sorted-large cells at a 50:50 ratio) showed a 50% decrease in cell viability compared to the unsorted or sorted-small groups.

[0013] Figure 4 Four weeks after injection, all injected cells failed to express the pluripotency marker OCT4, and only a small percentage of cells stained positive for Ki-67 and the neural progenitor cell marker SOX1. There were no significant differences between the unsorted and sorted groups.

[0014] Figure 5 : Four weeks after injection, transplanted cells showed neural stem cell markers (HuNu + / SOX2 + ) and maintain their spinal cord lineage (hNCAM + / HOXB4 + There was no significant difference between the unsorted group and the sorted group.

[0015] Figure 6 : (A) Four weeks after in vivo transplantation into intact rat spinal cord T9-10 segments, sorted-small cells showed a greater or equivalent degree of neuronal differentiation than unsorted cells. + / NeuN +(Indicating neuronal differentiation) cells. NeuN expression levels were similar in Experiment 2. (B) Further confirming that 4 weeks after in vivo transplantation into the intact rat spinal cord T9-10 segment, the sorted-small cells showed a greater or equivalent degree of neuronal differentiation than the unsorted cells. More hNCAM was observed in Experiment 1 + / NF + (indicating neuronal differentiation) cells, with a similar degree of NF expression as in Experiment 2.

[0016] Figure 7 :A small number of injected cells differentiate into astrocytes expressing hNCAM after 4 weeks in vivo + / GFAP + There was no significant difference between the unsorted group and the sorted group. DETAILED DESCRIPTION

[0017] The present disclosure teaches a method for removing undifferentiated cells from an input cell population using a curvilinear microfluidic channel. The input cell population can include differentiated cells and undifferentiated cells. The method can include: a) providing the input cell population to an inlet of a microfluidic device; b) flowing the input cell population through at least one curvilinear microfluidic channel coupled to the inlet, wherein the curvilinear microfluidic channel is configured to separate undifferentiated cells from the input cell population based on size; and c) collecting a first output cell population through one or more outlets of the curvilinear microfluidic channel coupled to the microfluidic device, wherein the first output cell population has a lower proportion of undifferentiated cells than the input cell population.

[0018] The present disclosure also teaches a method for purifying differentiated cells from a population of differentiated cells and undifferentiated cells, wherein the method comprises: a) providing an input population of differentiated cells and undifferentiated cells to an inlet of a microfluidic device; b) flowing the input cell population through at least one curvilinear microfluidic channel coupled to the inlet, wherein the curvilinear microfluidic channel is configured to separate differentiated cells from the input cell population based on size; and c) collecting a first output cell population through one or more outlets of the curvilinear microfluidic channel coupled to the microfluidic device, wherein the first output cell population has a higher proportion of differentiated cells than the input cell population.

[0019] Without being bound by theory, the inventors have developed a microfluidic size-based method for separating undifferentiated cells from a cell mixture. The method is based on the unexpected discovery that the size of undifferentiated cells is significantly different from that of differentiated cells, and can be separated using inertial focusing within a curved microfluidic channel. For example, the inventors found that undifferentiated multipotent cells can be larger than the progenitor cells (e.g., neural progenitor cells) from which they differentiate. The size difference can vary depending on the type and differentiation state of the differentiated and undifferentiated cells. However, the presence of a size difference generally allows for passive cell separation using inertial microfluidics.

[0020] Unlike conventional methods of cell separation such as FACS and MACS, the microfluidic method disclosed herein enables label-free, contactless, and high-throughput removal of undifferentiated cells from both differentiated and undifferentiated cell populations while minimizing the impact on cell viability and cell function. Another advantage of microfluidic-based cell separation is that, through careful selection of channel geometry and fluid parameters, it is suitable for resolving a wide range of size differences between various types of differentiated and undifferentiated cells. The microfluidic method disclosed herein can be automated and integrated into cell manufacturing processes to remove residual undifferentiated cells, thereby reducing the risk of tumor formation after cell implantation.

[0021] The method involves using a microfluidic device having at least one curvilinear microfluidic channel configured to separate undifferentiated or differentiated cells from a mixed cell population based on size. The separation is based on inertial concentration generated by the curved channel geometry, which causes cells of different sizes to locate and aggregate at equilibrium positions along the channel.

[0022] As used herein, the term "microfluidics" refers to a system or device having one or more fluid channels, conduits, or chambers that are typically fabricated on the millimeter to micrometer scale, e.g., typically having at least one cross-sectional size in the range of about 1 μm to about 1 mm.

[0023] As used herein, the term "channel" refers to a structure in which a fluid can flow. A channel can be a capillary tube, a conduit, a hydrophilic patterned stripe on a hydrophobic surface that restricts the flow of an aqueous fluid, or the like.

[0024] As used herein, "curvilinear microchannel" refers to a microchannel whose longitudinal axis along the direction of flow in the microchannel deviates from a straight line, for example, a spiral or sinusoidal channel.

[0025] It will be understood by those skilled in the art that the channels can be arranged in a variety of shapes (e.g., spiral, multi-loop, serpentine, including a mixture of curved and linear sections) and can have various cross-sectional shapes (e.g., rectangular, trapezoidal), so long as the dimensions of the channels are suitable for separating cells based on cell size along at least a portion of the cross-section of at least one curvilinear microchannel.

[0026] Without being bound by theory, when a fluid flows through a curved channel, it is affected by centrifugal force and inertia that push the fluid toward the outer wall of the curved channel. This forms a secondary flow pattern called Dean flow in the channel, in which the fluid moves radially. Cells suspended in the fluid are affected by these secondary flows and migrate laterally. The result is that the cells are pushed away from the channel wall and, depending on the cell size and flow conditions, eventually reach an equilibrium position along the channel cross section and gather in these areas in the channel. This cell aggregation and arrangement away from the channel wall contributes to cell separation based on size.

[0027] Without being bound by theory, when a fluid flows through a curved microchannel, especially a microchannel with a spiral shape or profile, the laminar Poiseuille flow is affected by centrifugal forces. The centrifugal force disrupts the parabolic fluid velocity distribution of the laminar flow, and the maximum fluid velocity position is offset from the center of the microchannel cross section to the outside of the microchannel, resulting in a sharp velocity gradient between the maximum fluid velocity position and the outside. The sharp velocity gradient increases the fluid pressure, and the local fluid velocity near the outside is not enough to balance this pressure difference. This imbalance is called Dean instability and causes the fluid to recirculate in the upper and lower halves of the microchannel in the form of two counter-rotating Dean vortexes. Each Dean vortex forms a cycle between the center and the outside of the cross section of the microchannel to balance the pressure difference. The Dean vortex or secondary flow is defined by a dimensionless parameter called the Dean number De, which represents the Dean force generated by the secondary flow in the microchannel. The Dean number is defined as De=Re(D h / 2r) 1 / 2 , where Re is the flow Reynolds number, D h is the hydraulic diameter of the microchannel (i.e., the minimum size of the microchannel, usually the cross-sectional height of the microchannel), r represents the average curvature radius of the channel, δ = D h / 2r is the curvature ratio. Hydraulic diameter D h For circular channels it is the cross-sectional diameter, while for rectangular channels it is D h =2wh / (w+h) (where w and h correspond to the width and height of the cross section, respectively).

[0028] Because lateral Dean vortex or secondary flow entrains cells along the flow direction of Dean vortex, the cells flowing in the curved microchannel are affected by Dean drag force. Therefore, when the fluid flows along the microchannel, the cells move repeatedly between the inside and outside sides of the microchannel. The speed of lateral migration of cells when flowing in the microchannel depends on the Dean number De. The lateral distance crossed by the cell can be defined by "Dean cycle". For example, if a cell is initially located near the inner wall of a curved microchannel and migrates to the outer wall of the microchannel at a certain distance downstream, it is considered that it has completed half a Dean cycle. If the cell returns to its original position near the inner wall of the microchannel further downstream, a Dean cycle is completed. Therefore, depending on the length of the microchannel, a cell can undergo one or more Dean cycles or partial Dean cycles.

[0029] In addition to Dean drag, cells may also be subject to inertial lift, which can be shear-induced due to the parabolic fluid velocity profile and / or wall-induced due to the interaction of cells with the sides or walls of the microchannel. Therefore, cells flowing in a microchannel are subject to a combination of inertial lift and Dean drag. As cell size increases, lift and Dean drag increase, but with varying magnitudes. L ), when close to the channel center, F L ∝a 4 ; When close to the channel wall, F L ∝a 6 , where the wall effect is dominant. As for the Dean resistance (F D ), F D ∝a. Therefore, larger cells are more affected by inertial lift, while smaller cells are more affected by Dean drag. Since the ratio of inertial lift to Dean drag varies for different particle sizes, cells can be balanced at different locations along the microchannel cross-section based on cell size. Specifically, the effect of Dean drag is greater than the inertial lift for smaller cells, and under its influence, smaller fluid particles begin to migrate along the Dean vortex and reach equilibrium near the outer wall of the curved microchannel. For larger cells, the effect of inertial lift is greater than the Dean drag, which prevents them from migrating sideways and causes them to reach equilibrium near the inner wall of the curved microchannel. In this way, cells of different sizes eventually reach equilibrium positions along the cross-section of the channel and aggregate in these areas within the channel. This aggregation and arrangement of cells facilitates size-based cell separation.

[0030] The confinement ratio is defined as CR = a / D h, where a is the particle diameter, is a key parameter to consider when performing size-based cell separation in curved microchannels. Typically, to allow cells to concentrate and balance at different locations along the microchannel cross-section, the CR should be at least 0.07 (CR ≥ 0.07).

[0031] The flow of a cell population through a curved microfluidic channel typically results in larger cells concentrating toward the inner channel wall (i.e., the radially inner wall), while smaller cells concentrating toward the opposite outer channel wall (i.e., the radially outer wall). Cells of different sizes can then be collected through channel outlets appropriately positioned along the channel cross-section.

[0032] Differentiated and undifferentiated cell input populations

[0033] In one embodiment, the cell input group comprises a suspension cell mixture. " Suspension cells " used herein refers to cells in a state of not closely adhering to the wall of a container (e.g., chamber) for its growth or maintenance. In the case where a cell type grows as an adherent cell, various reagents (such as trypsin) can be used to process it to provide suspension cells. When in suspension, most of the cells present can be introduced into a microfluidic device to separate according to size. In some embodiments, most or more than about 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% of the suspension cells exist as single cells, and preferably higher percentages.

[0034] The differentiated cells and undifferentiated cells in the input population can be derived from humans or any non-human animal. In some embodiments, the differentiated cells and undifferentiated cells are mammalian cells. In one embodiment, the differentiated cells and undifferentiated cells are human cells. The human cells can be cell lines or primary cells derived from the patient. In one embodiment, the differentiated cells include cells differentiated from undifferentiated cells in the cell input population.

[0035] As used herein, the term "undifferentiated cell" refers to a cell that lacks the specific morphological and functional characteristics that normal cells acquire during development (a process known in the art as "differentiation"). The differentiation of cells during development produces different tissues and cell types characterized by different sizes, shapes, metabolic activities, lineage or cell type specific markers and / or responsiveness to signals. Undifferentiated cells can be totipotent, pluripotent, multipotent, or unipotent. Undifferentiated cells can be part of a lineage that has never undergone differentiation (e.g., embryonic stem cells), or derived from differentiated cells by a process that eliminates the specific morphological and functional characteristics of differentiated cells (e.g., induced pluripotent stem cells).

[0036] In some embodiments, the undifferentiated cells are stem cells. As used herein, the term "stem cell" refers to a cell that has the ability to self-renew (i.e., generate identical copies of itself) and differentiate into one or more different cell lineages or cell types. Stem cells can be isolated from early developmental embryos, embryos, or adult tissues, or derived from differentiated cells. The stem cells herein can be totipotent stem cells (which can differentiate into cells of all three germ layers and extraembryonic tissues such as the placenta); pluripotent stem cells (which can differentiate into cells of all three germ layers); multipotent stem cells (which can differentiate into a subpopulation of cell types within a specific organ or tissue); oligopotent stem cells (which can differentiate into several related cell types); or unipotent stem cells (which can only differentiate into one specific cell type).

[0037] In some embodiments, the undifferentiated cells are pluripotent stem cells. As used herein, the terms "pluripotent cells" and "pluripotent stem cells" are used interchangeably and refer to stem cells that have the potential to differentiate into any one of the three germ layers (endoderm, mesoderm, or ectoderm). Examples include, but are not limited to, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Pluripotent stem cells can be identified by cell-specific protein markers such as OCT4, SOX2, and NANOG.

[0038] In some embodiments, the undifferentiated cells are induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs).

[0039] Human embryonic stem cells (hES) can be prepared from human blastocyst cells using techniques well known in the art. Induced pluripotent stem cells (iPSCs) can be generated from any vertebrate species, including humans and non-human primates, livestock, livestock, and other non-human mammals. iPSCs can be generated from any cell type, such as fibroblasts, tumor cells, bone marrow cells, gastric cells, hepatocytes, epithelial cells, follicular cells, connective tissue cells, muscle cells, bone cells, chondrocytes, gastrointestinal cells, spleen cells, kidney cells, lung cells, testicular cells, neural tissue cells, and blood cells. Such cells can be dedifferentiated by exposure to a combination of transcription factors, for example, such as OCT4, SOX2, KL4, and optionally, cMyc, NANOG, and / or LIN28. Exposure to transcription factors can be achieved by, for example, viral or non-viral application or transduction methods.

[0040] Induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) can be identified by suitable methods known in the art. For example, cells can be characterized by their ability to support teratoma formation in animal models as confirmation of pluripotency. Alternatively or additionally, cells can be identified by gene and / or DNA methylation profiles, or by expression of cell type-specific markers.

[0041] In some embodiments, the undifferentiated cells are neural stem cells. As used herein, "neural stem cells (NSC)" refer to cells that can generate nervous system cell types. NSCs can be characterized by the presence of the markers Nestin, SOX2, and CD133.

[0042] In some embodiments, the undifferentiated cells are mesenchymal stem cells. As used herein, "mesenchymal stem cells (MSCs)" refer to multipotent stem cells that can be isolated from various tissues and differentiated into various cell types, including osteoblasts (bone cells), chondrocytes (cartilage tissue cells), and adipocytes (fat tissue cells). MSCs are characterized by a variety of surface markers, including the expression of markers in the following list: CD29, CD44, CD73, CD105, CD106, CD166, and STRO-1.

[0043] In some embodiments, undifferentiated cells are hematopoietic stem cells. As used herein, "hematopoietic stem cell (HSC)" refers to such multipotent stem cells that can generate all types of blood cells, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (T cells, B cells, NK cells). People's HSC is described as CD34+CD133+Lin- cells.

[0044] The undifferentiated cells described herein (e.g., ESCs or iPSCs) can be differentiated into any cell type of interest, including endoderm (generating endoderm cells, which in turn give rise to internal tissues and organs, such as the digestive tract, intestines, digestive glands, respiratory system, and intestine / bladder), ectoderm (generating ectoderm cells, which in turn give rise to the nervous system, skin, and other outermost specialized tissues and organs), mesoderm (generating mesoderm cells; mesoderm is the middle germ layer of the embryo derived from the inner cell mass of the blastocyst; it gives rise to bone, muscle, connective tissue (including the dermis), vascular system, urogenital system (except bladder), and helps form some glands), neuroectoderm (generating any cell of the neuroectoderm, which in turn gives rise to neurons, supporting cells, ependyma of the central nervous system, and neural crest cells that form peripheral ganglia and a variety of other tissues), neural (generating any cell of the peripheral and central nervous systems; the autonomic and somatic nervous systems, including all neurons, supporting cells / glial cells, etc.).

[0045] In some embodiments, the undifferentiated cells differentiate into a cell population, such as a cobblestone-like cell line, a cardiomyocyte population, an epithelial cell population (such as a keratin-containing or intestinal epithelial cell population), a gastrointestinal cell population, a respiratory cell population, a hepatic cell population, a pancreatic cell population, an endocrine cell population, an epidermal cell population, a myogenic cell population, a chondrocyte population, a mucosal cell population, a skeletal cell population, a cartilage tissue cell population, a kidney cell population, a lymphocyte population, a spleen cell population, a neural cell population, a hematopoietic cell population, or a precursor cell of any of the foregoing.

[0046] As used herein, "differentiated cells" refers to cells that are at a later stage of differentiation than the undifferentiated cells in the cell input population. In preferred embodiments, the differentiated cells lack pluripotency, for example, these cells do not express one or more protein markers specific for pluripotent cells, such as OCT4, SOX2 and / or NANOG. The differentiated cells can be cells from any of the endoderm, mesoderm, or ectoderm lineages. For the avoidance of doubt, the differentiated cells in the present disclosure are capable of further differentiation into one or more lineages, or one or more cell types.

[0047] Cells of the endoderm, mesoderm, and ectoderm lineages are well known in the art and can be readily identified by those skilled in the art by, for example, the presence of cell-specific morphological or functional characteristics, or by the expression of lineage-specific markers. As non-limiting examples, endoderm cell markers may include SOX17, GATA4, FOXA2, CK8, CK18, CDX2, TTF-1, and HNF4α; mesodermal cell markers may include Brachyury, Nkx2.5, desmin, CD34, vimentin, MyoD, and the like; and ectoderm cell markers may include PAX6, nestin, NCAM, KRT10, KRT14, GFAP, and the like.

[0048] In one embodiment, the differentiated cells are cells of the endoderm lineage. Cells of the endoderm lineage include, but are not limited to, gastrointestinal epithelial cells, respiratory epithelial cells (e.g., alveolar cells and bronchial lining cells), thyroid and parathyroid cells, thymus and tonsil cells, hepatocytes (e.g., liver cells), pancreatic cells (e.g., beta cells), bladder and urethra lining cells, and the above-mentioned progenitor cells.

[0049] In one embodiment, the differentiated cell is a cell of mesodermal pedigree. The cell of mesodermal pedigree includes but is not limited to hematopoietic stem cell, mesenchymal stem cell, blood cell (for example, myeloid and lymphoid cells), vascular endothelial cell, dermal cell, muscle cell (for example, skeletal muscle cell, smooth muscle cell and cardiomyocyte), osteocyte (for example, osteoblast), chondrocyte (for example, cartilage tissue cell), connective tissue cell (for example, fibroblast, adipocyte etc.), male and female reproductive system cell, kidney cell (for example, renal epithelial cell), spleen cell and above-mentioned progenitor cell.

[0050] In one embodiment, the differentiated cell is a cell of the ectodermal lineage. Cells of the ectodermal lineage include, but are not limited to, neural stem cells, cells of the nervous system (e.g., neurons, glial cells), epidermal cells (e.g., keratinocytes), oral cells (e.g., buccal mucosal cells lining the mouth, nostrils, and anus), melanocytes, glandular cells of the mammary gland, sweat glands, and pineal gland, adrenal medullary cells, cells of the visual, auditory, and olfactory organs, and the progenitor cells described above.

[0051] In some embodiments, the differentiated cells are pluripotent cells. In some embodiments, the differentiated cells are oligopotent cells. In some embodiments, the differentiated cells are unipotent cells. In some embodiments, the differentiated cells are terminally differentiated cells. In some embodiments, the differentiated cells can be passaged in culture without an observable replication crisis, and passage times in cell culture can be up to and including several days, weeks, months, and years. In some embodiments, the differentiated cells cannot be passaged in culture without an observable replication crisis. In various cases, one of ordinary skill in the art can easily assess the viability and lineage potential of the derived cell population using methods known in the art.

[0052] Typically, it can be identified based on whether a specific stem cell or progenitor cell expresses one or more markers associated therewith. For example, markers for human embryonic stem cells (ESC) include, but are not limited to, OCT4, NANOG, TRA-1-81, SSEA-4, and SSEA-3. Markers for neural stem cells (NSC) and neural progenitor cells (NPC) include, but are not limited to, nestin, GFAP, Musashi-1, Sox1, Sox2, Pax6, and CD133. In addition, embryonic stem cells (ESC) that maintain an undifferentiated state typically do not express or express relatively low levels of differentiation indicator markers, such as Brachyury, Sox17, FoxA2, Pax6, Otx2, and Sox1. Markers such as SSEA-4, OCT4, Brachyury, and Sox17, as well as other markers indicating differentiation and undifferentiated states, are known in the art and are routinely used.

[0053] In one embodiment, the differentiated cells are spinal cord progenitor cells (SCPC), neural progenitor cells (NPC) and / or cells differentiated therefrom. NPC is distributed throughout the entire nervous system, including the brain and spinal cord, and has the potential to differentiate into various neural cell types including neurons and glial cells. NPC can be identified by cell markers including NF200, Sox1, NCAM, GFAP, etc. SCPC is a subpopulation of NPC located in the spinal cord, and its differentiation potential is more limited than NPC, mainly generating cell types present in the spinal cord, such as motor neurons, interneurons and glial cells that support spinal cord function. In addition, SCPC can also be identified by markers Olig2 and Nkx6.1, Nkx6.2 and HOX genes (e.g., HoxB4). For example, cells differentiated from SCPC or NPC include neurons and glial cells (e.g., astrocytes, oligodendrocytes, ependymal cells, microglia, Schwann cells, satellite cells, etc.).

[0054] In one embodiment, the differentiated cells are T cells, NK cells, or NKT cells. These cells can be differentiated from pluripotent cells or hematopoietic stem cells. T cells express T cell receptors (TCRs) and CD3, CD4, or CD8 on their cell surfaces. Markers including CD56 and CD16 can be used to identify and distinguish NK cells from other lymphocytes. NKT cells express TCRs and NK cell markers.

[0055] The size of differentiated cells and undifferentiated cells may vary depending on cell type, cell differentiation stage, and cell source. In some embodiments, the size of differentiated cells and undifferentiated cells in the input population is determined before microfluidic separation. Size can be used to adjust channel geometry and / or operating fluid parameters to improve the efficiency and / or resolution of cell sorting. The size ranges of various types of differentiated cells and undifferentiated cells are known in the art. Table 1 provides the size ranges of some cells. With regard to the methods herein, pluripotent stem cells (e.g., neural stem cells and mesenchymal stem cells) can be undifferentiated cells (e.g., if used to derive differentiated progeny) or differentiated cells (e.g., if differentiated from pluripotent cells). For example, those skilled in the art can also determine the size of differentiated cells and undifferentiated cells by measuring the cell size of pure or substantially pure differentiated cells or undifferentiated cell populations. Such cell populations can be separated and identified by methods known in the art, such as by immunolabeling of cell-specific markers.

[0056] In another embodiment, the average cell size of the cell input group is determined before microfluid separation. Size is used to adjust the first round of channel and microfluid parameters based on the separation of size. In the first round of separation, the mixed group of differentiated cells and undifferentiated cells is sorted into cell subpopulations with a limited size range. These subpopulations may comprise differentiated cells and undifferentiated cells in different proportions. Then determine the cell type in different subpopulations (for example, by morphology, functional assays and / or immune or other types of cell staining), and measure the size of different cell types. Then subpopulations are carried out second or subsequent rounds of sorting based on size, and operating parameters are adjusted based on the cell size measured. This embodiment can repeat cell separation, and there is no need to predetermine the size of differentiated cells and undifferentiated cells.

[0057] As a non-limiting example, the diameter of the undifferentiated cells may be about 7 μm to about 30 μm. The diameter of the undifferentiated cells may be about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, about 10 μm, about 10.5 μm, about 11 μm, about 11.5 μm, about 12 μm, about 12.5 μm, about 13 μm, about 13.5 μm, about 14 μm, about 14.5 μm, about 15 μm, about 15.5 μm, about 16 μm, about 16.5 μm, about 17 μm, about 17.5 μm, about 18 μm, about 19 μm, about 20 μm, about 21 μm, about 22 μm, about 23 μm, about 24 μm, about 25 μm, about 26 μm, about 27 μm, about 28 μm, about 29 μm, about 30 μm, about 31 μm, about 32 μm, about 33 μm, about 34 μm, about 35 μm, about 36 μm, about 37 μm, about 38 μm, about 39 μm, about 40 μm, about 41 μm, about 42 μm, about 43 μm, about 44 μm, about 45 μm, about 46 μm, about 47 μm, about 48 μm, about 49 μm, about 50 μm, about 51 μm, about 8.5 μm, about 19 μm, about 19.5 μm, about 20 μm, about 20.5 μm, about 21 μm, about 21.5 μm, about 22 μm, about 22.5 μm, about 23 μm, about 23.5 μm, about 24 μm, about 24.5 μm, about 25 μm, about 25.5 μm, about 26 μm, about 26.5 μm, about 27 μm, about 27.5 μm, about 28 μm, about 28.5 μm, about 29 μm, about 29.5 μm or about 30 μm.

[0058] As non-limiting examples, the diameter of the differentiated cells may be about 7 μm to about 30 μm. The diameter of the undifferentiated cells may be about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, about 10 μm, about 10.5 μm, about 11 μm, about 11.5 μm, about 12 μm, about 12.5 μm, about 13 μm, about 13.5 μm, about 14 μm, about 14.5 μm, about 15 μm, about 15.5 μm, about 16 μm, about 16.5 μm, about 17 μm, about 17.5 μm, about 18 μm, about 19 μm, about 20 μm, about 21 μm, about 22 μm, about 23 μm, about 24 μm, about 25 μm, about 26 μm, about 27 μm, about 28 μm, about 29 μm, about 30 μm, about 31 μm, about 32 μm, about 33 μm, about 34 μm, about 35 μm, about 36 μm, about 37 μm, about 38 μm, about 39 μm, about 40 μm, about 41 μm, about 42 μm, about 43 μm, about 44 μm, about 45 μm, about 46 μm, about 47 μm, about 48 μm, about 49 μm, about 50 μm, about 51 μm, about 8.5 μm, about 19 μm, about 19.5 μm, about 20 μm, about 20.5 μm, about 21 μm, about 21.5 μm, about 22 μm, about 22.5 μm, about 23 μm, about 23.5 μm, about 24 μm, about 24.5 μm, about 25 μm, about 25.5 μm, about 26 μm, about 26.5 μm, about 27 μm, about 27.5 μm, about 28 μm, about 28.5 μm, about 29 μm, about 29.5 μm or about 30 μm.

[0059] Table 1. Size range of differentiated and undifferentiated cells.

[0060]

[0061]

[0062] In some embodiments, the size difference between the undifferentiated cells and the differentiated cells is at least about 0.4 μm. For example, the size difference between the undifferentiated cells and the differentiated cells can be at least about 0.4 μm, at least about 0.6 μm, at least about 0.8 μm, at least about 1 μm, at least about 1.2 μm, at least about 1.4 μm, at least about 1.6 μm, at least about 1.8 μm, at least about 2 μm, at least about 2.2 μm, at least about 2.4 μm, at least about 2.6 μm, at least about 2.8 μm, at least about 3 μm, at least about 3.2 μm, at least about 3.4 μm, at least about 3.6 μm, at least about 3.8 μm, at least about 4 μm, at least about 4.2 μm, at least about 4.4 μm, at least about 4.6 μm, at least about 4.8 μm, at least about 5.6 μm, at least about 5.8 μm, at least about 5.9 μm, at least about 6.8 μm, at least about 7.9 μm, at least about 7.1 μm, at least about 7.2 μm, at least about 7.4 μm, at least about 7.6 μm, at least about 7.8 μm, at least about 7.9 μm, at least about 7.1 μm, at least about 7. At least about 5 μm, at least about 5.2 μm, at least about 5.4 μm, at least about 5.6 μm, at least about 5.8 μm, at least about 6 μm, at least about 6.2 μm, at least about 6.4 μm, at least about 6.6 μm, at least about 6.8 μm, at least about 7 μm, at least about 7.2 μm, at least about 7.4 μm, at least about 7.6 μm, at least about 7.8 μm, at least about 8 μm, at least about 8.2 μm, at least about 8.4 μm, at least about 8.6 μm, at least about 8.8 μm, at least about 9 μm, at least about 9.2 μm, at least about 9.4 μm, at least about 9.6 μm, at least about 9.8 μm or at least about 10 μm.

[0063] In some embodiments, the size difference between the undifferentiated cells and the differentiated cells is about 0.4 μm, about 0.6 μm, about 0.8 μm, about 1 μm, about 1.2 μm, about 1.4 μm, about 1.6 μm, about 1.8 μm, about 2 μm, about 2.2 μm, about 2.4 μm, about 2.6 μm, about 2.8 μm, about 3 μm, about 3.2 μm, about 3.4 μm, about 3.6 μm, about 3.8 μm, about 4 μm, about 4.2 μm, about 4.4 μm, about 4.6 μm, about 4.8 μm, or about 4.6 μm. In one embodiment, the size difference between the undifferentiated pluripotent cells and the differentiated SCPCs is about 4 μm.

[0064] The input cell population can be introduced or infused into the inlet using known means such as a syringe and / or a fluid pump, eg, piston pump, gear pump, peristaltic pump, piezoelectric micropump, or using a controllable pressure regulator.

[0065] In some embodiments, the methods remove at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the undifferentiated cells from the input population of cells. In some embodiments, the methods remove about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the undifferentiated cells from the input population of cells.

[0066] In some embodiments, the method purifies at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the differentiated cells from an input population of cells. In some embodiments, the method purifies about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the differentiated cells from an input population of cells.

[0067] In some embodiments, the viability of the differentiated cells in the input population of cells and the first output population of cells is comparable. For example, the method can result in a decrease in the viability of the differentiated cells by at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, or at most 1%. Cell viability can be measured by methods known in the art, such as using metabolic assays, proliferation assays, or colorimetric or fluorescent staining that differentially stains live and dead cells.

[0068] In some embodiments, the differentiation potential of the differentiated cells in the input population of cells and the first output population of cells is comparable. For example, the method can result in a decrease in the differentiation potential of the differentiated cells by at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, or at most 1%. The differentiation potential of the cells can be measured using a differentiation assay, for example, by exposing the differentiated cells to specific differentiation conditions or factors and determining the proportion of different cell types produced.

[0069] Dimensional properties of microfluidic channels

[0070] The curved channel for this paper method can have multiple curvatures. In one embodiment, the curved channel is spiral. The spiral microchannel can include one or more loops. In some aspects, each spiral microchannel can independently be 2-loop microchannel, 3-loop microchannel, 4-loop microchannel, 5-loop microchannel, 6-loop microchannel, 7-loop microchannel, 8-loop microchannel, 9-loop microchannel, 10-loop microchannel, etc. It should be understood that the spiral direction can be clockwise or counterclockwise. The entrance can be arranged on the outermost loop or the innermost loop of the spiral.

[0071] In some instances, the radius of curvature of spiral microchannel can be approximately 2.5mm to approximately 25mm. For example, the radius of curvature of spiral microchannel can be approximately 2.5mm, 3mm, approximately 4mm, approximately 5mm, approximately 6mm, approximately 7mm, approximately 8mm, approximately 9mm, approximately 10mm, approximately 11mm, approximately 12mm, approximately 13mm, approximately 14mm, approximately 15mm, approximately 16mm, approximately 17mm, approximately 18mm, approximately 19mm, approximately 20mm, approximately 21mm, approximately 22mm, approximately 23mm, approximately 24mm or approximately 25mm. In one embodiment, the radius of curvature of spiral microchannel decreases progressively to the approximately 5mm of innermost loop from the approximately 15mm of outermost loop.

[0072] The width of the curved channel for the disclosed method can be from about 100 μm to about 2000 μm, such as about 100 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, 900 μm, about 1000 μm, about 1100 μm, about 1200 μm, about 1300 μm, about 1400 μm, about 1500 μm, about 1600 μm, about 1700 μm, about 1800 μm, about 1900 μm or about 2000 μm. The width of the channel can be uniform over the entire channel length. In one embodiment, the width of the curved channel is about 800 μm.

[0073] The height of the curved channel may be about 20 μm to about 300 μm, for example, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, about 200 μm, about 210 μm, about 220 μm, about 230 μm, about 240 μm, about 250 μm, about 260 μm, about 270 μm, about 280 μm, about 290 μm, or about 300 μm.

[0074] In some embodiments, the cross-sectional height of the curved channel is uneven. For example, the channel can have a trapezoidal cross section limited by a radial inner wall, a radial outer wall, a bottom wall and a top wall, wherein the height of the radial inner wall and the radial outer wall are unequal. The height of the radial inner wall can be less than or greater than the height of the radial outer wall. The microfluidic channel preferably has a trapezoidal cross section, because the maximum velocity is asymmetric along the channel cross section, thereby forming a stronger Dean vortex core tilted to the deeper channel side. Even in the single inlet setting without sheath flow, this can also increase the separation distance between cells of different sizes. In a trapezoidal channel, as shown in WO2014 / 046621, particles are concentrated near the inner wall of the channel (similar to the channel with a rectangular cross section) at low flow rates, and when exceeding a certain threshold flow rate, particles switch to the equilibrium position located at the outer half. Other factors also can affect the concentrated position and separation efficiency, such as the width of the microchannel, the inside and outside height of the microchannel cross section, the spiral curvature radius and the inclination angle.

[0075] In one embodiment, the curved channel has a trapezoidal cross-section in which the height of the radially inner wall is less than the height of the radially outer wall. The radially inner height may be from about 20 μm to about 200 μm, such as about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, or about 200 μm. The height of the radially outer side may be about 50 μm to about 300 μm, such as about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, about 200 μm, about 210 μm, about 220 μm, about 230 μm, about 240 μm, about 250 μm, about 260 μm, about 270 μm, about 280 μm, about 290 μm or about 300 μm.

[0076] The angle of inclination is the angle between the top of the shorter inner wall and the top of the taller outer wall. The angle of inclination can be from about 2 degrees to about 60 degrees. Therefore, the angle of inclination can be about 2 degrees, about 4 degrees, about 6 degrees, about 8 degrees, about 10 degrees, about 12 degrees, about 14 degrees, about 16 degrees, about 18 degrees, about 20 degrees, about 22 degrees, about 24 degrees, about 26 degrees, about 28 degrees, about 30 degrees, about 32 degrees, about 34 degrees, about 36 degrees, about 38 degrees, about 40 degrees, about 42 degrees, about 42 degrees, about 46 degrees, about 48 degrees, about 50 degrees, about 52 degrees, about 54 degrees, about 56 degrees, about 58 degrees or about 60 degrees. The angle of inclination of the trapezoidal channel affects the focusing behavior in two ways: (i) the relationship between the threshold flow rate required to capture cells in the Dean vortex and the particle size; (ii) the position of the Dean vortex core. Larger tilt angles (e.g., about 10 degrees to about 60 degrees) will produce strong Dean flow on the outside, thereby improving cell capture. Larger tilt angles can also reduce the threshold flow rate required to capture cells of a given size in the Dean vortex.

[0077] As used herein, "aspect ratio" is the ratio of a channel's height divided by its width, providing an appropriate channel cross-section to separate cells in an input population based on cell size along at least a portion of the cross-section of at least one curvilinear microchannel.

[0078] The aspect ratio of the channel can be from about 0.1 to about 1, such as about 0.1, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.2, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 0.30, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about 0.40, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, about 0.48, about 0.49, about 0.50, about 0.51, about 0.52, about 0.53, about 0.54, about 0.55, about 0.56, about 0.57, about 0.58, about 0.59, about 0.60, about 0.61, about 0.62, about 0.63, about 0.64, about 0.65 .3, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about 0.4, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, about 0.48, about 0.49, about 0.5, about 0.51, about 0.52, about 0.53 , about 0.54, about 0.55, about 0.56, about 0.57, about 0.58, about 0.59, about 0.6, about 0.61, about 0.62, about 0.63, about 0.64, about 0.65, about 0.66, about 0.67, about 0.68, about 0.69, about 0.7, about 0.71, about 0.72, about 0.73, about 0.74, about 0.75, about 0.76, about 0.77, about 0.78, about 0.79, about 0.8, about 0.81, about 0.82, about 0.83, about 0.84, about 0.85, about 0.86, about 0.87, about 0.88, about 0.89, about 0.9, about 0.91, about 0.92, about 0.93, about 0.94, about 0.95, about 0.96, about 0.97, about 0.98, about 0.99, or about 1.

[0079] The length of the curved channel can be equal to or greater than about 3 cm, for example, about 5 cm, about 10 cm, about 15 cm, about 20 cm, about 25 cm, about 30 cm, about 35 cm, about 40 cm, about 45 cm, about 50 cm, about 55 cm, about 60 cm, about 65 cm, about 70 cm, about 75 cm, about 80 cm, about 85 cm, about 90 cm, about 95 cm or about 100 cm.

[0080] In some embodiments, the method includes flowing an input population of cells through first and second curvilinear microfluidic channels that are sequentially connected and in fluid communication, wherein the first channel couples an inlet and the second channel couples one or more outlets. The first channel and the second channel can have different geometries, thereby generating different confinement ratios (CRs), to achieve more efficient size-based cell separation.

[0081] In some embodiments, the first curved microfluidic channel has a smaller cross-sectional area than the second curved microfluidic channel. For example, when both channels have rectangular cross-sections, the width and / or height of the first channel may be smaller than the width and / or height of the second channel. In another embodiment, when the first channel has a rectangular cross-section and the second spiral microchannel has a trapezoidal cross-section, the cross-sectional area of the first spiral microchannel may be smaller than the cross-sectional area of the second channel.

[0082] Cells entering the first microchannel with a smaller cross-section can have a relatively large CR due to the small size or cross-section of the first spiral microchannel. This can cause cells to aggregate in a band-like region near the channel wall as they pass through the first microchannel. Subsequently, this aggregated cell stream enters the second microchannel. Due to the increased channel size, the CR value of the cells in the second microchannel decreases, causing the equilibrium position to shift along the channel cross-section and causing cells to form aggregate bands at different equilibrium positions depending on their size.

[0083] In some embodiments, the first and second curvilinear channels are both spiral channels.

[0084] In one embodiment, the first spiral microchannel and the second spiral microchannel are nested together, for example, to form a Fermat spiral. The transition region can be S-shaped. The spiral of the first spiral microchannel can be in a clockwise or counterclockwise direction, and the spiral of the second spiral microchannel can be in the same direction as or opposite to the first spiral microchannel. For example, the first spiral microchannel can be in a counterclockwise direction, change direction in the transition region (for example, in the S-shaped transition region), and then the second spiral microchannel can be in a clockwise direction (for example, see Figure 1 Alternatively, the first spiral microchannel may spiral in a clockwise direction, change direction in a transition region, and then the second spiral microchannel may spiral in a counterclockwise direction.

[0085] In one embodiment, the second spiral microchannel is parallel to the first spiral microchannel. In another embodiment, the second spiral microchannel is located above or below the first spiral microchannel.

[0086] Depending on the structure of the spiral microchannel, the inlet can be located at the circumference or periphery (outside of the spiral) of the first spiral channel, or at the interior or center of the first channel. In addition, depending on the structure of the spiral microchannel, one or more outlets can be located at the circumference (outside of the spiral) of the second channel, or at the interior of the second channel. In some embodiments of nested first and second spiral microchannels, the inlet and outlet are located on the circumference of the channel.

[0087] The first and second spiral microchannels can each independently have uniform cross-sectional height or non-uniform cross-sectional height.For example, the cross section of the first and second microchannels can be rectangular.In another example, the cross section of the first and second microchannels can be non-rectangular, for example, and the cross section of two microchannels can be trapezoidal.In another example, the cross section of the first microchannel is rectangular, and the cross section of the second microchannel is non-rectangular (for example, trapezoidal).The microfluidic system with non-rectangular cross section is described in for example WO2014 / 046621, and its content is incorporated herein by reference.By designing appropriate channel geometry parameters, minicells can be concentrated to the outer wall of bending or spiral microchannel, and larger cells can be concentrated to the inwall.

[0088] Fluid properties of microfluidic channels

[0089] As mentioned above, the inertial lift force F L and Dean Drag F D Varies with cell size. Specifically, the force F L and F D The level changes with the size of the cell, and F L and F D The superposition of determines the equilibrium position of the cells within the channel cross section. Therefore, by calibrating the microchannel dimensions (e.g., hydraulic radius D) according to the particle size of the target differentiated cells and / or undifferentiated cells, the microchannel size can be adjusted. h and curvature radius r) and fluid velocity, those skilled in the art can estimate the F L and F D , and determine the approximate equilibrium position for a cell of a specific size. Using computational flow models may help estimate the equilibrium position. L and F D By superposition of the target cells, the target cells can be balanced at different positions along the cross-section of the microchannel, thereby facilitating the separation and isolation of undifferentiated cells.

[0090] In some embodiments, the cell density of the input population of cells is about 10 5 to about 10 7 For example, the cell density can be about 10 5 cells / ml, about 10 6 cells / ml or about 10 7 Higher cell densities may lead to cell-cell interactions and interactions between cells and the channel walls, thus affecting the focusing behavior.

[0091] The flow velocity of fluid by microfluidic device will depend on channel geometry, cell size etc. and change.Exemplary flow velocity can be about 0.5ml / minute to about 100ml / minute, such as about 0.5ml / minute to about 20ml / minute, or about 0.5ml / minute to about 10ml / minute.Flow velocity can be about 0.5ml / minute, about 1 ml / minute, about 2ml / minute, about 3ml / minute, about 4ml / minute, about 5ml / minute, about 6ml / minute, about 7ml / minute, about 8ml / minute, about 9ml / minute, about 10ml / minute, about 11ml / minute, about 12ml / minute, about 13ml / minute, about 14ml / minute, about 15ml / minute, about 16ml / minute, about 17ml / minute, about 18ml / minute, about 19ml / minute or about 20ml / minute.Higher flow velocity can be used for separating larger cell mass, and lower flow velocity can be used for processing smaller cell mass.

[0092] Collection export

[0093] Cells can be collected at one or more outlets at the end of a curved channel. The outlets can be configured to output cell populations within a specific size range. For example, larger cells may be concentrated near the inner wall of a curved or spiral channel and thus collected from an outlet near the inner wall; smaller cells may be concentrated near the outer wall of the channel and thus collected from an outlet near the outer wall.

[0094] In a preferred embodiment, the device has at least two outlets. One outlet provides a first cell output population in which undifferentiated cells are relatively reduced compared to the cell input population. Depending on the channel geometry and the relative size difference between undifferentiated cells and differentiated cells, the outlet can be located closer to the inner wall or the outer wall. As a non-limiting example, when the cell input population contains undifferentiated cells that are larger than differentiated cells, and the device comprises a spiral channel with a trapezoidal cross-section, and wherein the inner wall is smaller than the outer wall, the first output population (which is enriched in smaller differentiated cells) can be collected from the outlet near the outer wall of the channel.

[0095] In some embodiments, the methods described herein comprise collecting a second output population of cells through one or more outlets of a microfluidic device, wherein the second output population of cells has a higher proportion of undifferentiated cells relative to the input population of cells.

[0096] In other embodiments, the methods described herein comprise repeating steps (a) to (c) at least once, wherein the first cell output population is provided as the cell input population in the first and subsequent repetitions. Such cell recycling can further deplete the undifferentiated cell population and / or aggregate differentiated cells.

[0097] In certain embodiments, the apparatus comprises a system for closed-loop recirculation of cells. The closed-loop recirculation system may include a syringe in fluid communication with an inlet of the curvilinear channel and a first outlet providing a first output population; a first check valve located between and in fluid communication with the first outlet and the syringe; a syringe in fluid communication with the first outlet and the inlet of the curvilinear channel; and a second check valve located between and in fluid communication with the inlet of the curvilinear channel.

[0098] The check valve only allows fluid flow in one direction, while fluid flow in the opposite direction is blocked, for example, by an inner membrane. The first check valve allows flow from the first outlet to the syringe and blocks flow from the syringe to the first outlet. The first check valve may include an inner membrane that blocks flow from the syringe to the first outlet when the syringe is actuated to inject fluid into the channel inlet. The second check valve allows flow from the syringe to the microchannel inlet and blocks flow from the microchannel inlet to the syringe. The second check valve may include an inner membrane that blocks flow from the microchannel inlet to the syringe when the syringe is actuated to draw fluid from the first outlet into the syringe.

[0099] Thus, for a device with closed-loop recirculation, the method may include: providing differentiated cell and undifferentiated cell input populations to an inlet of a microfluidic device; flowing the cell input population through at least one curvilinear microfluidic channel coupled to the inlet, wherein the curvilinear microfluidic channel is configured to separate undifferentiated cells from the cell input population based on size; collecting a first cell output population through a first outlet of the curvilinear microfluidic channel coupled to the microfluidic device, wherein the first cell output population has a lower proportion of undifferentiated cells relative to the cell input population; and recirculating the first cell output population by actuating a syringe to extract the first output population from the first outlet and injecting the first output population into the inlet of the curvilinear microchannel.

[0100] As described herein, actuation of a syringe can refer to an extraction motion (e.g., extracting fluid from one of the outlets) and / or an infusion motion (e.g., injecting fluid into the inlet of a curved channel). The reciprocating motion of the syringe and / or syringe pump (in other words, extraction and infusion motion) causes the cell population to recirculate from the first outlet to the inlet of the microchannel. Whenever all or substantially all cells in the first outlet are recirculated in the device, a recirculation cycle is considered to be completed. The cell population collected after being guided through the device (whether after the first passage through the device or after one or more recirculation cycles) may be referred to herein as the "final cell output population". The methods described herein may not include a recirculation cycle, or may include one or more recirculation cycles. In certain aspects, the method requires one, two, three, four, five, six, seven, eight, nine, or ten recirculation cycles. The number of recirculation cycles depends on many factors, including but not limited to the cell separation required in the final output population, the cell purity required in the final output, the cell concentration required in the final output, the operation time, the number of devices, etc.

[0101] Microfluidic devices

[0102] Disclosed herein is a device for removing undifferentiated cells from a population of differentiated and undifferentiated cells, the device comprising: a) an inlet for receiving an input population of differentiated and undifferentiated cells; b) at least one curvilinear microfluidic channel connected to the inlet, wherein the curvilinear microfluidic channel is configured to separate undifferentiated cells from the input population of cells based on size; and c) one or more outlets connected to the curvilinear microfluidic channel and configured to collect an output population of cells, wherein the output population of cells has fewer undifferentiated cells than the input population of cells.

[0103] The present invention also discloses a device for purifying differentiated cells from a population of differentiated and undifferentiated cells, the device comprising: a) an inlet for receiving an input population of differentiated and undifferentiated cells; b) at least one curved microfluidic channel connected to the inlet, wherein the curved microfluidic channel is configured to separate differentiated cells from the input cell population based on size; and c) one or more outlets connected to the curved microfluidic channel and configured to collect an output cell population, wherein the output cell population has a higher proportion of differentiated cells than the input cell population.

[0104] According to embodiments of the present invention, microchannels and devices taught in PCT Application No. PCT / US2019 / 061479, including spiral microchannels, may be used; the entire contents of the PCT application are incorporated herein by reference.

[0105] As will be readily appreciated by those skilled in the art, microfluidic devices can be manufactured or produced using polycarbonate (PC) materials by injection molding using a suitable master mold. Alternatively, the device can be manufactured using polydimethylsiloxane (PDMS) using standard microfabrication soft lithography techniques known in the art. Other materials that can be used to manufacture microfluidic devices include, but are not limited to, glass, polystyrene (PS), polymethyl methacrylate (PMMA), and cyclic olefin copolymer (COC).

[0106] It will be understood by those skilled in the art that the microfluidic device may further comprise other components located upstream, downstream or inside the device. For example, one or more microfluidic devices may further comprise one or more collection devices (e.g., reservoirs), flow devices (e.g., syringes, pumps, pressure gauges, thermometers), analytical devices (e.g., 96-well microtiter plates, microscopes), filtration devices (e.g., membranes), for example for upstream or downstream analysis (e.g., immunostaining, polymerase chain reaction (PCR), such as reverse transcription PCR, quantitative PCR, fluorescence, such as fluorescence in situ hybridization (FISH)), sequencing, etc. The imaging system may be connected to the device to capture images from the device and / or may receive light from the device to visualize the separation process in real time and / or count the separated cells in real time. In one example, when the device is mounted on a microscope slide, the imaging system may inspect and / or digitize the images obtained by the microscope. For example, the imaging system may include a digitizer and / or camera connected to a microscope, an inspection monitor, and a computer processor. In some aspects, the device includes a pump, such as a syringe pump, a pressure pump, a peristaltic pump, or a combination thereof. In some aspects, the device is portable.

[0107] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as situations where combinations are not included when interpreted as an alternative (or).

[0108] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof.

[0109] Throughout this specification and in the claims that follow, unless the context requires otherwise, the word "comprise" or variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0110] Reference in this specification to any previous publication (or information obtained therefrom) or any known matter is not and should not be taken as an admission or any form of suggestion that the previous publication (or information obtained therefrom) or known matter forms part of the common general knowledge in the field to which this specification relates.

[0111] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the present invention includes all such variations and modifications that fall within the spirit and scope of the invention. The present invention also includes all steps, features, compositions and compounds referred to or indicated in the specification, either singly or in combination, and any and all combinations of any two or more of the steps or features.

[0112] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0113] Certain embodiments of the present invention will now be described with reference to the following examples, which are for illustrative purposes only and are not intended to limit the general scope described above.

[0114] Example

[0115] Example 1: Fabrication of a Multidimensional Double Helix (MDDS) Sorting Device

[0116] The plastic spiral device was manufactured using a standard injection molding method. Plastic spiral devices with specific channel dimensions were designed using 3D CAD software (SolidWorks 2020). For the injection molding of the plastic spiral device, top (flat, with or without protruding connection ports) and bottom (channel side) master molds were manufactured using a micro-milling process (RnD Factory, South Korea), and duralumin was used as the mold material. The top master mold with protruding connection ports was used to manufacture plastic devices for pipe connection as the top layer of stacked devices (or when used alone as a non-stacked single device); the top master mold without connection ports was used to manufacture plastic devices for device stacking, while the same bottom master mold was used for both connection types to emboss the same spiral channel. The plastic device material was polycarbonate resin. Double-sided adhesive film (IS-00726-01, RnD Factory, South Korea) was used to seal the channel side of the plastic spiral device. The process of cutting the film into the same size as the plastic device and punching holes in the film for fluid entry is accurately and automatically performed using a film cutting machine (GSP32-32, Gu Sung Machine, South Korea). In a clean room space, the manufactured plastic spiral device is bonded to a double-sided adhesive film using an alignment guide plate through a manual film connection process. In order to manufacture multiple (multiplexed) spiral devices, a plastic device with protruding connection ports is used as the top layer of the stacking device for pipe connection, while a plastic device without connection ports is used for device stacking. In order to accurately and quickly align the stacking, an aluminum pin fixer is manufactured by a standard milling process. In order to prevent fluid leakage, the multiple spiral devices are tightened and fixed with bolt joints through top and bottom stainless steel plates (5 mm thick).

[0117] Example 2: Size-Based Isolation of Spinal Cord Progenitor Cells (SCPCs) from Induced Pluripotent Stem Cells (iPSCs)

[0118] Materials and methods

[0119] Differentiation of human iPSCs into spinal cord progenitor cells (SCPCs)

[0120] Human induced pluripotent stem cells (iPSCs) were maintained in StemMACS iPS-Brew XF cell culture medium on Matrigel matrix (Corning, USA). iPSCs derived from umbilical cord lining epithelial cells (CLECs) were developed by Cell Research Corporation and are hypothesized to have the immune privileged properties of CLECs. TMiPSCs were routinely passaged every 5-7 days using a passage reagent (Stemcell Technologies, Canada). iPSCs were then differentiated into SCPCs based on a modified protocol to generate spinal motor neurons. Briefly, when the iPSC confluence reached 70-80%, iPSCs were dissociated into single cell suspensions using Accutase (Nacalai Tesque Inc, Japan). The dissociated iPSCs were then counted and re-seeded into neural induction medium supplemented with the ROCK inhibitor Y-27632 (5 μM) in 6-well plates at a density of 800,000 cells / well. Neural induction medium contains DMEM / F12 (50%, Biological Industries, Israel), neural culture medium (50%), NeuroBrew-21 (1X), N2 (1X), non-essential amino acids (1X, Thermo Fisher Scientific, USA), Glutamax (0.5X, Thermo Fisher Scientific), LDN-193189 (0.5 μM) and CHIR-99021 (4.25 μM). On day 3, retinoic acid (RA, 10 μM, Sigma-Aldrich, USA) was added to the culture medium. On day 4, cells were re-seeded in the same culture medium at a lower density (2 million cells / 100 mm culture dish) and the culture medium was changed daily until day 10. On day 10, the cells were characterized and named SCPCs and then frozen for storage or used in subsequent experiments. Unless otherwise stated, all other culture components were purchased from Miltenyi Biotech (Germany).

[0121] SCPC sorting using MDDS sorting equipment

[0122] Before sorting, the MDDS sorter was incubated with 70% ethanol for at least 30 minutes for sterilization. The cells were then rinsed with 1X phosphate buffered saline (PBS) and culture medium. A 50 mL syringe was loaded with cells at a concentration of 500,000 to 1,000,000 cells / mL and an injection device (PHD ULTRA syringe pump, Harvard Apparatus, USA) was used. The flow rate in the low-speed mode was 5 mL / min, and the flow rate in the high-speed mode was 9 mL / min. In order to implement the recirculation strategy, a double check valve (Quosina, USA) was used to extract the sorted cells from the outlet reservoir at a flow rate of 10 ml / min using a syringe pump to retract the sorted cells into the syringe.

[0123] Cell groups of different sizes were placed into the cell counting chip, and several 10x bright field images were collected for each group. The images were then analyzed using a self-developed MATLAB code to obtain the size of each group of cells.

[0124] Engraftment studies of sorted and unsorted cells

[0125] Female Sprague-Dawley rats (7-9 weeks, 230-250 g) were obtained from InVivos Pte Ltd (Singapore). Rats were housed under temperature-controlled conditions with a normal 12 / 12 hour light / dark cycle and free access to food and water. Rats (n=16) underwent 7 days of adaptive feeding and were injected with cyclosporin A (10 mg / kg, once a day, intraperitoneal injection) 3 days before surgery until infusion. Rats were anesthetized with intraperitoneal injection of ketamine (50 mg / kg) and xylazine (5 mg / kg). The surgical area was shaved and cleaned with 70% ethanol and povidone iodine. Subsequently, the skin and muscles above the thoracic vertebrae were incised to expose the vertebrae of the T8-T11 segment. Dorsal laminectomy was performed on T9-T10. Afterwards, the dura mater was opened and a local anesthetic (lidocaine, 20 mg / mL) was applied to the spinal cord. The animals were placed in a stereotaxic system (KOPF5000 with a microinjection unit). Hamilton syringes (model 701N) were filled with SCPC (1 x 10 6 Cells / μL: unsorted n=4; sorted - small n=4; sorted - large n=4; mixed n=4) and growth factors bFGF (10 μg / mL), VEGF (10 μg / mL), BDNF (50 μg / mL) and calpain inhibitor (50 μM) were connected to a microinjection unit. 8 μl of SCPC (4 μl on each side of the spinal cord) were injected at a flow rate of 0.5 μl / min. After two minutes, the syringe was gently withdrawn to avoid backflow and the injured area was covered with a polycaprolactone film (3 mm x 3 mm, approximately 50 μm thick). TM The muscle layer was closed with 4-0 sutures, and the skin was then closed with wound clips. Animals were monitored regularly and received subcutaneous buprenorphine (0.65 mg / kg) twice daily and meloxicam (1 mg / kg) once daily for 5 days after injury.

[0126] Immunohistochemical analysis

[0127] Four weeks after in vivo cell injection, animals were first infused with 0.9% saline followed by 4% PFA. After infusion, the spinal cord containing the implanted cells was removed, fixed with 4% PFA for 24 hours, and then transferred to a 15% sucrose solution for an additional 24 hours. Afterwards, the samples were transferred to a 30% sucrose solution and stored at 4°C until sectioning. Spinal cord samples were cut longitudinally into 20 μm thick slices using a cryostat (Leica CM1950) and mounted directly onto slides. Immunofluorescence staining was used to assess in vivo cell viability and cell differentiation.

[0128] Briefly, cryosections were permeabilized with 0.3% TritonX-100 for 15 minutes and then incubated in blocking buffer (1% BSA diluted in 0.1 PBST) for at least 1 hour. Primary antibodies were then added and incubated overnight at 4°C. The following primary antibodies were used: Oct4 (1:500) as an iPSC marker; SOX1 (1:500) as a neuronal progenitor cell marker; SOX2 (1:200) as a neural stem cell marker; HoxB4 (1:250) as a spinal cord identity marker; NF200 (1:500) for identifying neurofilaments; GFAP (1:1000) as an astrocyte marker; HuNu (1:200) as a human nuclear marker; hNCAM (1:200) as a human neural adhesion marker; NeuN (1:500) as a mature neuronal marker, and Iba1 (1:1000) as an inflammatory marker. Samples were washed three times with PBS and incubated with species-specific secondary antibodies conjugated to 488 or 555 (1:1000) for 2 hours at room temperature. Cell nuclei were counterstained with DAPI (1:1000). Sections were mounted with mounting medium and visualized using a Zeiss inverted confocal microscope (LSM800) and epifluorescence. Images were analyzed and quantified using FIJI / ImageJ software.

[0129] result

[0130] In this study, iPSCs derived from umbilical cord lining endothelial cells (CLECs) were differentiated into spinal cord progenitor cells (SCPCs) over a 10-day differentiation process. Analysis of the size of the resulting SCPC populations revealed that the large group primarily contained cells with residual pluripotency markers (e.g., Oct4), while the small group contained relatively few such cells.

[0131] Size-based label-free separation was performed using an inertial microfluidics-based device, which was used to remove Oct4-positive undifferentiated cells. The device has the advantages of label-free, contactless and high-throughput sorting (e.g., up to 3 million cells / minute, and even higher when stacked), without affecting cell viability and function. Approximately 33 million cells can be sorted into small and large cell populations within 30 minutes. Flow cytometric analysis showed that 50% of all Oct4-positive cells in the unsorted cell population were sorted into a large cell population that contained approximately 16% of the original cell population.

[0132] The cell population flowed through a double-helix microfluidic sorter to separate differentiated and undifferentiated cells based on size. The device sorted cells into two populations through two outlets, called the "small cell" outlet and the "large cell" outlet. Cells from the small and large cell outlets were mixed in a 50-50 ratio to obtain a mixed cell group. Quantitative analysis of cell size showed that the average cell size of the small and large cell groups was significantly different ( Figure 2 A and 2B).

[0133] When comparing the unsorted cell group, the sorted-small cell group, and the sorted-large cell group, cell viability was not significantly affected by the MDDS sorter as shown by trypan blue dye exclusion. Figure 2 C). The in vivo cell viability of injected SCPCs was assessed by analyzing the intracellular location of the HuNu marker (nuclear location identifies live SCPCs, while cytoplasmic location identifies dead SCPCs). Four weeks after implantation, the results showed that all rats (n=4) that received either unsorted cells or sorted-small cells had live SCPCs (evidenced by dense accumulation of nuclear HuNu+ cells within the implantation site, while sorted-large cells were mostly dead (evidenced by cytoplasmic or no HuNu signal). The viability of the mixed cell group was 50% ( Figure 3 ).

[0134] Immunohistochemical staining was used to phenotype the pluripotency, proliferation, and progenitor cell markers of the injected cells in the unsorted and sorted groups. The results showed that the injected cells were no longer pluripotent and most of them had differentiated into other cell types. Specifically, 4 weeks after injection, all cells did not express the pluripotency marker OCT4, and only a very small number of HuNu+ cells co-localized with Ki-67 and the neural progenitor cell marker SOX1 ( Figure 4 Furthermore, as shown by the expression of spinal cord markers HoxB4 and SOX2 ( Figure 5 ), the injected cells retained their spinal cord properties after transplantation.

[0135] Immunostaining for the pan-neuronal markers NF200 (immature) and NeuN (mature neuronal marker) showed that although most living HuNu+ cells differentiated into NeuN+, after 4 weeks of in vivo transplantation, sorted-small cells exhibited a more extensive or equivalent degree of neuronal differentiation than unsorted cells ( Figure 5 In addition, the colocalization of human neural adhesion marker (hNCAM) and NF200 also confirmed the differentiation of SCPC into neurons ( Figure 6 ).

[0136] Finally, colocalization of hNCAM and GFAP indicated that a small population of SCPCs differentiated into astrocytes in vivo ( Figure 7 ). However, there were no differences between the unsorted and sorted groups in both experimental groups.

Claims

1. A method for removing undifferentiated cells from a population of differentiated and undifferentiated cells, wherein: The method comprises: a) providing an input population of differentiated cells and undifferentiated cells to an inlet of a microfluidic device; b) flowing the input population of cells through at least one curvilinear microfluidic channel coupled to the inlet, wherein the curvilinear microfluidic channel is configured to separate undifferentiated cells from the input population of cells based on size; and c) collecting a first cell output population through one or more outlets connected to a curvilinear microfluidic channel of the microfluidic device, wherein the first cell output population has a lower proportion of undifferentiated cells than the cell input population.

2. The method according to claim 1, wherein The at least one curvilinear microfluidic channel is a spiral microfluidic channel.

3. The method according to claim 1 or 2, wherein: The differentiated cells and undifferentiated cells are mammalian cells.

4. The method according to any one of claims 1 to 3, wherein The undifferentiated cells are stem cells.

5. The method according to any one of claims 1 to 4, wherein The undifferentiated cells are pluripotent cells.

6. The method according to claim 5, wherein: The pluripotent cells are induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs).

7. The method according to any one of claims 1 to 6, wherein The differentiated cells are spinal cord progenitor cells (SCPCs), neural progenitor cells (NPCs) and / or cells differentiated therefrom.

8. The method according to any one of claims 1 to 7, wherein The cross-section of the curved channel is highly non-uniform.

9. The method according to any one of claims 1 to 8, wherein Step (b) comprises flowing the input population of cells through first and second curvilinear microfluidic channels that are serially connected and in fluid communication, wherein the first channel is coupled to an inlet and the second channel is coupled to one or more outlets.

10. The method according to claim 8, wherein The first curvilinear microfluidic channel has a smaller cross-sectional area than the second curvilinear microfluidic channel.

11. The method according to claim 8 or 9, wherein: The first and second curved microfluidic channels are spiral channels.

12. The method according to any one of claims 1 to 10, wherein The method further includes collecting a second output population of cells through one or more outlets of the microfluidic device, wherein the second output population of cells has a higher proportion of undifferentiated cells than the input population of cells.

13. The method according to any one of claims 1 to 11, wherein The method further comprises repeating steps (a) to (c) at least once, wherein the first output population of cells is provided as the input population of cells in the first and subsequent repeats.

14. The method according to any one of claims 1 to 12, wherein The viability of the differentiated cells in the input population of cells and the first output population of cells is comparable.

15. A method for purifying differentiated cells from a population of differentiated and undifferentiated cells, wherein the method comprises: a) providing an input population of differentiated cells and undifferentiated cells to an inlet of a microfluidic device; b) flowing the input population of cells through at least one curvilinear microfluidic channel coupled to the inlet, wherein the curvilinear microfluidic channel is configured to separate differentiated cells from the input population of cells based on size; as well as c) collecting a first cell output population through one or more outlets connected to a curvilinear microfluidic channel of the microfluidic device, wherein the first cell output population has a higher proportion of undifferentiated cells than the cell input population.

16. The method according to claim 13, wherein: The method includes removing undifferentiated cells from the input population of cells.

17. A device for removing undifferentiated cells from a population of differentiated cells and undifferentiated cells, wherein the device comprises: a) an inlet for receiving an input population of differentiated and undifferentiated cells; b) at least one curvilinear microfluidic channel coupled to the inlet, wherein the curvilinear microfluidic channel is configured to separate undifferentiated cells from the input population of cells based on size; as well as c) connecting one or more outlets of the curved microfluidic channel for collecting an output cell population, wherein the output cell population has a lower proportion of undifferentiated cells than the input cell population.

18. A device for purifying differentiated cells from a population of differentiated cells and undifferentiated cells, wherein the device comprises: a) an inlet for receiving an input population of differentiated and undifferentiated cells; b) at least one curvilinear microfluidic channel coupled to the inlet, wherein the curvilinear microfluidic channel is configured to separate differentiated cells from the input population of cells based on size; as well as c) connecting one or more outlets of the curved microfluidic channel to collect a cell output population, wherein the cell output population has a higher proportion of differentiated cells than the cell input population.

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