Microfluidic systems and methods of using them

Abstract

The present invention provides a microfluidic system and method for reprogramming, proliferating, preserving, and optionally differentiating cells. [Solution] An integrated microfluidic system is provided that utilizes microfluidic chip technology to accept a patient sample containing cells, grow the cells, reprogram the grown cells, and then store the reprogrammed cells in a microfluidic chip. These microfluidic chips containing the reprogrammed cells can then be used in situations of genetic differentiation to specific cell types. Overall, this system and workflow is suitable as a hospital-based device that enables the creation of any patient-derived iPSC for downstream diagnostic or therapeutic applications.

Description

[Technical Field]

[0001] Cross-reference with related applications This application claims priority under U.S. Patent Application No. 62 / 424,208, filed November 18, 2016, 35 U.S.C § 119(e), the entire contents of which are incorporated herein by reference.

[0002] Field of Invention The present invention generally relates to the field of microfluidic devices, and more specifically to microfluidic systems and methods for reprogramming, growing, preserving, and optionally differentiating cells. [Background technology]

[0003] Background information Microfluidic systems are crucial in medical diagnostics and biotechnology research. Components of such systems include networks of very small wells and channels through which fluids can deliver and combine precisely controlled amounts of chemicals, cells, and molecules. The systems are used for a variety of tasks, including reagent mixing, biomolecule isolation and testing, and the isolation and sorting of living cells. To achieve the required high-precision movement, mixing, and accurate measurement, microfluidic chips and substrates require complex control mechanisms, such as microvalves and pumps integrated within the chip, as well as pneumatic actuators, electronic solenoids, robotic control devices, and the complex computer programs and systems needed to control these devices.

[0004] The use of microfluidic devices offers numerous advantages over standard benchtop methods, including unparalleled economies of scale and a high degree of parallelization and integration. With technological advancements, microfluidic devices are becoming increasingly smaller and capable of performing an increasing number of tasks. For example, microfluidic approaches have been proposed separately for cell separation and isolation, cell culture, cell differentiation, and screening of cell reprogramming factors.

[0005] Stem cells are cells whose ability to self-regenerate through cell division over long periods of time is not yet fully understood, and which can be induced to differentiate into cells with specific functions, i.e., differentiated cells. These properties make stem cells highly promising for therapeutic applications in replacing cells and tissues damaged in various disease conditions. Embryonic stem (ES) cells originate from the blastocyst of an early embryo and have the ability to develop into the endoderm, ectoderm, and mesoderm (three germ layers) (i.e., ES cells are "pluripotent"). In vitro, ES cells tend to spontaneously differentiate into various types of tissues, and controlling their differentiation direction can be difficult. Ethical issues associated with destroying embryos to harvest human ES cells remain unresolved, and these issues limit their availability for research and therapeutic applications.

[0006] Adult stem (AS) cells are found in differentiated tissues. Stem cells derived from adult tissues typically have a more limited ability to form a range of cells (i.e., "pluripotency") and usually differentiate only into the cell type of the tissue in which they were found. However, recent reports have shown that certain types of AS cells have some plasticity. Their proliferative capacity is also generally limited.

[0007] Induced pluripotent stem cells (iPSCs) are created from differentiated adult cells using experimental techniques. iPSCs are widely recognized as an important tool, for example, for conducting medical research. Previous iPSC creation techniques were time-consuming and labor-intensive. Differentiated adult cells, such as fibroblasts, are reprogrammed and cultured to form individual colonies that constitute a single clone. Until now, identifying these cell types has been extremely difficult because the majority of cells were iPSC clones that were not fully reprogrammed. For iPSC clones, the standard practice is to select desired colonies with well-defined boundaries, including cells with a high nucleus-to-cytoplasm ratio, based on cell morphology. Once clones are identified, they are manually extracted using very thin glass instruments and cultured on a cell "feeder" layer, typically mouse embryonic fibroblasts (MEFs). This process is usually performed 14–21 days after the introduction of the reprogramming vector. The clones are then grown for another 14–21 days or longer, followed by molecular analysis.

[0008] Other researchers are focusing on developing techniques for the rapid and more accurate identification and analysis of fully reprogrammed adult fibroblasts and their downstream differentiation potential (Bock et al., 2011, Cell 144: 439-452 (Non-Patent Literature 1); Boulting et al., 2011, Nat Biotechnol 29: 279-286 (Non-Patent Literature 2)). See also, for example, the shared U.S. Patent No. 13 / 159,030 (Patent Literature 1), filed June 13, 2011, which describes the use of fluorescence-activated cell sorting (FACS) for identification and the in-situ sorting of unique subpopulations identified by unique expression patterns of surface proteins.

[0009] Therefore, stem cells are an attractive source of cells for therapeutic applications, medical research, and pharmaceutical trials. The use of patient-specific stem cells and reprogrammed somatic cells represents an immunologically compatible cell replacement strategy that is highly desirable for several medical treatments, including the treatment of cancer and neurological disorders, to name a few. However, there remains a long-standing need in this field for improved microfluidic devices and methods for processing patient-specific cells, utilizing an integrated approach that performs multiple tasks using automated, rapid approaches, from processing the initial patient's blood to personalized treatment of the same patient using patient-specific stem cells and reprogrammed somatic cells. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] U.S. Patent No. 13 / 159,030 [Non-patent literature]

[0011] [Non-Patent Document 1] Bock et al., 2011, Cell 144: 439-452 [Non-Patent Document 2] Boulting et al., 2011, Nat Biotechnol 29: 279-286 [Overview of the project]

[0012] This invention provides microfluidic-based systems and methods that utilize systems for processing biological samples to provide time- and cost-effective workflows in laboratory and / or medical-based environments. The entire workflow has the capability to provide patient-specific treatment.

[0013] Accordingly, in one embodiment, a microfluidic system for processing biological samples is provided. The system comprises one or more microfluidic units operable to perform steps for processing several samples, thereby allowing cells from or derived from the sample to be preserved, cataloged, and ultimately used to treat the patient from whom the sample was taken. The microfluidic units are operable to isolate cells from the sample, grow the isolated cells, and reprogram the cells. The system also includes microfluidic functionality to differentiate the reprogrammed cells into cell types desirable for use in treating the patient. At any point in the processing, the system includes functionality for preserving and cataloging cells. In addition, the system is operable to perform cell analysis and qualitative and quantitative evaluation of cells at any stage of processing.

[0014] In one embodiment, the system includes one or more computer memory modules containing instructions for controlling processing functions, together with one or more computer processor modules configured to execute the instructions.

[0015] In another aspect, the present invention provides a method for processing a biological sample using a microfluidic-based system of the present disclosure. The method includes the steps of applying the sample to the system and performing a processing step on the sample to generate reprogrammed cells and / or cells of a desired cell type derived from the reprogrammed cells.

[0016] In another aspect, the present invention provides a method for treating a disease or disorder in a subject using a microfluidic-based system of the present disclosure. The method includes (a) obtaining a sample from a subject; (b) applying the sample to a system; (c) processing the sample in the system; and (d) administering the processed cells to the subject to treat a disease or disorder in the subject.

[0017] In yet another aspect, the present invention provides a pharmaceutical composition comprising cells or cellular fractions thereof processed by a microfluidic system, optionally comprising a pharmaceutically acceptable excipient.

[0018] In yet another aspect, the present invention provides a cell bank. The cell bank comprises one or more populations of cells processed by the system of the present disclosure. In embodiments, each cell population is cataloged and stored at a temperature suitable for future use. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] [Figure 1] It is a schematic diagram of a microfluidic system in an embodiment of the present invention. [Figure 2] It is a schematic diagram of a microfluidic system in an embodiment of the present invention. [Figure 3] It shows the steps for obtaining a fibroblast cell bank in an embodiment of the present invention. [Figure 4] It shows the steps for obtaining a stem cell array from a fibroblast cell bank in an embodiment of the present invention. <0000-097> [Figure 5] It is a flowchart showing the steps in a system for generating iPSCs in an embodiment of the present invention. [Figure 6A] It shows an example of the flow of a patient sample through a multi-well tissue culture plate during an automated reprogramming process in an embodiment of the present invention. [Figure 6B-1] It shows an example of the flow of a patient sample through a multi-well tissue culture plate during an automated reprogramming process in an embodiment of the present invention. [Figure 6B-2] It shows an example of the flow of a patient sample through a multi-well tissue culture plate during an automated reprogramming process in an embodiment of the present invention. [Figure 6C] It shows an example of the flow of a patient sample through a multi-well tissue culture plate during an automated reprogramming process in an embodiment of the present invention. [Figure 7A]An example of an equipment configuration for achieving the workflow in one embodiment of the present invention is shown. [Figure 7B] An example of an equipment configuration for achieving the workflow in one embodiment of the present invention is shown. [Figure 7C] An example of an equipment configuration for achieving the workflow in one embodiment of the present invention is shown. [Modes for carrying out the invention]

[0020] Detailed explanation This invention provides an integrated microfluidic system that utilizes microfluidic chip technology to process patient samples and produce patient-specific reprogrammed cells and, optionally, differentiated cells of specific cell types derived from reprogrammed cells. The system of this invention significantly improves the efficiency and reproducibility of producing standardized iPSC lines. Typically, researchers produce iPSCs manually, which limits the usefulness of these cells due to inter-researcher variability and the inability to produce large quantities of cells. The system avoids these challenges by providing a fully automated system from receiving tissue or cell samples to creating a bank of clearly defined iPSC line stocks. The system enables consistency and invariance in the production of large quantities of cells from multiple donors, which facilitates the use of iPSC technology.

[0021] The system utilizes one or more microfluidic units, which may be in the form of one or more individual microfluidic chips, to process a sample, generate reprogrammed cells, and retain these cells in a microfluidic chip (hoteling) format for subsequent harvesting and differentiation. Differentiation and proliferation are then carried out to produce cells of the desired cell type. Overall, the workflow methodology and system are ideal as a laboratory or hospital-based system enabling the generation of pluripotent cells from any patient for downstream diagnostic or therapeutic applications.

[0022] Before describing the compositions and methods, it should be understood that this invention is not limited to the specific compositions, methods, and experimental conditions described, and that such compositions, methods, and conditions may be modified. Since the scope of the invention is limited only to the appended claims, it should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

[0023] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural nouns unless otherwise explicitly indicated. Thus, for example, a reference to “the method” includes one or more methods and / or processes of the type described herein, which would be obvious to a person skilled in the art who has read this disclosure.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described herein.

[0025] This disclosure provides a microfluidic system for processing biological samples. The system comprises one or more microfluidic units that are operable to perform several sample processing steps, thereby allowing cells from a sample or cells derived from a sample, such as iPSCs, to be stored, cataloged, and ultimately used to treat the patient from whom the cells were taken. In an embodiment, iPSCs produced from adult cells isolated from a patient sample are differentiated into a desired cell type suitable for use in treating the patient.

[0026] As used herein, “adult” means an organism from the fetal stage onward, that is, from the neonatal period until the end of its life, and includes, for example, cells obtained from postpartum placental tissue, amniotic fluid, and / or umbilical cord blood.

[0027] As used herein, the term “adult differentiated cells” encompasses a broad range of differentiated cell types obtained from the adult stage of an organism and capable of accepting the production of iPSCs using the automated system described herein. These adult differentiated cells are preferably “fibroblasts.” Fibroblasts (also called “fibrous cells” in less active forms) are derived from the mesenchyme. Their functions include the secretion of precursors of extracellular matrix components, such as collagen. Histologically, fibroblasts are highly branched cells, while fibrous cells are generally described as smaller and more spindle-shaped. Fibroblasts and fibrous cells of any tissue origin can be used as starting materials in the automated workflow system according to this invention.

[0028] As used herein, the term “induced pluripotent stem cell,” or iPSC, means that the stem cells are created from induced or altered, i.e., reprogrammed, differentiated adult cells into cells capable of differentiating into all three germ layers: mesoderm, endoderm, and ectoderm. Created iPSCs mean that they are not naturally occurring cells.

[0029] Examples of mammalian somatic cells useful in the present invention include, but are not limited to, adult stem cells, Sertoli cells, endothelial cells, granular epithelial cells, neurons, islet cells, epidermal cells, epithelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, fibroblasts, cardiomyocytes, other known muscle cells, and essentially all living somatic cells. In certain embodiments, fibroblasts are used. As used herein, the term somatic cell is also intended to include adult stem cells. Adult stem cells are cells capable of giving rise to all cell types of a particular tissue. Examples of adult stem cells include hematopoietic stem cells, neural stem cells, and mesenchymal stem cells.

[0030] One of the advantages of the present invention is the ability to supply isogenic, i.e., human cells with identical genes, essentially without limitation, suitable for use in transplantation, drug discovery assays, or disease modeling. iPSCs can be specifically tailored to a patient while avoiding immune rejection. Therefore, iPSCs eliminate significant problems associated with current transplantation methods, such as rejection of transplanted tissue that can occur due to host-versus-graft or graft-versus-host rejection. When used in drug discovery, these cells exhibit the response to chemicals specific to each patient in drug discovery, or the disease symptoms of individual patients in disease models. Several types of iPSCs, or fully differentiated somatic cells prepared from iPSCs derived from human somatic cells, can be stored in an iPSC bank as a cell library, and one or more types of iPSCs in that library can be used to prepare somatic cells, tissues, or organs that do not cause rejection in patients receiving stem cell therapy.

[0031] The iPSCs of the present invention may be differentiated into several different cell types and used to treat various disorders by methods known in the art. For example, iPSCs may be induced to differentiate into hematopoietic stem cells, muscle cells, cardiomyocytes, hepatocytes, chondrocytes, epithelial cells, urinary tract cells, neuronal cells, etc. The differentiated cells may then be transplanted back into the patient's body for the prevention or treatment of the disease, or used in advanced medical research or drug discovery assays. Accordingly, the method of the present invention can be used as a treatment for or in the development of treatments for subjects suffering from myocardial infarction, congestive heart failure, stroke / attack, ischemia, peripheral vascular disease, alcoholic liver disease, cirrhosis, Parkinson's disease, Alzheimer's disease, diabetes, cancer, arthritis, wound healing, immunodeficiency, aplastic anemia, anemia, Huntington's disease, amyotrophic lateral sclerosis (ALS), lysosomal storage disease, multiple sclerosis, spinal cord injury, genetic disorders, and similar diseases, where enhancement or replacement of specific cell types / tissues, or dedifferentiation of cells, is desired.

[0032] The term "totipotent" refers to all cells in an adult body, as well as cells capable of developing extraembryonic tissues, such as the placenta. A fertilized egg (zygote) is totipotent, as are the cells (blastomeres) of a morula (up to the 16-cell stage after fertilization).

[0033] As used herein, the term “pluripotent” refers to a cell that, under differentiation conditions, has the developmental capacity to differentiate into cell types exhibiting characteristics of all three germ layers: endoderm (e.g., gastrointestinal tissue), mesoderm (e.g., blood, muscle, and blood vessels), and ectoderm (e.g., skin and nerves). The developmental capacity of pluripotent cells is lower than that of totipotent cells. The ability of a cell to differentiate into all three germ layers can be determined, for example, by a teratoma formation assay in nude mice. In some embodiments, evidence of pluripotency can also be confirmed by the expression of embryonic stem (ES) cell markers, but a preferred test for the pluripotency of a cell or a population of cells produced using the compositions and methods described herein is to demonstrate that the cell has the developmental capacity to differentiate into cells of each of the three germ layers. In some embodiments, pluripotent cells are named “undifferentiated cells.” Thus, as used herein, the terms “pluripotency” or “pluripotent state” refer to the developmental capacity of a cell that results in the cell’s ability to differentiate into all three germ layers (endoderm, mesoderm, and ectoderm). Those skilled in the art will recognize the germ layer or cell lineage that gives rise to a given cell type. Cells in a pluripotent state typically have the ability to divide in vitro over long periods, for example, more than one year, or over more than 30 passages.

[0034] The term "multipotent," as used in reference to "pluripotent cells," refers to cells that have the developmental capacity to differentiate into cells of one or more germ layers, rather than all three. Therefore, pluripotent cells can also be called "partially differentiated cells." Pluripotent cells are well known in this field, and examples of pluripotent cells include adult stem cells, such as hematopoietic stem cells and neural stem cells. "Pluripotency" indicates that a cell can form many types of cells of a given cell lineage (rather than other cell lineages). For example, pluripotent hematopoietic cells can form many different types of blood cells (red, white, platelets, etc.), but cannot form neurons. Therefore, the term "pluripotency" refers to a state of cells that have a certain range of developmental capacity, but is less than totipotent and pluripotent.

[0035] As used herein, the terms “stem cell” or “undifferentiated cell” refer to a cell that is undifferentiated or partially differentiated, possessing the characteristic of self-renewal and the developmental ability to differentiate into multiple cell types, but without any specific meaning regarding its developmental ability (i.e., totipotency, pluripotency, multipotency, etc.). Stem cells can proliferate and produce more such stem cells while maintaining their developmental ability. In principle, self-renewal can occur by one of two main mechanisms. Stem cells may divide asymmetrically, which is known as inevitable asymmetric differentiation. In this case, one daughter cell retains the developmental ability of the parent stem cell, while the other daughter cell exhibits several other distinct specific functions, phenotypes, and / or developmental ability derived from the parent cell. The daughter cell itself can be induced to proliferate and produce progeny, which then differentiate into one or more mature cell types while maintaining one or more cells with parental developmental ability. Differentiated cells may originate from multipotent cells, and multipotent cells themselves may originate from multipotent cells, etc. Each of these pluripotent cells can be considered a stem cell, and the range of cell types that each of these stem cells can produce—that is, their developmental potential—can be considerably diverse. Alternatively, some stem cells in a population may symmetrically divide into two stem cells, a phenomenon known as probabilistic differentiation. In this case, some of the stem cells in the population are fully maintained, while the others produce only differentiated progeny. Thus, the term "stem cell" refers to a small population of cells that, under certain circumstances, possesses the developmental potential to differentiate into more specialized or differentiated phenotypes, and under certain circumstances, retains the ability to proliferate substantially without differentiation. In some embodiments, the term stem cell generally refers to naturally occurring parent cells whose offspring (progeny) are specialized, often in different directions, by fully acquiring individual characteristics through differentiation, such as in the progressive diversification of embryonic cells and tissues. Some differentiated cells also possess the ability to produce cells with high developmental potential. Such ability may be naturally occurring or artificially induced by processing various factors.Cells that have emerged as stem cells may progress toward a differentiated phenotype, but they can be induced "in the reverse direction" to re-express the stem cell phenotype, a process often referred to by those skilled in the art as "dedifferentiation," "reprogramming," or "retrodifferentiation."

[0036] As used herein, the term “embryonic stem cell” refers to naturally occurring pluripotent stem cells of the inner cell mass of a blastocyst (see, for example, U.S. Patent Applications No. 5,843,780; No. 6,200,806; No. 7,029,913; and No. 7,584,479, incorporated herein by reference). Such cells can also be obtained from the inner cell mass of a blastocyst derived from somatic cell nuclear transfer (see, for example, U.S. Patent Applications No. 5,945,577, No. 5,994,619, and No. 6,235,970, incorporated herein by reference). Embryonic stem cells are pluripotent and, during development, give rise to derivatives of all three primary germ layers (ectoderm, endoderm, and mesoderm). In other words, embryonic stem cells, given a given sufficient and essential stimulus to a particular cell type, can develop into each of the more than 200 adult cell types. Embryonic stem cells do not contribute to the extraembryonic membrane, i.e., the placenta; in other words, they are not totipotent.

[0037] As used herein, the characteristic properties of embryonic stem cells are defined as the “embryonic stem cell phenotype.” Therefore, a cell has the embryonic stem cell phenotype if it possesses one or more properties unique to embryonic stem cells that distinguish it from other cells that do not have the embryonic stem cell phenotype. Examples of characteristic embryonic stem cell phenotypic properties include, but are not limited to, the expression of specific cell surface or intracellular markers (including proteins and microRNAs), gene expression profiles, methylation profiles, deacetylation profiles, proliferative capacity, differentiation capacity, karyotype, and responsiveness to specific culture conditions. In some embodiments, the determination of whether a cell has the “embryonic stem cell phenotype” is made by comparing one or more characteristics of the cell with one or more characteristics of embryonic stem cell lines cultured in the same laboratory.

[0038] As used herein, the term “somatic stem cell” refers to any pluripotent or multipotent stem cell derived from tissues other than embryonic tissues, such as embryonic, juvenile, and adult tissues. Naturally occurring somatic stem cells have been isolated from a variety of adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Each of these somatic stem cells can be characterized by gene expression, factor responsiveness, and morphology in culture. Examples of naturally occurring somatic stem cells include, but are not limited to, neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells, and pancreatic stem cells. In some aspects described herein, “somatic pluripotent cells” refers to somatic cells or their offspring whose developmental potential has been altered, i.e., increased to a pluripotent state, by contacting or inducing one or more reprogramming factors using the compositions and methods described herein.

[0039] As used herein, the term “progenitor cell” refers to a cell with high developmental potential, that is, a cell with a more primitive phenotype (e.g., one in an earlier stage of development or progression) than a cell that could be produced by differentiation. Progenitor cells often have significant or very high proliferative capacity. Depending on the developmental pathway and the environment in which the cell develops and differentiates, a progenitor cell may give rise to multiple distinct cells with lower developmental potential, i.e., differentiated cell types, or to a single differentiated cell type.

[0040] As used herein, the term “somatic cell” refers to all cells other than germ cells, cells present in or derived from a preimplantation embryo, or cells resulting from the in vitro proliferation of such cells. In other words, somatic cells, as opposed to germline cells, refer to all cells that make up the body of an organism. In mammals, germline cells (also known as “gametes”) are sperm and eggs, which fuse during fertilization to produce a cell called a zygote. The entire mammalian embryo develops from this zygote. Each other cell type in the mammalian body (other than sperm and eggs, the cells from which they are made (germ cells), and undifferentiated, pluripotent embryonic stem cells) is a somatic cell (internal organs, skin, bone, blood, and connective tissue made from somatic cells). In some embodiments, somatic cells are “non-embryonic somatic cells,” meaning that the somatic cell is not present in or derived from an embryo, and is not resulting from the in vitro proliferation of embryonic cells. In some embodiments, somatic cells are “adult somatic cells,” meaning that the cells are present in or obtained from organs other than the embryo or fetus, or are produced by the in vitro proliferation of such cells. Unless otherwise specified, the compositions and methods described herein for reprogramming somatic cells can be carried out both in vivo and in vitro (in vivo, the somatic cells are present in the body of the subject; in vitro, the methods are carried out using isolated somatic cells maintained in culture).

[0041] The term “differentiated cell” is defined herein to encompass all somatic cells that are not pluripotent in their native form. Accordingly, the term “differentiated cell” also encompasses partially differentiated cells, such as pluripotent cells or stable, non-pluripotent, partially reprogrammed cells, or partially differentiated cells produced using any of the compositions and methods described herein. In some embodiments, differentiated cells are stable, intermediate cells, such as non-pluripotent, partially reprogrammed cells. It should be noted that placing a large number of primary cells in culture can result in the loss of some of the fully differentiated characteristics. Therefore, simply culturing such differentiated or somatic cells will not result in them becoming undifferentiated cells (e.g., undifferentiated cells) or pluripotent cells. Transition of differentiated cells (including intermediates of stable, non-pluripotent, partially reprogrammed cells) to pluripotency requires a reprogramming stimulus that goes beyond the stimulus that induces the loss of partially differentiated properties when placed in culture. Reprogrammed cells, in some embodiments, also have the characteristic of being able to be passaged for long periods without losing growth ability, compared to parent cells that have lower developmental capacity and can usually only divide a limited number of times. In some embodiments, the term “differentiated cell” also refers to a more specialized cell type (i.e., a cell type with lower developmental capacity) derived from a less specialized cell type (i.e., a cell type with high developmental capacity) (e.g., an undifferentiated cell or a reprogrammed cell) when the cell is in the process of cell differentiation.

[0042] In various embodiments, the system is configured to perform a series of processes in a directional workflow. The processes performed by the system include isolating cells, growing the isolated cells, reprogramming the grown cells, differentiating the reprogrammed cells into a desired cell type, and preserving the cells.

[0043] In various embodiments, the system is configured to isolate cells from a biological sample. This includes the isolation and separation of specific cell types. In embodiments, the biological sample may include pre-isolated cells, in which case the isolation step does not need to be performed. Isolation and / or separation techniques performed using microfluidics are known in the art and may be utilized in the implementation of the present invention. Such techniques include, but are not limited to, methodologies for cell capture and separation.

[0044] A “biological sample” is a sample of biological material taken from a patient or subject, containing intact cells. Biological samples include samples taken from body fluids and tissues (e.g., from biopsy material) or tissue preparations (e.g., tissue sections, homogenates, etc.). “Body fluid” is any liquid obtained from or derived from a subject suitable for use according to the present invention. Such liquids include whole blood, blood fractions, e.g., serum and plasma, urine, sweat, lymph, feces, ascites, semen, sputum, papillary aspirate, postoperative seroma, wound drainage fluid, saliva, synovial fluid, ascites fluid, bone marrow aspirate, cerebrospinal fluid, nasal secretions, amniotic fluid, bronchoalveolar lavage fluid, pleural fluid, peripheral blood mononuclear cells, total leukocytes, lymph node cells, spleen cells, and tonsillar cells. In embodiments, the sample contains leukocytes or is a sample of isolated leukocytes.

[0045] As used herein, the term “isolated cells” refers to cells that have been removed from the organism from which they were originally found, or to the offspring of such cells. Optionally, cells are cultured in vitro, for example, in the presence of other cells. Optionally, cells are then introduced into a second organism or reintroduced into the original organism from which they were isolated (or the cells or cell population from which they were derived).

[0046] As used herein, the term “isolated population” in relation to an isolated population of cells refers to a population of cells that have been taken out and separated from a mixed or heterogeneous population of cells. In some embodiments, the isolated population is a population of cells that is “substantially pure” compared to the original heterogeneous population from which the cells were isolated or concentrated. In some embodiments, the isolated population is a population of isolated pluripotent cells that includes a population of pluripotent cells that is substantially pure compared to the heterogeneous population of somatic cells from which the pluripotent cells are derived.

[0047] In various embodiments, the system is also configured to carry out a cell proliferation process using cells isolated from the sample. This ensures that there are enough cells to carry out downstream processing.

[0048] After the cells have been proliferated, the system includes functionality to reprogram the proliferated cells, for example, to produce iPSCs. As used herein, the term “reprogramming” refers to the process of reversing the developmental potential of a cell or cell population (e.g., somatic cells). In other words, reprogramming refers to the process of inducing a cell to a state of higher developmental potential, i.e., a state of being returned to a more undifferentiated state. The cells being reprogrammed may be partially differentiated or fully differentiated before reprogramming. In some embodiments of the aspects described herein, reprogramming includes the complete or partial reversal of a differentiated state to a pluripotent state, i.e., an increase in the cell’s developmental potential. In some embodiments, reprogramming includes inducing a somatic cell to a pluripotent state so that the cell has the equivalent developmental potential of an embryonic stem cell, i.e., exhibits the phenotype of an embryonic stem cell. In some embodiments, reprogramming also includes a partial reversal of a cell’s differentiated state or a partial improvement in its developmental potential, e.g., a change in a somatic cell or unipotent cell to a pluripotent state. Reprogramming also includes partial reversal of the cell's differentiation state, which makes it more susceptible to complete reprogramming to a pluripotent state when further operations such as those described herein are performed. Such operations may induce endogenous expression of specific genes that contribute to or maintain reprogramming in the cell or its progeny. In certain embodiments, reprogramming of cells using the synthetically modified RNA and methods described herein results in cells (e.g., pluripotent cells) that are assumed to be in a pluripotent state. In some embodiments, reprogramming of cells (e.g., somatic cells) using the synthetically modified RNA and methods described herein results in cells that are assumed to be in a pluripotent-like state or embryonic stem cell phenotype. Hereinafter, the resulting cells are referred to as “reprogrammed cells,” “somatic pluripotent cells,” and “RNA-induced somatic pluripotent cells.”In this specification, the term “partially reprogrammed somatic cells” refers to cells that have been reprogrammed from cells with low developmental potential by the methods disclosed herein. Partially reprogrammed cells are not fully reprogrammed into a pluripotent state and are in a stable, intermediate state that is not pluripotent. Such partially reprogrammed cells are defined herein as cells that have differentiation potential but is lower than that of pluripotent cells. Partially reprogrammed cells, for example, can differentiate into one or two of the three germ layers but not into all three germ layers.

[0049] As used herein, the term “reprogramming factor” is defined herein as a factor that alters developmental potential. Such factors include, for example, genes, proteins, RNA, DNA, or small molecules whose expression contributes to reprogramming cells, such as somatic cells, into a less differentiated or undifferentiated state. Reprogramming factors may include any gene, protein, RNA, or small molecule, such as transcription factors that reprogram cells into a pluripotent state (e.g., SOX2, OCT3 / 4, KLF4, NANOG, LIN-28, c-MYC), which can be replaced by one or more of these in an in vitro cell reprogramming method. In some embodiments, the expression of exogenous reprogramming factors using the synthetically modified RNA and method described herein induces the expression of one or more endogenous reprogramming factors such that the expression of one or more exogenous reprogramming factors is no longer required to stably maintain cells in a reprogrammed or partially reprogrammed state. As used herein, “reprogramming to a pluripotent state in vitro” refers to a method of reprogramming in vitro that does not require and / or involves nuclear or cytoplasmic translocation or cell fusion with, for example, oocytes, embryos, germ cells, or pluripotent cells. Reprogramming factors may also be referred to as “dedifferentiation factors.” Dedifferentiation factors refer to factors that alter developmental potential, and are defined herein as, for example, proteins or RNAs that dedifferentiate cells and induce a less differentiated phenotype. In other words, dedifferentiation factors enhance the developmental potential of cells.

[0050] Methods for transmuting and transforming or reprogramming adult cells to form iPSC lines are generally known, for example, Takahashi et al., 2007 Cell, 131: 861-872, 2007, Yu et al., 2007, Science, vol. 318, pp. 1917-1920. iPSCs are induced from somatic cells by reprogramming factors, which are assumed to include, for example, transcription factors. Methods for reprogramming adult cells include, for example, introducing and expressing specific combinations of transcription factors, such as Oct3 / 4, Sox2, Klf4, and the c-Myc gene. Other researchers have shown that other combinations of transcription factors may be used to transform or reprogram adult cells. Other such transcription factors include, for example, Lin28, Nanog, hTert, and SV40 large T antigen, as described in Takahashi et al., 2006 Cell, 126: 663-676 and Huiqun Yin, et al. 2009, Front. Agric. China 3(2): 199-208 (incorporated herein by reference).

[0051] iPSCs can also be generated by directly introducing RNA into cells that, when translated, yield one or more desired proteins. Higher eukaryotic cells have evolved cellular defense mechanisms against foreign "non-self" RNA. Such defense mechanisms ultimately lead to an overall inhibition of intracellular protein synthesis, resulting in cytotoxicity. This response is partly accompanied by the production of type I or type II interferons and is commonly referred to as the "interferon response" or "cellular innate immune response." This cellular defense mechanism typically recognizes synthetic RNA as a foreign substance and induces this cellular innate immune response. In certain embodiments where the ability to achieve sustained or repeated expression of exogenous induced proteins using RNA is hindered by the induction of this innate immune response, it is desirable to use synthetic RNA that has been modified in a manner that circumvents or reduces this response. By circumventing or reducing the innate immune response, sustained expression from exogenous introduced RNA becomes possible, for example, to modify the phenotype relating to cell development. In one embodiment, sustained expression is achieved by repeatedly introducing the synthetically modified RNA into target cells or their progeny. The methods of the present invention include native RNA or synthetic RNA.

[0052] In one embodiment, native, modified, or synthetic RNA can be introduced into cells to induce the exogenous expression of a target protein within the cell. The ability of the modified or synthetic RNA described herein to induce the exogenous expression of a target protein is useful, for example, in treating disorders caused by endogenous genetic defects in cells or organisms in which the ability of the cell or organism to produce the target protein is impaired or hindered. Accordingly, in some embodiments, compositions and methods comprising the RNA described herein can be used for gene therapy purposes.

[0053] The RNAs described can be conveniently used to alter cell fate and / or developmental potential. The ability to express proteins from exogenous RNA allows for either altering or reversing cell developmental potential—that is, reprogramming cells and differentiating cells toward more differentiated phenotypes. A key aspect of altering cell developmental potential is the need for one or more developmental potential-altering factors to be expressed sustained and over a long period in the cell or its direct progeny. To date, such sustained expression has been achieved by introducing DNA or viral vectors into cells. These methods are limited to therapeutic applications due to the risk of generating insertional mutations.

[0054] One of the areas that will most benefit from the ability to express one or more desired proteins over extended periods from the exogenous RNAs described herein is the generation of pluripotent or multipotent cells from cells that initially possess a differentiated phenotype. In this embodiment, RNA encoding reprogramming factors is used to reprogram cells into a less differentiated phenotype, i.e., cells with higher developmental potential.

[0055] In this embodiment and several embodiments of all such embodiments described herein, the synthetically modified RNA molecule comprises at least two modified nucleosides. In one such embodiment, the two modified nucleosides are 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2'-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2'-fluorouridine, pseudouridine, 2'-O-methyluridine (Um), 2'-deoxyuridine (2'dU), 4-thiouridine (s4U), 5-methyluridine (m5U), and 2'-O-methyluridine. The modified nucleosides are selected from the group consisting of adenosine (m6A), N6,2'-O-dimethyladenosine (m6Am), N6,N6,2'-O-trimethyladenosine (m62Am), 2'-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2'-O-methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2,N2,7-trimethylguanosine (m2,2,7G), and inosine (I). In this embodiment and all such embodiments described herein, in one such embodiment, at least two modified nucleosides are 5-methylcytidine (5mC) and pseudouridine. (See, for example, U.S. Patent No. 2012 / 0046346 granted to Rossi et al., incorporated herein by reference).

[0056] The genes, proteins, or RNAs used in the methods of the present invention include, but are not limited to, OCT4, SOX1, SOX2, SOX3, SOX15, SOX18, NANOG, KLF1, KLF2, KLF4, KLF5, NR5A2, c-MYC, 1-MYC, n-MYC, REM2, TERT, and LIN28.

[0057] It has also been found that a single transcription factor can be used in conjunction with specific small molecule pathway inhibitors to reprogram adult fibroblasts into iPSCs. Such pathway inhibitors include, for example, the transforming growth factor-beta (TGFb) pathway inhibitors SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]-benzamide) and A-83-01 [3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide]. PD0325901 (N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide), an inhibitor of the extracellular signal-regulated kinase (ERK) and microtubule-associated protein kinase (MAPK / ERK) pathways, and CHIR99021 [6-((2-((4 [(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazole-2-yl)pyrimidine-2-yl)amino)ethyl)amino)nicotinonitrile], Parnate (also known as tranylcypromine), a lysine-specific demethylase 1; PS48 [(2Z)-5-(4-chlorophenyl)-3-phenyl-2-pentenoic acid], a small molecule activator of 3'-phosphoinositide-dependent kinase-1 (PDK1); sodium butyrate and valproic acid, histone deacetylase (HDAC) inhibitors; small molecules that regulate mitochondrial oxidation (e.g., 2,4-dinitrophenol), glycolysis (fructose 2,6-bisphosphate and oxalate), and activation of the HIF pathway (N-oxaloylglycine and quercetin) (the whole of which is incorporated herein by reference, Zhu (et al., 2010, Cell Stem Cell 7: 651-655) is one example. Zhu et al. have shown that the combination of Oct4 and Parnate with CHIR99021 is sufficient for reprogramming adult human epidermal keratinocytes.

[0058] Although individual protocols differ, reprogramming protocols generally consist of growing differentiated adult cells derived from tissue samples, such as skin biopsy material, and contacting them with the reprogramming factors described above, for example, infecting them, that is, transforming them with expression vectors such as viral constructs containing transcripts of pluripotent transcription factors. Fibroblasts are obtained by methods known in the field, for example, by mechanically disrupting the tissue and then dissociating it using enzymes to release fibroblasts, and these fibroblasts are then cultured by methods known in the field, for example, the method described in Dimos et. al., 2008, Science Vol. 321 (5893): 1218-1221.

[0059] In exemplary embodiments of the present invention, vectors, such as viral vectors and plasmid vectors, are used, but in some embodiments, a vector is not required for translocation techniques, which include transferring mRNA molecules into cells.

[0060] Translocation of fibroblasts using an expression vector is performed according to the instructions provided with the desired vector. After a certain period (e.g., approximately 2 to 10 days after translocation), the cells are isolated and CD13 NEG SSEA4 POS and Tra-1-60 POS The cells are brought into contact with a fluorescently labeled antibody against the surface marker. The isolated and antibody-labeled cells are then resuspended in phosphate-buffered saline, and the process moves to automated sorting and isolation of iPSC clones. Surface marker-positive cells are sorted by the color or presence or absence of the label and placed directly into sterile tubes containing tissue medium, or into multi-well (6-96 well) tissue culture plates coated with MEF or a cell-free biological matrix, and cultured until colony formation is visually apparent.

[0061] Subsequently, the resulting colonies are observed under a microscope to further confirm that they are iPSCs, or, if necessary, clones labeled with fluorescently tagged antibodies are observed under a fluorescence microscope. In certain embodiments, a green fluorescent protein (GFP) expression marker is also inserted by one or more vectors, if necessary, to facilitate sorting and identification. Multiple individual colonies with morphological features consistent with pluripotent ES cell lines are harvested from the culture and grown individually to form monoclonal cultures.

[0062] In a preferred embodiment of the system of the present invention, treated cells are subjected to genetic analysis to confirm and identify them as iPSCs at an early stage. Preferably, this genetic analysis is performed by Southern blotting, but other methods known in the art, including but not limited to microarrays, NanoString, quantitative real-time PCR (qPCR), whole-genome sequencing, immunofluorescence microscopy, and flow cytometry, may be used. The clone can also be confirmed as an iPSC by detection of alkaline phosphatase enzymatic activity, positivity of the cell membrane surface markers SSEA3, SSEA4, Tra-1-60, and Tra-1-81, and expression of the transcription factors KLF4, Oct3 / 4, Nanog, and Sox2 in reprogrammed human fibroblasts. It is preferable that all markers are present.

[0063] Any transduction vector known in the field can be used as a reprogramming factor, and these include RNA such as mRNA, microRNA, siRNA, antisense RNA, and combinations thereof. Other expression vectors that can be used include, for example, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses, Sindbisviruses, poxviruses, baculoviruses, bacterial phages, Sendai viruses, and combinations thereof. It is preferable that the vector used is a non-replicating vector, such as a non-replicating Sendai virus vector. A preferred Sendai virus vector is non-replicating while simultaneously maintaining the ability to productively express the nucleic acid encoding the protein it holds, thereby preventing any possibility of the vaccine spreading uncontrolled to other cells or into the body. This type of Sendai vector is commercially available as the CytoTune®-iPSC Sendai Virus Vector Kit (DNAVEC, DV-0301).

[0064] To insert such vectors into adult fibroblasts, any translocation method known in the art, such as electroporation or gene guns, may be used. If necessary, chemical translocation may also be carried out by means of translocation agents, such as polymers, calcium phosphate, cationic lipids, or lipofection. Cell-permeable peptides may also be used as needed to deliver vectors or other agents to adult fibroblasts. Briefly, cell-permeable peptides include protein-derived peptides, such as protein transduction domains and / or amphiphilic peptides that deliver vectors or other agents (including peptides) to cells. A comprehensive list of cell-permeable peptides is provided, for example, by Heitz et al., 2009 British Journal of Pharmacology, 157: 195-206, which is incorporated herein by reference in its entirety. Other cell-permeable peptides are also known in the art and disclosed by Heitz (above). Other cell-permeability techniques, such as liposomes and nanoparticles, may also be used in the methods of the present invention. Liposomes and nanoparticles are also described in Heitz (see above).

[0065] Antibodies can also be used to identify transformed cells. Four antibodies against stem cell-specific surface proteins—SSEA3, SSEA4, Tra-1-60, and Tra-1-81—are commonly used to identify and characterize populations of human pluripotent stem cells. Developmental stage-specific embryonic antigens 3 and 4 (SSEA3 and SSEA4) are two monoclonal antibodies that recognize contiguous regions of gangliosides present in human 2102Ep cells (Henderson et al., 2002 Stem Cells 20: 329-337; Kannagi et al., 1983, Embo J 2: 2355-2361). Tra-1-60 and Tra-1-81 antibodies were initially produced against human embryonic carcinoma (EC) cells (PW et al., 1984, Hybridoma 3: 347-361), but were later found to specifically recognize the carbohydrate epitope of keratan sulfated glycoprotein, which was identified as podocalyxin (a member of the CD34-related family of sialomucin) (Badcock et al., 1999, Cancer Research 59: 4715-4719; Nielsen et al., 2007, PLoS ONE 2: e237; Schopperle and DeWolf, 2007, Stem Cells 25: 723-730). Several other surface markers have been shown to be expressed in ES cells, including CD326 or EpCam (Sundberg et al., 2009, Stem Cell Res 2: 113-124), CD24 (thermally stable antigen), and CD133 (Barraud et al., 2007, Journal of Neuroscience Research 85, 250-259) (Gang et al., 2007, Blood 109: 1743-1751).Chan et al. (2009, see above) reported that the identification of true iPSCs from fibroblasts through reprogramming via transduction of four retroviral factors can be achieved by live-cell imaging, and that long-term observation shows that fibroblasts lose the expression of cell surface markers CD13 and D7Fib and acquire the expression of pluripotent stem cell markers SSEA4 and Tra-1-60 (Chan et al., 2009, see above).

[0066] Accordingly, the present invention further provides iPSCs generated using the method described herein, as well as populations of such cells. The reprogrammed cells of the present invention, having the ability to differentiate into diverse cell types, have diverse applications and therapeutic uses. The fundamental properties of stem cells, their ability to self-regenerate indefinitely and their ability to differentiate into any cell type in the body make them ideal for therapeutic applications.

[0067] In various embodiments, the system further includes functionality for differentiating reprogrammed cells into desired cell types. The primary goal of stem cell technology is to differentiate stem cells into desired cell types, i.e., to generate cells through directed differentiation or transdifferentiation. The compositions and methods described herein are useful not only for cell reprogramming but can also be applied to directed differentiation and transdifferentiation of cells to desired phenotypes. That is, the same techniques described herein for reprogramming can be directly applied to the differentiation of reprogrammed cells, or even any other stem or progenitor cells, into desired cell types.

[0068] A wide variety of further cell types may be created by differentiation, transdifferentiation, and dedifferentiation. In the context of cell-organ development, the terms “differentiating” or “being differentiated” are relative terms referring to a developmental process in which, by its developmental process, a cell progresses to a developmental pathway further downstream than its direct progenitor cell. Thus, in some embodiments, a reprogrammed cell, as defined herein, can subsequently differentiate into a lineage-limited progenitor cell (such as a mesodermal stem cell) that can differentiate into other types of progenitor cells further downstream in its pathway (such as tissue-specific progenitors, e.g., cardiomyocyte progenitors), and then into a final-stage differentiated cell that plays a characteristic role in a particular tissue type and may or may not retain the ability to proliferate further.

[0069] Differentiation is typically carried out by contacting iPSCs with one or more differentiation factors. As used herein, the term “differentiation factor” refers to a factor that alters the developmental potential of a cell, such as a protein, RNA, or small molecule, which induces the cell to differentiate into a desired cell type, as defined herein; that is, the differentiation factor reduces the developmental potential of the cell. In some embodiments, the differentiation factor may be a cell type-specific polypeptide, but this is not essential. Differentiation into a particular cell type may require the simultaneous and / or sequential expression of two or more differentiation factors. In some embodiments described herein, the developmental potential of a cell or cell population is first enhanced by reprogramming or partial reprogramming using synthetically modified RNA as described herein, and then the cell or progenitor cell produced by such reprogramming is induced to differentiate and the developmental potential of the cell or progenitor cell is reduced by contacting or introducing one or more synthetically modified RNA encoding a differentiation factor.

[0070] As used herein, the term "without forming pluripotent intermediate cells" means that one cell type is converted to another, preferably in a single step; therefore, a method for modifying a differentiated phenotype or the developmental potential of a cell without forming pluripotent intermediate cells does not require the cell to be first dedifferentiated (or reprogrammed) and then differentiated into another cell type. Instead, the cell type is simply "switched" from one cell type to another without passing through a less differentiated phenotype. Thus, conversion refers to a change in the developmental potential of a cell, which is induced to become different cells with similar developmental potential, for example, from liver cells to pancreatic cells, or from pancreatic α-cells to pancreatic β-cells. The systems and methods of the present invention are well suited for cell conversion.

[0071] Exemplary genes encoding differentiation factors useful for differentiating, dedifferentiating, or transdifferentiating cells in various aspects include OCT4, NANOG, SOX2, SOX17, HNF4, GATA4, HHEX, CEBPβ, CEBPδ, PRDM16, MYOD1, NKX2.5, MEF2c, myocaldin, RUNX2, PDX, NGN, SALL4, or SOX9, or combinations thereof. Transcription factors that are encoded include Oct4, NANOG, Sox2, Sox9, Sox17, HNF4α2, HNF4α4, HNF4α7, HNF4α8, HNF4γ, GATA4, Hhex, CEBPβ, CEBPδ, PRDM16, MyoD1, Nkx2.5, Mef2c, myocaldin, Runx2-I, Pdx1, Ngn3, Sall4, or Runx2-II. For example, the differentiation of mesoderm or fibroblasts into adipocytes, chondrocytes, osteocytes, and muscle cells may be carried out using chimeric proteins containing the following transcription factors: CEBPβ / CEBPδ (adipocytes), Sox9 (chondrocytes), Runx2 (osteocytes), and MyoD1 (muscle cells).

[0072] Cell differentiation techniques performed using microfluidics are known in the art and can be utilized in the implementation of the present invention. Such techniques include those described in WO2013 / 188748, incorporated herein by reference. WO2013 / 188748 describes a microfluidic apparatus for transdifferentiating cells from one cell type to another. Cells are cultured concurrently or sequentially with one or more vector-free gene regulatory oligonucleotides and then harvested when cellular markers or morphology of the culture indicate completion of transdifferentiation. Suitable gene regulatory oligonucleotides include microRNAs and messenger RNAs encoding differentiation factors. Conditions for transdifferentiation are optimized by separating cells into different culture chambers of the microfluidic apparatus. Cells are cultured with different additives in each chamber and then compared.

[0073] After differentiation is complete, the system has the functionality to proliferate the differentiated cells and produce a sufficient number of the desired cell type for downstream use.

[0074] At any stage of processing, the system of the present invention has functionality for preserving cells. For example, isolated cells may be preserved, proliferated cells may be preserved, reprogrammed cells may be preserved, or differentiated cells may be preserved. Preservation may be carried out under any conditions suitable for extending cell lifespan, for example, by freezing the cells at about -80°C or below.

[0075] Furthermore, the system also includes functionality to analyze cells at any stage of processing to perform qualitative and quantitative evaluations of the cells. The analysis may include any type of cell analysis known in the art, such as image analysis, cell count analysis, cell morphology analysis, polymerase chain reaction (PCR) analysis, sequence analysis, DNA analysis, RNA analysis, gene expression profiling, proteome analysis, metabolome analysis, immunoassay, nuclear exclusion analysis, or a combination thereof.

[0076] Figure 1 illustrates an embodiment of a system including a single microfluidic unit 100 configured to perform each of the processing steps. The system is also shown to include a single computer module 140, which includes a computer memory module containing instructions for controlling the processing steps and a computer processor module configured to execute the instructions.

[0077] It is understood that the processing steps may be carried out via one or more microfluidic units. For example, Figure 2 shows an embodiment of a system having a first microfluidic unit 100 and a second microfluidic unit 200. Unit 100 is operable to perform cell isolation, cell proliferation, cell reprogramming, and optionally cell preservation. Unit 200 is operable to perform cell differentiation and preservation. Each unit is controlled by a single computer module 150.

[0078] It is conceivable that any number of processing steps may be performed by a single microfluidic unit. For example, each processing step may be performed by a different microfluidic unit. In the context of the present invention, a microfluidic unit may be formatted as a microfluidic chip designed to perform specific tasks, such as cell isolation, proliferation, reprogramming, and differentiation, on a chip. A single microfluidic chip may also include multiple microfluidic units, with various units aligned at different positions on the chip. In embodiments, the chip may be separable so that the microfluidic units may be separated at several stages of the processing steps. For example, reprogramming may be performed by a microfluidic unit located on a first region of the chip, and the preservation of the reprogrammed cells may be performed by a microfluidic preservation unit located on a second region of the chip. These two regions may be separable from each other so that the reprogramming region is separated from the preservation region, and only the preservation region can be frozen.

[0079] To achieve a specific processing task, the microfluidic unit is designed to include multiple channels through which the fluid flow is directed, and these channels are formed from a non-porous substrate. The term “non-porous substrate” means a solid support or matrix on which the microfluidic unit of the present invention is fabricated using photolithography or other suitable processing. This material is typically polydimethylsiloxane (PDMS) or polymethyl methacrylate (PMMA) or other suitable material known in the art.

[0080] In various embodiments, the width of the flow channel may be from about 5 μm to about 1000 μm. In wider flow channels, it may be about 100 μm, from about 100 μm to about 150 μm, from about 150 μm to 200 μm, from about 200 μm to 250 μm, from about 250 μm to about 300 μm, from about 300 μm to about 350 μm, from about 350 μm to about 400 μm, from about 400 μm to about 450 μm, from about 450 μm to about 500 μm, from about 500 μm to about 550 μm, from about 550 μm to 600 μm, from about 600 μm to about 650 μm, from about 650 μm to about 700 μm, from about 700 μm to about 750 μm, from about 750 μm to 800 μm, from about 800 μm to about 850 μm, from about 850 μm to about 900 μm, from about 900 μm to about 950 μm, or from 950 μm to 1000 μm. In many applications, a range of flow channel widths from about 75 μm to about 2 125 μm is preferred. However, in certain examples, the channel width may exceed 1000 μm. In narrower channels, the width may be from about 5 μm to about 100 μm. The channel width may be from about 10 μm to about 75 μm, from about 15 μm to about 50 μm, and from about 20 μm to about 40 μm. In some embodiments, the channel width is about 5, 10, 1, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 μm. The height may be from about 5 μm to about 100 μm, from about 10 μm to about 75 μm, from about 15 μm to about 50 μm, and from about 20 to about 40 μm. In some embodiments, the height of the channel is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 μm. The cross-sectional area may be from about 20 to about 13000 μm 2 ², from about 50 to about 10000 μm 2 ², from about 200 to about 8000 μm 2 ², from about 250 to about 5000 μm 2 ², from about 500 to about 3000 μm 2 ², and in many embodiments, from about 1400 to about 1600 μm 2It is preferable that the cross-sectional area is about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or about 2000 μm. 2 The cross-sectional shapes of the individual channels in the matrix apparatus of the present invention may be the same or different, and may be of various shapes such as square, rectangular, other polygonal, circular, elliptical, semicircular, and semi-elliptical. The cross-sectional shapes and areas may differ within the same channel and can be prepared by previously described manufacturing techniques and manufacturing techniques known in the art. Square or rectangular channel shapes are generally preferred.

[0081] The present invention is described in part with respect to functional components and various processing steps. Such functional components and processing steps may be recognized by any number of components, operations, and techniques configured to perform specific functions and achieve various results. For example, the present invention may utilize various biological samples, biomarkers, elements, materials, computers, data sources, storage systems and media, information acquisition techniques and processing, data processing standards, statistical analysis, and regression analysis to perform diverse functions. In addition, although the present invention is described in the context of medical diagnostics, it may be carried out in conjunction with any number of applications, environments, and data analyses; the systems described are merely illustrative applications of the present invention.

[0082] Methods for processing according to various aspects of the present invention may be carried out in any suitable form, for example, by computer program operations on a computer system. Exemplary systems according to various aspects of the present invention are carried out in conjunction with a computer system, for example, a typical computer system including a processor and random access memory, such as a remotely accessible application server, network server, personal computer, or workstation. The computer system may also suitably include further memory devices or information storage systems, such as a mass storage system and a user interface, such as a typical monitor, keyboard, and tracking device. On the other hand, the computer system may include any suitable computer system and associated equipment, and may be configured in any suitable form. In one embodiment, the computer system includes a standalone system. In another embodiment, the computer system is part of a computer network including servers and databases.

[0083] The software required to receive, process, and analyze information may be implemented on a single device or on multiple devices. The software may be accessible via a network so that the storage and processing of information can be performed remotely to the user. Systems and various elements of the present invention provide functions and operations that facilitate biomarker analysis, such as data collection, processing, analysis, reporting, and / or diagnosis. The system can hold information related to a sample and also facilitate analysis and / or diagnosis. For example, in this embodiment, a computer system runs a computer program that can receive, store, retrieve, analyze, and report information related to cell analysis. The computer program may include multiple modules that perform various functions or operations, such as a processing module for processing raw data and creating supplemental data, and an analysis module for providing a system that analyzes the raw and supplemental data to perform specific tasks.

[0084] The system may also provide various additional modules and / or individual functions. For example, the system may include reporting functions that provide information related to processing and analysis functions. The system may also provide various management and operational functions, such as restricting access and implementing other administrative functions.

[0085] It is understood that all or any part of the processes required to produce iPSCs or differentiated cells derived therefrom may be carried out using a microfluidic unit, or a similar automated process operably connected to a microfluidic unit in the system of the present invention.

[0086] In various embodiments, the systems of this disclosure may utilize or be operablely linked with one or more systems (Systems 1-8) described in the following workflow systems, such as those disclosed in U.S. Patent Application Publication 2013 / 0345094, which is incorporated herein by reference in its entirety.

[0087] Workflow System The workflow system is divided into four units, each operated independently. (1) Isolation and growth of somatic cells in an isolation ward (System 1); (2) Isolation Station Assay (System 2); (3) Thawing, infection and identification (systems 3, 4, and 5); and (4) Maintenance, QC, propagation, and freezing (systems 6, 7, and 8)

[0088] In addition, the system includes an automated -80 storage and retrieval system for preserving fibroblasts, and the final clones in screw-cap tubes containing 1.4 mL of matrix. The systems that operate each unit, as well as the processes and operations, are described below.

[0089] System 1, Part A: Somatic cell isolation and growth workflow in isolation chamber, Mycoplasma preliminary testing for biopsy processing. 1. The technician will plate 40 biopsy samples per week into 6 well dishes; Maintain the 2.6-well plates in an isolation incubator capable of holding 200 plates; 3. Regularly check the culture density using an integrated Cyntellect Celigo hemocytometer.

[0090] System components that may be used to carry out these automated processes include, but are not limited to, a STARlet Manual Load, a Modular Arm for 4 / 8 / 12ch. / MPH and 8-channel pipetting with 1000 μl pipetting channels, and an iSWAP plate handler, all available from Hamilton Science Robotics. If centrifugation is required or desired, an Agilent VSpin microplate centrifuge may be used. The software may be Celigo API software. The incubator may be a Cytomat incubator. For plate handling, a Cytomat 24 barcode reader, a Cytomat 23mm stand, and a Cytomat 400mm mobile station may be used. A Multi Flex tilt module may be used to tilt the plates. The system controller may be a Dell PG running on a Windows XP operating system. The carrier package may be a Q Growth carrier package.

[0091] System 1, Part B: Isolation Colony Growth Workflow, Mycoplasma Testing 1. Return the culture medium from the incubator to the deck of the isolation cell Growth STARlet, and collect the medium from the wells into plates for mycoplasma testing using ELISA. Manually transfer the 2.96-well assay plate to the isolation assay STARlet.

[0092] System 1, Part C: Isolation Colony Growth Workflow After Mycoplasma Testing 1. Distribute the proliferated fibroblasts into multiple freezing vials, seal them, and transfer them to a SAM at -80°C.

[0093] The system components used to implement these automated processes can be selected from the same components used in the isolation growth workflow, except that the STARlet Auto Load may be used. A Spectramax L reader can be used as the spectrum acquisition device.

[0094] System 2: Workflow for Isolation Assay 1. Testing using the Lonza MycoAlert with the glow luminescence method. 2. Measurement of the luminescence plate using a spectral collection device.

[0095] System components that may be used to perform these automated processes include, all available from Hamilton Science Robotics, a STARlet Manual Load, a Modular Arm for 4 / 8 / 12ch. / MPH and 8-channel systems with 1000 μl pipetting channels, and an iSWAP plate handler. For luminescence assays, a BioTek Synergy HT reader may be used. The system controller may be a Dell PG that can run on a Windows XP operating system. The carrier package may be a Q Growth carrier package.

[0096] Systems 3, 4, and 5: Melting, infection, and identification Melting module and infection module 1. Retrieve the freezing tubes from the SAM at -80°C (61, 190). 2. Melt on a heating block (122) 3. Remove the lid (Hamilton Capper Decapper) (126) 4. Add the culture medium to dilute the cryoprotective agent (122) 5. Spin (128) 6. Resuspend in the plating data (122) Plate one sample per well of the 7.6 wells (62, 122). 8. Move to the incubator (130, 132) 9. Allow fibroblasts to recover for approximately 3-4 days. 10. Confirm the culture density using a Cyntellect Celigo hemocytometer (124) 11. Passage all wells of fibroblasts on the day of reprogramming (122) 12. In the batch, pass the trypsin through the wells (122). 13. Counting cells using the Cyntellect Celigo hemocytometer (124) Plate a certain number of cells into 1 to 3 wells of a 14.24-well plate, and fix the sample to a minimum number of 24-well plates (64, 122). 15. Return the plates to the incubator overnight (130, 132). 16. Collect the plate, thaw the virus in the tube form, and add it to each well of the fibroblasts in the 24-well plate (130, 122). 17. Replace a portion of the culture medium daily. (122)

[0097] Magnetic separation module 18. Collect the culture using accutase and prepare a single-cell suspension (134). 19. Dilute with staining buffer (134) 20. Staining of fibroblast surface markers with magnetic beads (134) 21. Washing process (134) 22. Apply magnetically (for Dynal beads) or to a column (for Miltenyi systems) (134, 136) 23. Collect the non-magnetic fraction and transfer it to a new well (134). 24. Counting cells using the Cyntellect Celigo hemocytometer (124) 25. Dilute the cells to an appropriate density by transferring 1 to 10 cells from the subculture medium to each well of a 96-well plate (66, 134). Retrieve the 96-well plate coated with fresh Matrigel or matrix from the 26.4°C incubator (142). 27. Based on the number of cells counted, for example, distribute 2 cells per plate representing one infection into a 96-well matrix plate (66, 134). 28. Return the plate to the incubator (132) 29. Replace a portion of the culture medium daily. (122)

[0098] Colony Identification Module Return the 30.96-well plate from the incubator to the liquid handling machine for colony identification (66, 132, 138). 31. Perform live cell staining using pluripotent surface markers (138) 32. Image taken with a Cyntellect Celigo hemocytometer (140) 33. Identify wells containing colonies with clearly defined boundaries and positive for a single marker (140). 34. The technician reconfirms the selection, chooses six samples from the initial sample for passage, and retrieves the IDs for the plate and positive wells. 35. Select a well containing a single marker-positive colony (138) Retrieve the 96-well plates coated with fresh Matrigel or matrix from the 36.4°C incubator (68, 142). 37. Collect the selected wells and subculture them into a new 96-well matrix plate, fix the clones to the minimum number of plates, and plate each in subculture medium (68, 138). 38. Replace a portion of the culture medium daily (122)

[0099] The system components that may be used to carry out these automated processes can be selected from the same ones used in the isolation cell growth workflow, with the only addition being one or more CORE 96 PROBEHEAD II (1000 μl model) probe heads.

[0100] Systems 6, 7, and 8: Maintenance, QC, Propagation, and Freezing Maintenance module 39. Pass the colonies in successive 1:1 stages in new plates coated with 96-well matrix until the colony density is sufficiently high (68-72, 160). 40. By replacing approximately 75% of the culture medium daily using 96 tip heads, all plates are nourished. (160) 41. Regularly monitor colony density and growth rate using the Cyntellect Celigo hemocytometer (166) 42. Duplicate the plate and create a plate for clone QC (74-86, 160). 43. The ultimate goal is to grow the clones on multiple plates for use in multiple QC assays to eliminate poor-quality clones and retain clones that are 2-3 times higher quality than the initial sample. 44. Select the clones that passed the QC process, realign them, and fix them to the minimum number of plates. At the same time, remove the clones that did not pass (80, 86, 160). 45. Provide nutritional support daily while this process is underway. (160)

[0101] QC Module 46. ​​Collect the cells (74, 150) 47. Counting cells (164) 48. Plate a certain number of cells (ranging from 5,000 to 10,000 cells per well) onto a V-bottom plate. Prepare 2 to 6 copies per cell strain (84, 150). 49. Return to incubator (1g agglomeration) (172) 50. Replace the culture medium after 2 days (150) 51. Incubate for an additional 12 days in the incubator (172) 52. Replace a portion of the culture medium every 2.2 days (150) 53. Transfer to the nucleic acid preparation station to remove the culture medium from the wells while leaving the embryoid bodies in the wells (84, 192). 54. Resuspend the RNA in RNA lysis buffer to combine the replicates of each sample and prepare a plate that can be used for analysis in the Nanostring nCounter assay (84, 192).

[0102] Freezing module 55. Start with a 96-well plate after subculturing and propagation (88) 56. Incubate in an incubator for 6 days (172) 57. Replace a portion of the culture medium daily. (154) 58. Remove the plate from the incubator (88, 162) 59. Remove the culture medium (remove it completely) (154) 60. Add chilled pre-frozen medium (Matrigel diluted with growth medium) (154) 61. Incubate in an incubator for 1 hour (172) 62. Remove the culture medium (remove it completely) (154) 63. Add a small amount of chilled frozen culture medium (154). 64. Seal the plate (88, 164) 65. Transfer the sample to a storage chamber at -80°C and freeze it. (190) 66. Store in liquid nitrogen in the gas phase.

[0103] Storage of frozen vials 67. Start with a 96-well plate after subculturing and propagation (90) Incubate for 68.6 days (172) 69. Replace a portion of the culture medium daily. (154) 70. Pass the wells into a 24-well plate at a 1:1 ratio (92, 154). Incubate for 71.6 days (172) 72. Replace a portion of the culture medium daily (154) 73. Pass the wells into a 6-well plate at a 1:1 ratio (94, 154). Incubate for 74.4 to 6 days (172) 75. Replace a portion of the culture medium daily (154) 76. Remove the plate from the incubator (162) 77. Replace a portion of the culture medium with pre-frozen medium (154) 78. Incubate in an incubator for 1 hour (172) 79. In the case of normal subculturing, cells are harvested for freezing (154) 80. Transfer to matrix tubes. Use 2-3 tubes per well (96, 154). 81. Spin to remove the culture medium (168, 154) 82. Add the frozen culture medium (154) 83. Put the cap on the tube (170) 84. Transfer the sample to a storage chamber at -80°C (190)

[0104] Figure 3 shows the steps performed by System 1, including plating (2) of the biopsy material, propagation and subculturing (4) (rotational culture using a liquid handling robot), QC (6) (automated mycoplasma testing), and (8) automated freezing using a liquid handling robot.

[0105] Figure 4 shows the steps carried out by systems 2, 3, and 4. Fibroblasts are plated by an automated system (10), reprogramming factors are introduced by an automated system (12), iPSCs are isolated by automated sorting and isolation (14), desired clones are selected and propagated by an automated system (16), pluripotency status is automatically quality-checked by marker assays and embryoid body assays (QC) (18), and then the desired cells are automatically frozen and stored (20).

[0106] Figure 5 is a flowchart showing the steps (22) to (60) included in System 1.

[0107] Figure 5 illustrates an example workflow and a decision tree from biopsy material to fibroblast generation. The workflow is divided into an isolation stage (58) and a clean stage (60). Once the biopsy material is placed in the equipment, a technician plates the biopsy material into a 6-well plate (22) and collects the plate in an automated incubator (24). After a predetermined time has elapsed and the biopsy material has adhered to the plate, a liquid handling robot removes the plate from the automated incubator to supply nutrients and check the growth density with an automated microscope (26). The plate is returned to the incubator for growth (28). A liquid handling machine removes the plate from the incubator and replaces the culture medium with a medium that does not contain antibiotics or antifungals (30). The robot returns the plate to the incubator and incubates it for another 5 days (32). After that, the robot removes the plate and collects the culture medium into a daughter plate for mycoplasma testing (34). For mycoplasma testing, transfer the daughter plates to the isolation assay system (36). Select based on the positive signal of the assay (38). If the mycoplasma assay results show all wells of a 6-well plate are positive (40), discard them. If all wells of a 6-well plate are negative and do not contain mycoplasma, transfer the plate from the isolation to the clean growth system (46). If some wells are positive and some are negative, maintain the negative wells in the isolation (42). Pass the negative wells onto a new plate (44), transfer to the incubator, and discard the original plate containing the positive wells. Proceed with these cultures until mycoplasma retesting (24, 26, 28, 30, 32, 34, 36, 38). Monitor the growth of the clean cultures (50), pass them (52), and freeze them in freezing vials (54, 56).

[0108] Figures 6A, 6B1, 6B2, and 6C illustrate examples of the flow of patient samples through multi-well tissue culture plates during an automated reprogramming process. The upper part of each figure is a flowchart illustrating the flow of procedures performed by each step of the workflow (70, 88, 98). The multi-well cell culture plate in the lower part of each figure is shown along with a plate map. For example, the gray wells or groups of wells labeled with sample names represent wells containing samples (61-68, 72-86, 88-96). The movement of samples through the procedure, between plates or between wells, proceeds from left to right, as indicated by the arrows. As shown in Figure 6A, the automated iPSC induction process begins when patient samples and control fibroblast samples (61) are plated into individual wells of a 6-well plate (62). These samples are passaged in individual wells of a 24-well plate with a predetermined number of cells for infection with a virus encoding a reprogramming factor or for other means of introducing the reprogramming factor into cells (64). In the next step, unreprogrammed cells are removed from the reprogrammed samples by cell sorting or, preferably, concentration using magnetic beads, and plated at the same density into multiple wells of a 96-well plate (66). This example shows two such plates. In this example, six wells (indicated by dots drawn in the center of the wells) are identified by immunofluorescence assay using an automated imaging device as being pluripotent surface marker positive and containing a single clone (66). These clones are passaged and selected and rearranged on a minimum number of 96-well plates (68). The example figure shows six clones for each starting material and demonstrates that clones from 16 different starting materials can be arranged on a 96-well plate. To expedite plate processing, this selection step can be performed over multiple passages to fix the clones on a minimum number of plates. As shown in Figures 6B1 and 6B2, these clones are sequentially passaged until the culture density of stem cell colonies in the well is similar to that of their respective starting materials (72).Next, the samples on each plate are duplicated on a duplicate plate (74-86) to enable quality control (6) and selection of clones exhibiting appropriate stem cell characteristics. To initiate the QC process, this system generates one plate for the pluripotency quality control assay required to determine the pluripotency status of individual clones (74), and another plate for further passage (76). The plate for further passage is then passaged into three more plates for further quality control and proliferation (78, 80, 82). One plate is collected for a QC assay to analyze karyotype and genetic diversity (78). The second plate (82) is passaged into a v-bottom plate to form embryoid bodies for a QC assay to evaluate the differentiation potential of iPS clones (84). The final plate (80) continues to proliferate. Individual clones that do not pass the quality control by the aforementioned pluripotency QC assay (indicated by "×" in the wells in Figure 6) are not further processed. In the example shown in Figure 6B2, a single fixed 96-well plate (86) contains up to 32 iPS cell lines (or differentiated cell lines) as three iPS clones each, or up to 96 iPS cell lines as one clone each. The remaining clones are fixed to as few plates as possible until only 1 to 3 clones remain (86-92). As shown in Figure 6C, these are grown for cryopreservation while attached to the plates (88), or grown further (92-94) and then cryopreserved in freezing vials (96). Any or all information from screening using pluripotency markers is shown in Figure 6A (70). The quality control assays shown in Figure 6B1 can be used alone or in combination to determine which clones to select for fixation and alignment in an automated process.

[0109] Figures 7A, 7B, and 7C illustrate examples of equipment configurations required to achieve the workflow in one embodiment of the present invention. Figure 7A shows a system configuration for automated proliferation and quality control of a fibroblast bank. Figure 7B shows a system configuration for automated cell sorting using MultiMACS, automated introduction of reprogramming factors into patient samples, e.g., fibroblasts, and automated colony identification and rearrangement, for the automated thawing of patient samples, e.g., fibroblasts. Figure 7C shows a system configuration for automated proliferation, automated embryoid body production, and automated freezing of iPS clones.

[0110] As described herein, cells processed using the systems of the Disclosure may be stored for downstream use. For example, the processed cells of the Invention may be used to treat a subject. For example, reprogrammed or differentiated cells may be used to treat a disease or disorder in a subject. Accordingly, the Invention provides a method for treating a disease or disorder in a subject using the microfluidic-based systems of the Disclosure. The method comprises (a) obtaining a sample from a subject; (b) applying the sample to the system; (c) processing the sample in the system; and (d) administering the processed cells to the subject.

[0111] In the embodiments, the subject is healthy when the sample is obtained. The sample is processed to produce reprogrammed cells, which are cataloged and stored. After the subject is diagnosed with a disease and in need of medical treatment, the reprogrammed cells may be further processed to produce differentiated cells of the desired cell type to be subsequently used to treat the subject. In some treatments, reprogrammed cells may be used to treat the subject. Appropriate differentiated cells (ectoderm, mesoderm, or endoderm) may be derived from iPSCs produced by the method of the present invention. The mode of administration can be determined by those skilled in the art depending on the type of organ / injury to be treated. For example, iPSCs or differentiated cells derived therefrom may be administered by injection (as a suspension) or transplanted onto a biodegradable matrix.

[0112] The terms “healthy,” “normal,” or “clinically normal” mean that the subject does not have any known, apparent, or currently detectable disease or dysfunction that is associated with disease.

[0113] In another embodiment, the subject from which the sample is obtained is diagnosed with or at risk of having a disease or disorder. The sample is processed to generate reprogrammed cells, which are optionally stored. After the medical treatment of the subject has been determined, the reprogrammed cells may be further processed to generate differentiated cells of the desired cell type, which will then be used to treat the subject. In some treatments, the reprogrammed cells may be used to treat the subject.

[0114] The “subject” is any animal species, preferably a member of a mammalian species, and may be a human. Thus, the methods and compositions described herein are applicable to both human and livestock diseases. Furthermore, the subject is preferably a living organism, but the inventions described herein may also be used in postmortem analysis. The preferred subject is a human, most preferably a “patient,” where “patient” as used herein refers to a living human being receiving medical care for a disease or condition. This includes a person without a clear disease who is being examined for signs of symptoms. The subject may be a clearly healthy individual, an individual suffering from a disease, or an individual undergoing treatment for a disease.

[0115] Furthermore, compositions containing iPSCs or differentiated cells, such as compositions containing an effective amount of cells prepared by the system, which are used as research tools or pharmaceutical compositions, are also intended to be within the scope of the present invention.

[0116] In addition, the present invention relates to a method for testing a pharmaceutical agent by contacting iPSCs, differentiated cells derived therefrom, or differentiated cells with, for example, one or more pharmaceutical agents of interest, and subsequently detecting the effect of the pharmaceutical agent used on the contacted cells. To increase efficiency, the pharmaceutical agent is used with a series of iPSCs or differentiated cells derived therefrom. The cells may be diverse in terms of tissue source, differentiated cell type, or allele source, in order to enable the identification of cell or tissue types that respond preferably or unpredictably to one or more pharmaceutical agents of interest.

[0117] Furthermore, iPSCs produced by the automated system of the present invention may be used as a medium for introducing genes to correct genetic defects, such as osteogenesis imperfecta, diabetes mellitus, neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease), and various motor neuron disorders (MNDs) (e.g., amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA)).

[0118] The automated system of the present invention can also be used to provide specific cell types for biomedical research, and similarly, it can produce specific cell types for cell assays, such as cytotoxicity studies (studies to determine the effects of a test compound on cytotoxicity), either directly or as a precursor, and the teratogenicity or carcinogenicity of a test compound can also be determined by treating cells with the compound and observing and / or recording the effects of the compound on the cells, such as its effect on cell differentiation.

[0119] Although the present invention has been described with reference to the above embodiments, it will be understood that modifications and alterations are included within the spirit and scope of the invention. Accordingly, the present invention is limited only by the appended claims.

Claims

1. A microfluidic system for processing biological samples, (a) One or more microfluidic units capable of processing the sample, wherein the processing is (i) A step of isolating cells from the sample, (ii) Optionally, a step of growing isolated cells to produce a population of grown cells. (iii) A step of reprogramming isolated or proliferated cells, and A step to preserve cells derived from one or more of (iv), (i), (ii), or (iii). Microfluidic units including; and (b) One or more computer memory modules containing instructions for controlling one or more of the processes (i) to (iv); and (c) One or more computer processor modules configured to execute instructions Microfluidic systems, including

2. The system according to claim 1, further operable to perform analysis of (i) isolated cells, (ii) proliferated cells, (iii) reprogrammed cells, or combinations thereof.

3. The system according to claim 1, wherein one or more microfluidic units are further operable to differentiate iPSCs and generate cells of a desired cell type.

4. The system according to claim 3, further operable to perform analysis on cells of a desired cell type.

5. The system according to claim 3, further operable to store cells of a desired cell type.

6. The system according to claim 1, wherein the isolated cells are somatic cells.

7. The system according to claim 1, wherein the isolated cells are leukocytes.

8. The system according to claim 1, wherein the sample is selected from whole blood, blood fraction, serum, plasma, urine, sweat, lymph, feces, ascites, semen, sputum, papillary aspirate, postoperative seroma, wound drainage fluid, saliva, synovial fluid, ascites fluid, bone marrow aspirate, cerebrospinal fluid, nasal secretions, amniotic fluid, bronchoalveolar lavage fluid, pleural fluid, peripheral blood mononuclear cells, skin cells, total leukocytes, lymph node cells, spleen cells, skin biopsy material, umbilical cord blood, umbilical cord tissue, and tonsil cells.

9. The system according to claim 2, wherein the analysis includes image analysis, cell count analysis, cell surface marker analysis, cytokine secretion analysis, cell morphology analysis, polymerase chain reaction (PCR) analysis, sequence analysis, DNA analysis, RNA analysis, gene expression profiling, proteome analysis, metabolome analysis, immunoassay, nuclear exclusion analysis, or a combination thereof.

10. The system according to claim 1, wherein the step of preserving cells includes freezing cells.

11. The system according to claim 1, further comprising a graphical user interface.

12. The system according to claim 1, comprising at least two, three, or four microfluidic units.

13. The system according to claim 12, wherein the microfluidic units are connected wirelessly or electrically.

14. The system according to claim 12, wherein microfluidic units are fluidically connected.

15. The system according to claim 1, wherein the microfluidic units are arranged in a single housing container.

16. The system according to claim 1, wherein cells are stored on a microfluidic chip.

17. The system according to claim 1, further comprising a storage unit for storing cells at -80°C or below.

18. The system according to claim 1, further operable to process and store sample data related to stored cells.

19. The system according to claim 1, wherein cells are stored as a master cell bank and a working cell bank.

20. The system according to claim 1, wherein the step of reprogramming cells includes contacting the cells with one or more nuclear reprogramming factors.

21. The system according to claim 21, wherein the nuclear reprogramming factor is a polynucleotide, polypeptide, or small molecule.

22. The system according to claim 3, wherein differentiation includes contacting a cell with one or more differentiation factors.

23. The system according to claim 22, wherein one or more differentiation factors are polypeptides, vector-free gene regulatory oligonucleotides, microRNAs, messenger RNAs encoding differentiation factors, or oligonucleotides that affect the expression of differentiation factors by cells.

24. A method for processing biological samples, (a) the step of applying the sample to the system according to any one of claims 1 to 23; and (b) (i) Isolating cells from the sample, (ii) Optionally, growing isolated cells to produce a population of grown cells. (iii) Reprogramming isolated or proliferated cells, and Preserving cells from any of (iv), (i), (ii), or (iii) A process that includes processing a biological sample and thereby processing the sample in a system. Methods that include...

25. The method according to claim 24, further comprising the step of differentiating reprogrammed cells to generate cells of a desired cell type.

26. The method according to claim 25, further comprising the step of analyzing (i) isolated cells, (ii) proliferated cells, (iii) reprogrammed cells, cells of a desired cell type, or combinations thereof.

27. The method according to claim 26, wherein the analysis includes image analysis, cell count analysis, cell morphology analysis, polymerase chain reaction (PCR) analysis, sequence analysis, DNA analysis, RNA analysis, gene expression profiling, proteome analysis, metabolome analysis, immunoassay, nuclear exclusion analysis, or a combination thereof.

28. The method according to claim 24, further comprising the step of obtaining the sample from the target.

29. The method according to claim 28, wherein the subject has a disease or disability, or is at risk of having one.

30. The method according to claim 29, further comprising the step of treating a target with cells processed by the system.

31. The method according to claim 28, wherein the subject is a healthy individual.

32. The method according to claim 30, further comprising the step of preserving cells derived from the sample or cells processed from the sample until the subject is diagnosed with a disease or disorder.

33. The method according to claim 32, further comprising the step of treating a disease or disorder using stored cells.

34. A method for treating a disease or disorder in a subject, (a) The process of obtaining a sample from the subject; (b) the step of applying the sample to the system according to any one of claims 1 to 23; (c) (i) Isolating cells from the sample, (ii) Optionally, growing isolated cells to produce a population of grown cells. (iii) Reprogramming isolated or proliferated cells, (iv) Differentiating reprogrammed cells into desired cell types, and (v) Preserve cells derived from any of (i), (ii), (iii), or (iv) Including the process of processing the sample in a system; and (d) A step of administering any of the cells described in (i) to (v) to a target to treat a disease or disorder in the target. Methods that include...

35. The method according to claim 34, further comprising the step of analyzing (i) isolated cells, (ii) proliferated cells, (iii) reprogrammed cells, cells of a desired cell type, or combinations thereof.

36. The method according to claim 35, wherein the analysis includes image analysis, cell count analysis, cell morphology analysis, polymerase chain reaction (PCR) analysis, sequence analysis, DNA analysis, RNA analysis, gene expression profiling, proteome analysis, metabolome analysis, immunoassay, nuclear exclusion analysis, or a combination thereof.

37. The method according to claim 34, further comprising the step of diagnosing a disease or disorder in the subject.

38. A pharmaceutical composition comprising cells or a cell fraction thereof treated by the system according to any one of claims 1 to 23.

39. A cell bank comprising one or more populations of cells processed by the system described in any one of claims 1 to 23.

40. The cell bank according to claim 39, wherein each group is derived from a different sample.

41. The system according to claim 1, comprising at least one, two, three, or four microfluidic units operably linked to a second device.

42. The system according to claim 41, wherein the second device is a cell culture device, a bioreactor, a diagnostic device, an imaging device, a sequencing device, a nucleic acid amplification device, a nucleic acid or protein isolation device, a genome analysis device, a cell isolation device, a cell fractionation device, or any combination thereof.

43. The system according to claim 41, wherein the second system is a non-microfluidic device capable of performing one or more of (i) to (iv).

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