Automated electronic tissue stem cell counter

Abstract

An automated electronic tissue stem cell counter is configured to receive a tissue cell sample and incubate it at a required temperature for cells to multiply. The instrument can determine differential counts of vertebrate tissue stem cells based on the mitotic index (MI) of tissues or tissue cell samples in which stem cells are present. The instrument uses computer-implemented algorithms to calculate the differential tissue stem cell count of tissues or tissue cell samples from an input of the MI of tissues or cell samples.

Description

AUTOMATED ELECTRONIC TISSUE STEM CELL COUNTERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from and benefit of U.S. Provisional Patent Application Serial No. 63 / 730,555, filed on December 11, 2024, titled “Automated Electronic Tissue Stem Cell Counter,” which is hereby incorporated by reference herein in its entirety.FIELD

[0002] The present invention relates generally to cell counters, and more specifically, to an automated electronic tissue stem cell counter that can determine various measures of a cultured stem cell sample.BACKGROUND

[0003] Many research, clinical, and industrial investigators who perform tissue cell analyses maintain serially-transferred cell cultures for their cell analyses and cell experiments. These “stock” cell cultures are typically maintained in culture flasks with loose caps that are maintained in cell culture incubators. Cell culture incubators are typically maintained at 37 °C with a humidified, 5% carbon dioxide atmosphere, which serves to maintain sodium bicarbonate pH buffer systems in cell culture media. Culture vessels are kept “open” (e.g., loose caps, unsealed culture plate covers), so that their culture medium equilibrates with the incubator atmosphere.

[0004] The purpose of stock cell cultures is to have actively growing cells ready for expansion into more culture flasks to achieve greater cell numbers needed for use in cell analyses and cell experiments that may be performed in culture flasks, culture plates, culture dishes, or other project-specific devices.

[0005] Stock cell cultures can be used to culture stem cells. The stem cells of the organs and tissues of vertebrate animals, including humans, are responsible for the continuous replacement of expired and lost mature, differentiated cells throughout the vertebrate lifespan. Because of this function, tissue stem cells are used in transplantation therapies to restore damaged or lost stem cells. Tissue stem cell process applications support many other fields of cell research, tissue engineering, cell biomanufacturing, toxicology, and drug development.

[0006] Many investigators in academic cell research, medical, tissue and cell4908-1847-6928 1 Page 1 of 53 068661-000100USPTbiomanufacturing, pharmaceutical, toxicological, and cultured food disciplines have an interest in accurately and precisely quantifying the number of stem cells in tissue cell preparations. For example, in hematopoietic stem cell transplantation (HSCT) medicine, samples with too few stem cells result in HSCT failure in treated patients.

[0007] Obtaining cell proliferation rate data may also be beneficial to culturing and assessing the quality of stem cell samples.

[0008] A major challenge in the research and use of tissue stem cells is difficulty quantifying them accurately in the isolated and manipulated tissue cell samples in which they are found. Tissue stem cells are difficult to distinguish from the immediate progeny cells produced by their division, which are called committed progenitor cells. Although committed progenitor cells have lost the ability to replenish differentiated tissue cells continuously like their stem cell parents are able to do, they are otherwise indistinguishable by currently known morphological and molecular properties.

[0009] Currently available automated, electronic, general cell counters do not provide a differential stem cell count of samples. Human and animal tissue cell samples are always cell- heterogeneous. They contain varying subfractions of stem cells, non-stem committed progenitor cells, and differentiated cells. A differential stem cell count gives the number of stem cells in a sample without including the numbers of the other cell subtypes present in the sample. Currently used tests for stem cells, like the “CD34 count,” greatly over-estimate the number of stem cells in samples, because they also include the number of non-stem committed progenitor cells, which are a significant subfraction of the cells in tissue cell samples that typically greatly exceeds in number the subfraction of stem cells. Likewise, using present techniques, obtaining a differential tissue stem cell count-a count of the number of stem cells in a sample without confounding with the number of committed progenitor cells in the sample- is possible by only two methods. Both previous methods have significant technical challenges that preclude or limit their utility for routine differential tissue stem cell counting.

[0010] One method is the SCID mouse repopulating cell assay method. The SCID mouse repopulating cell assay method is only applicable to blood hematopoietic stem cells. It requires injections of tested cell samples into 30-40 immunodeficient SCID mice, maintenance of the injected mice for at least 16 weeks, and then examination of their organs and tissues to establish that continuous production of differentiated human blood cells occurred in them. The SCID mouse assay method can be expensive, cumbersome, time consuming, and imprecise, making it impractical for many applications that require routine differential counting of stem cells. In4908-1847-6928 1 Page 2 of 53 068661-000100USPTaddition, the SCID mouse repopulating cell assay only works for blood stem cells.

[0011] Another method is the kinetic stem cell counting method. The kinetic stem cell counting method uses computational simulation to compute the number of stem cells in a sample based on how the total number of cells in the sample changes during cell culture. It provides a precise differential count of stem cells from any type of organ or tissue. However, the method requires at least three days of cell culture and precise cell counting to compute differential tissue stem cell counts.

[0012] Additionally, evaluations of cell proliferation rates require expensive equipment (e.g., incubators, microscopes, digital cameras, microplate readers, flow cytometers), expensive instruments (e.g., automated, electronic, general cell counters; computers and special software), expensive reagents (e.g., colorimetric and fluorometric dyes), and well- salaried, trained personnel.

[0013] This extended culture analyses period, and the high cost of human-operated equipment are not ideally compatible with existing research, medical, and industrial workflows. Thus, there exists a need for more rapid, accurate and precise, automated stem cell counters and methods, as well as devices and methods that can be used to count stem cells, calculate proliferation rates, and more in various types of animal tissue.

[0014] The present disclosure provides a solution for these and other problems.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The disclosure, and its advantages and drawings, will be better understood from the following description of representative embodiments together with reference to the accompanying drawings. These drawings depict only representative embodiments and are therefore not to be considered as limitations on the scope of the various embodiments or claims.

[0016] FIG. 1 is a perspective view illustrating an automated electronic instrument, according to certain aspects of the present disclosure.

[0017] FIG. 2 is a top view of a disposable cell culture cartridge with a sliding lid, according to certain aspects of the present disclosure.

[0018] FIG. 3 is a perspective view of another disposable cell culture cartridge with a clip lid, according to certain aspects of the present disclosure.

[0019] FIG. 4 is a side view of the clip lid disposable cell culture cartridge, according to certain aspects of the present disclosure

[0020] FIG. 5 is a perspective view illustrating a rotary array of slots for inserting, heating,4908-1847-6928 1 Page 3 of 53 068661-000100USPTimaging, and counting prepared triplicate cell culture holders, according to an aspect of the present disclosure.

[0021] FIG. 6 shows data depicting mitotic index moving average values over time for Viall-Vial4 during experimental configurations of certain aspects of the present disclosure.

[0022] FIG. 7 shows data depicting a relationship between mitotic index values and stem cell counts over time for Viall-Vial4 during experimental configuration of certain aspects of the present disclosure.

[0023] FIG. 8 shows images captured by an optical assembly of the automated electronic instrument for various light configurations, according to certain aspects of the present disclosure.

[0024] FIG. 9 is an example graphical user interface of the automated electronic instrument, according to certain aspects of the present disclosure.

[0025] FIG. 10 is a diagram depicting operations between the automated electronic instrument, a computing system, and a database, according to certain aspects of the present disclosure.

[0026] FIG. 11 is a flowchart of an example method of determining a cell proliferation rate of a tested cell culture, according to certain aspects of the present disclosure.

[0027] FIG. 12 is a flowchart of an example method of determining a differential stem cell count of a tested cell culture, according to certain aspects of the present disclosure.DETAILED DESCRIPTION

[0028] Various embodiments are described with reference to the attached figures, where like reference numerals are used throughout the figures to designate similar or equivalent elements. The figures are not necessarily drawn to scale and are provided merely to illustrate aspects and features of the present disclosure. Numerous specific details, relationships, and methods are set forth to provide a full understanding of certain aspects and features of the present disclosure, although one having ordinary skill in the relevant art will recognize that these aspects and features can be practiced without one or more of the specific details, with other relationships, or with other methods. In some instances, well-known structures or operations are not shown in detail for illustrative purposes. The various embodiments disclosed herein are not necessarily limited by the illustrated ordering of acts or events, as some acts may occur in different orders and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events are necessarily required to implement certain aspects and features of4908-1847-6928 1 Page 4 of 53 068661-000100USPTthe present disclosure.

[0029] For purposes of the present detailed description, unless specifically disclaimed, and where appropriate, the singular includes the plural and vice versa. The word “including” means “including without limitation.” Moreover, words of approximation, such as “about,” “almost,” “substantially,” “approximately,” and the like, can be used herein to mean “at,” “near,” “nearly at,” “within 3-5% of,” “within acceptable manufacturing tolerances of,” or any logical combination thereof. Similarly, terms “vertical” or “horizontal” are intended to additionally include “within 3-5% of’ a vertical or horizontal orientation, respectively. Additionally, words of direction, such as “top,” “bottom,” “left,” “right,” “above,” and “below” are intended to relate to the equivalent direction as depicted in a reference illustration; as understood contextually from the object(s) or element(s) being referenced, such as from a commonly used position for the object(s) or element(s); or as otherwise described herein.

[0030] Referring generally to FIGs. 1-8, various embodiments are disclosed below to describe features of a cell counter. For example, Embodiments 1 and 2 below illustrate features related to an automated electronic tissue stem cell counter.

[0031] Exemplary Embodiment 1 - Automated Electronic Instrument for Mitotic Index Measurement, Cell Proliferation Rate Determination, and Differential Stem Cell Count Determination. Basic computer vision algorithms were successful in counting both adherent and suspension cells. This finding supported the creation of a custom microscope that would allow for the evaluation of different imaging modalities and allow for the creation of consumable cell culture cartridge designs.

[0032] Dark field imaging can provide improved contrast and perform well with computer vision-based cell counting algorithms. The automated electronic instrument can be used for automated stem cell counting based on tissue stem cell counting algorithms.

[0033] FIG. 1 is a perspective view illustrating an automated electronic instrument 100, according to certain aspects of the present disclosure. The automated electronic instrument 100 includes an adjustable stand arm 101, a heating block 102, an illumination source 103, one or more closed cell culture holders 104, motion stages 105, an imaging apparatus 106, and one or more cartridge holders 109. The heating block 102 may be an aluminum heating block with a resistive film heater to maintain a temperature in the closed cell culture holders 104. The heating block 102 may also include thermocouples or other devices configured to generate heat. The heating block 102 may heat the closed cell culture holders 104 from the bottom surface of the closed cell culture holders 104. In this scenario, the closed cell culture holders4908-1847-6928 1 Page 5 of 53 068661-000100USPT104 may be made of a material that is thermally conductive, and that allows for heat from the heating block 102 to heat the stored samples in the closed cell culture holders 104 to the desired temperature. In other embodiments, the heating block 102 may be configured to heat any surface of the closed cell culture holders 104. For example, the heating block 102 may heat a top surface or a lid of the closed cell culture holders 104. In further configurations, the heating block 102 is configured to heat each sample holder in each of the closed cell culture holders 104 separately, allowing granular control of temperature across samples. In some configurations, this temperature is 37 °C. The heating block 102 may also be configured to control the temperature of the closed cell culture holders 104 using a working fluid heated to a given temperature by the heating block 102 and configured to flow against a surface of the closed cell culture holders 104 to control the temperature of the samples inside the closed cell culture holders 104. For example, the heating block 102 may heat the closed cell culture holders 104 using heated air, heated water, or heated mineral oil as a working fluid.

[0034] In other configurations, the temperature is controlled by a computing system operably coupled with the automated electronic instrument 100. For example, an external computing system may be operably coupled with the automated electronic instrument 100 via a communications interface such as the internet. In other example embodiments, the automated electronic instrument 100 includes one or more processors. The imaging apparatus 106 may include a microscope 107 with interchangeable illumination sources to achieve either brightfield, phase contrast, or darkfield imaging. The imaging apparatus 106 also includes a digital camera 108. The imaging apparatus 106 can also include a two-axis polar scanning system to move across the entire growing surface area of the closed cell culture holders 104. In some example embodiments, the imaging apparatus 106 includes one or more lenses included in the microscope 107 or optically coupled to the digital camera 108. These lenses may be a bright field, dark field, phase contrast, or another type of lens. In some example embodiments, no lens may be used by the imaging apparatus 106.

[0035] The adjustable stand arm 101 is an arm, holder, stand, or other structure configured to accept the one or more closed cell culture holders 104. For example, the adjustable stand arm 101 may be fixedly secured to a mounting structure. The mounting structure is shaped to accept one or more closed cell culture holders 104. In some example embodiments, the adjustable stand arm 101 may move with respect to the automated electronic instrument 100. This may allow for the one or more closed cell culture holders 104 to move accordingly with respect to automated electronic instrument 100. For example, the adjustable stand arm 101 may4908-1847-6928 1 Page 6 of 53 068661-000100USPTinclude a telescoping mechanism that moves the one or more closed cell culture holders 104 in a vertical direction with respect to the imaging apparatus 106.

[0036] In other configurations, the one or more closed cell culture holders are fixedly secured to the heating block 102. In this scenario, the one or more closed cell culture holders may not be secured to the adjustable stand arm 101.

[0037] The one or more closed cell culture holders 104 may be fixedly secured with the automated electronic instrument 100 via one or more cartridge holders 109. Each cartridge holder 109 is shaped or configured to accept a corresponding closed cell culture holder 104. The cartridge holder 109 may fixedly secure the corresponding closed cell culture holder 104 using clips, latches, a sliding track, screws, or another structure for fastening the closed cell culture holder 104 to the cartridge holder 109. In some example embodiments, the cartridge holder 109 is configured to be movably positioned relative to the automated electronic instrument 100, thereby allowing the closed cell culture holder 104 to be movably positioned. In further configurations, the cartridge holder 109 may be moved according to instructions executed by a processor of the automated electronic instrument 100.

[0038] The one or more closed cell culture holders are configured to culture one or more biological samples, In some embodiments, the biological sample is a tissue sample and / or a heterogeneous population of cells.

[0039] In some embodiments, the automated electronic instrument 100 may be manufactured by a method of assembly involving integrating the electronic components and structural hardware. This method typically includes securing the heating block 102 and motion stages 105, mounting the imaging apparatus 106 and the digital camera 108, and interfacing these components via a computing system, such as computing system 1002 within a housing, ensuring alignment and calibration of the optical path and temperature control systems.

[0040] The methods and systems provided herein comprise a step of culturing a heterogeneous population of cells from a specific tissue comprising tissue stem cells and transiently amplifying committed progenitor cells and terminally differentiated non-dividing cells. In some embodiments of any of the aspects, the cells are a vertebrate population of cells. In some embodiments of any of the aspects, the cells are mammalian or human cells.

[0041] The heterogeneous cell population can be isolated from tissue of an adult mammal, preferably a human. Cells can be obtained from donor tissue, such as donor skin or other organs, by dissociation of individual cells from the connecting extracellular matrix of the tissue. Tissue is removed using a sterile procedure, and the cells are dissociated using any method4908-1847-6928 1 Page 7 of 53 068661-000100USPTknown in the art including treatment with enzymes such as trypsin, collagenase, and the like, or by using physical methods of dissociation such as with a blunt instrument. The heterogeneous cell population may also be obtained from bodily fluids; including, but not limited to, blood, umbilical cord blood, amniotic fluid, spinal fluid, pleural fluid, and lymphatic fluid.

[0042] In some embodiments, the heterogeneous population of cells is obtained from organ tissue. Tissue can be obtained from any organ, including but not limited to: organs of the musculoskeletal skeletal system, e.g. bone, cartilage, fibrous joints, cartilaginous joints, synovial joint, muscle, tendon, or diaphragm; organs of the cardiovascular system, e.g. artery, vein, lymphatic vessel, or heart; organs of the lymphatic system, e.g. primary (bone marrow, thymus), secondary (spleen and lymph node), CNS equivalent (cerebral spinal system); organs of the nervous system, e.g. brain, spinal cord, nerve; organs of the sensory system, e.g. ear, cochlea, eye; organs of the integumentary system, e.g. skin, subcutaneous tissue, breast (mammary gland), hair; organs of the immune system, e.g. myeloid (myeloid immune system) or lymphoid (lymphoid immune system); organs of the respiratory system, e.g. upper (e.g. nose, nasopharynx, larynx) or lower system (e.g. trachea, bronchus, lung); organs of the digestive system, e.g. mouth (salivary gland, tongue), upper gastrointestinal (GI; oropharynx laryngopharynx, esophagus, stomach), lower GI (e.g. small intestine, appendix, colon, rectum, anus) or accessory GI (e.g. liver, biliary tract, pancreas); organs of the urinary system, e.g. genitourinary system: e.g. kidney, ureter, bladder, and urethra; organs of the reproductive system, female (uterus, vagina, vulva, ovary, placenta) male (scrotum, penis, prostate, testicle, seminal vesicle); organs of the endocrine system, e.g. pituitary, pineal gland, thyroid, parathyroid, adrenal, or islets of Langerhans; and perinatal organs and tissues, e.g., umbilical cord, amniotic membrane, or placenta. Cells can be of the mesoderm, endoderm, or ectoderm origin.

[0043] Also useful in methods provided herein are cell culture systems that contain a heterogeneous population of cells including stem cells, transient cells, and terminally differentiated cells.

[0044] Somatic stem cells, also known as adult stem cells, are derived from tissues of a fetal or neonatal organism or from a post-natal adult organism, in contrast to other sources of stem cells such as embryonic stem cells, which may originate from a variety of sources of embryonic, pre-fetal tissues. Somatic stem cells are particularly attractive for a range of therapies in light of the ongoing controversies surrounding the use of embryonic stem cells. An4908-1847-6928 1 Page 8 of 53 068661-000100USPTadult stem cell is physiologically and phenotypically distinct from an embryonic stem cell not only in markers it does or does not express relative to an embryonic stem cell, but also by the presence of epigenetic differences, e.g. differences in DNA methylation patterns, and as provided herein, the cell kinetics properties of the cells, i.e., their patterns of division with respect to the cells that they produce. The properties of somatic stem cells are described, e.g., in US Patent Nos. 7,883,891 B2 and 7,867,712 B2, the contents of each of which is incorporated herein by reference in their entireties.

[0045] In some embodiments of any of the aspects, the cells are cultured in a standard suspension culture or an adherent cell culture. In some embodiments of any of the aspects, cells are cultured in a 3 -dimensional cell culture or a microcarrier cell culture. The type of cell culture method can be determined by one of skill in the art for a given application of the counting method provided herein.

[0046] Any medium can be used that is capable of supporting cell growth. For example, the medium can include but is not limited to: HEM, DMEM, RPMI, F-12, and the like. The medium can contain supplements or agents which are required for cellular metabolism such as glutamine and other amino acids, vitamins, minerals and useful proteins such as transferrin and the like. Medium may also contain antibiotics to prevent contamination with yeast, bacteria and fungi such as penicillin, streptomycin, gentamicin and the like. In some cases, the medium may contain serum derived from bovine, equine, chicken and the like. Serum can contain xanthine, hypoxanthine, or other compounds that enhance guanine nucleotide biosynthesis, although generally at levels below the effective concentration to suppress asymmetric cell kinetics. In one embodiment, for the cell culture the medium and serum contain levels below the effective concentration to suppress asymmetric cell kinetics. In some embodiments, the culture medium is a defined culture medium comprising a mixture of DMEM, F12, and a defined hormone and salt mixture.

[0047] The culture medium can be supplemented with a proliferation-inducing growth factor(s) or agents as defined herein. As used herein, the term “growth factor” refers to a protein, peptide or other biological molecule having a growth, proliferative, differentiating, or trophic effect on stem cells or other cell types. Growth factors that may be used include any trophic factor that promotes cells to proliferate, including any molecule that binds to a receptor on the surface of the cell to exert a trophic, or growth-inducing effect on the cell. Examples of proliferation-inducing growth factors include, but are not limited to, EGF, amphiregulin, acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2),4908-1847-6928 1 Page 9 of 53 068661-000100USPTtransforming growth factor alpha (TGF-alpha), and combinations thereof. Growth factors are usually added to the culture medium at concentrations ranging between about 1 fg / ml to 1 mg / ml. Concentrations between about 1 to 100 ng / ml are usually sufficient. Simple titration experiments can be easily performed to determine the optimal concentration of a particular growth factor. In one preferred embodiment, epidermal growth factor is used. In addition to proliferation-inducing growth factors, other growth factors may be added to the culture medium that influence proliferation and differentiation of the cells including NGF, platelet-derived growth factor (PDGF), thyrotropin releasing hormone (TRH), transforming growth factor betas (TGFPs), insulin-like growth factor (IGF-1) and the like.

[0048] Conditions for culturing should be close to physiological conditions. The pH of the culture medium should be close to physiological pH, preferably between pH 6-8, e.g., between about pH 7 to 7.8, with pH 7.4 being most preferred. Physiological temperatures range between about 30° C. to 40° C. Cells are preferably cultured at temperatures between about 32° C. to about 38° C., and more preferably between about 35° C. to about 37° C. Culture is often performed in ambient oxygen, but it may also be performed with regulated oxygen concentration, including hypoxic conditions.

[0049] The methods provided herein comprise a step of serially passaging the heterogeneous population of cells described herein wherein the total number of live and dead cells in the population of cells are counted. Methods of passaging cells are known in the art, see, e.g., US Pat Pg 2003 / 0133918 Al, the contents of which are incorporated herein by reference in its entirety.

[0050] Conventional serial passaging involves growing a cell culture until the culture vessel is replete with cells, e.g., greater than 70%, greater than 80%, or more confluency for adherent cultures or at saturation when nutrients become limiting in suspension cell cultures. Confluence of an adherent cell culture occurs when the culture vessel's maximum cell capacity is reached before dilution with additional cell culture medium and adherent surface. Saturation of a suspension cell culture occurs when the rate of cell proliferation begins to decrease before dilution with additional cell culture medium. When replete, the cells are harvested; and a fixed fraction of the harvested cells is transferred to a new culture vessel. The new culture is allowed to grow until replete again, and the dilution process is performed again. There are well known examples of such serial passaging schedules for both human cells and rodent cells. See, e.g., Hayflick, L. (1965) “The Limited In Vitro Lifetime of Human Diploid Cell Strains,” Exp. Cell Res. 37, 614-636; and Todaro, G. J. and Green, H. (1963) “Quantitative Studies of the Growth4908-1847-6928 1 Page 10 of 53 068661-000100USPTof Mouse Embryo Cells in Culture and Their Development into Established Lines,” J. Cell Biol. 17, 299-313, the contents of each of which are incorporated herein by reference in their entireties.

[0051] In the case of human tissue cell cultures, this serial process inevitably leads to a complete stoppage in new cell production. At the endpoint, the cultures are predicted to contain only terminal cells. This outcome results from first dilution of tissue stem cell number to zero, followed by completion of the remaining transient cells differentiation and production of terminal cells.

[0052] In some embodiments of any of the aspects, the cells described herein are passaged every 8 hours or more, 12 hours or more, 16 hours or more, 20 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 72 hours or more, or 96 hours or more, irrespective of their state of repletion.

[0053] In some embodiments of any of the aspects, a constant fraction of cells is serially passaged irrespective of their state of repletion. In some embodiments of any of the aspects, a constant number of cells are serially passaged, irrespective of their state of repletion. In some embodiments of any of the aspects, the cells are serially passaged until the number of total cells after two consecutive passages does not increase.

[0054] In some embodiments of any of the aspects, the cells are passaged on an irregular schedule based on when they achieve saturation density or confluence.

[0055] In some embodiments of any of the aspects, the method provided herein further comprises contacting the cells with an agent. The term “agent” refers to any entity to be administered to or contacted with a cell, tissue, organ or subject that is normally not present or not present at the levels being administered to the cell, tissue, organ, or subject. Agents can be selected from a group comprising: chemicals; small molecules; nucleic acids; nucleic acid analogues; proteins; peptides; peptidomimetics; peptide derivatives; peptide analogs; aptamers; antibodies; intrabodies; biological macromolecules; or functional fragments thereof.

[0056] The adjustable stand arm 101 may also include an illumination source or other lighting device, such as illumination source 103. The illumination source 103 extends from the adjustable stand arm 101 in the direction of the one or more closed cell culture holders 104. The illumination source 103 may be configured to illuminate at least a portion of the one or more closed cell culture holders 104. For example, the illumination source 103 may include a light source that is configured to illuminate a cell sample in one of the one or more closed cell culture holders 104. The illumination source 103 may also be configured to illuminate an image4908-1847-6928 1 Page 11 of 53 068661 -000100USPTof the one or more closed cell culture holders 104 that is captured by the imaging apparatus 106. The illumination source 103 may be placed over one or more closed cell culture holders 104. In this position, the illumination source 103 emits light in the direction of the imaging apparatus 106. The illumination source 103 may also be placed underneath the one or more closed cell culture holders 104. In this position, the illumination source 103 emits light upwards, away from the position of the imaging apparatus 106. In some example embodiments, the illumination source 103 may be integrated into the imaging apparatus 106.

[0057] The motion stages 105 are components of the automated electronic instrument 100 that are configured to move the one or more closed cell culture holders vertically, horizontally, and rotationally with respect to the illumination source 103 and the imaging apparatus 106. The motion stages 105 include one or more motors, servos, and other mechanical components that can move the structure of the automated electronic instrument 100 that is holding the closed cell culture holders 104. In some example embodiments, the motion stages 105 are operably coupled with the one or more processors of the automated electronic instrument 100 and / or the computing system operably coupled with the automated electronic instrument 100. The automated electronic instrument 100 may use the motion stages 105 to focus or otherwise adjust the images or video data captured by the imaging apparatus 106. For example, the motion stages 105 may move the closed cell culture holders 104 closer or farther from a lens of the imaging apparatus 106. In further configurations, the motion stages 105 are configured to move the closed cell culture holders 104 to mix or agitate a solution or sample contained in the closed cell culture holders 104.

[0058] In some example embodiments, the imaging apparatus 106 has a focusing axis that uses an auto-focusing algorithm to currently find the plane the cells are growing on. This autofocusing algorithm can cause the digital camera 108 or the microscope to move along one or more axes.

[0059] The automated electronic instrument 100 may also include a computing system, such as the computing system illustrated in FIG. 6. The computing system may be a part of the automated electronic instrument 100, or it may be communicatively coupled to the automated electronic instrument 100 by a network. In some configurations, the computing system is a small personal computing system, such as a New Unit of Computing (NUC) computer. The computing system can also be communicatively coupled to the automated electronic instrument by a wired or wireless local connection protocol such as Wi-Fi or LabJack. The computing system may control the automated electronic instrument 100 and allow for automated scanning.4908-1847-6928 1 Page 12 of 53 068661 -000100USPT

[0060] In some example embodiments, the functions of the automated electronic instrument 100 may be controlled by the computing system. For example, the adjustable stand arm 101 may include a motor operably coupled with the computing system. The motor may be used to control a position of the adjustable stand arm 101 according to instructions executed by the computing system. Likewise, the computing system may control the illumination source 103, the imaging apparatus 106, the digital camera 108, the automated cell culture holders 104, and / or the heating block 102.

[0061] The automated electronic instrument 100 includes one or more closed cell culture holders 104. These closed cell culture holders 104 may include one or more sample holders. The sample holders are configured to hold a sample of cells. The cells may be cultured or otherwise grown. The cells may be from a plant, an animal tissue, human tissue such as stem cell tissue, and / or synthetic or artificial tissue. The closed cell culture holders 104 may be fixedly secured to the automated electronic instrument 100 by the methods and techniques described herein. The automated electronic instrument 100 may also be configured to rotate the closed cell culture holders 104. Rotating the closed cell culture holders 104 move given portions of the closed cell culture holders 104 into a view of the imaging apparatus 106. By rotating different portions of the closed cell culture holders 104 into view of the imaging apparatus 106, the automated electronic instrument 100 may assess and analyze different cell samples in the one or more sample holders of the closed cell culture holders 104.

[0062] FIG. 2 is a top view of a disposable cell culture cartridge with sliding lid, according to certain aspects of the present disclosure. In some example embodiments, the closed cell culture holders 104 are lid-design cartridges 200, as shown in FIG. 2. The lid-design cartridges 200 include a machined body 202. The machined body 202 may be a polycarbonate material, a metallic material, a glass material, a plastic material, or another material that defines one or more lid sample holders 206 covered by a lid. This lid can slide to move vent ports 204 and inlet ports 208 in and out of a sealed area. The inlet port 208 allows for material to be injected or otherwise inserted into the corresponding sample holder 206. For example, sample material may be injected into the corresponding sample holder 206 via the inlet port 208. In some example embodiments, various contacting agents may be injected into the inlet port 208. For example, growth factors, cytokines, nutrients, metabolites, toxins, and drug candidates may be injected into the inlet port. The vent port 204 allows for material to escape or be removed from the corresponding sample holder 206.

[0063] Cell samples may be injected into at least one of the lid sample holders 206. In some4908-1847-6928 1 Page 13 of 53 068661-000100USPTembodiments, each lid sample holder 206 may accept at least one independent cell injection sample to provide cell cultures analysis. The holders require a small number of cells (<10,000) and a small amount of cell culture medium (<2 mLs per cell holder culture). Other populations of cells may be used in the sample holders 206. Non-adherent or adherent cells can be injected. Adherent cells attach to the bottom surface of the holders’ cell culture wells; and non-adherent cells settle onto the same bottom surface without adhering. Because the holder cultures are completely filled with cell culture medium and closed, they may not require a 5% carbon dioxide atmosphere, even when using sodium bicarbonate pH buffer systems.

[0064] In other example embodiments, the lid is a film that is held onto the machined body 202 of the lid-design cartridges 200 by an adhesive, magnet, or other mechanism. The film lid covers the inlet ports 208 and the vent ports 204, and can be peeled off to allow access to the vent ports 204 and the inlet ports 208. The film lid may be disposable, or it may be reusable, with an adhesive, magnet, or other mechanism that may be used multiple times.

[0065] FIG. 3 is a top view of another disposable cell culture cartridge with a latch lid, according to certain aspects of the present disclosure. In FIG. 3 a latch-lid cartridge 300 is shown. Latch-lid cartridge 300 includes a molded body 302 that defines one or more cell culture holders 304. The molded body 302 can be manufactured by injection molding. Other molding techniques may also be used to manufacture the molded body 302. In some configurations, the molded body 302 is made of polystyrene, polycarbonate, or another substance capable of being injection molded. For example, the molded body may be made of polyethylene, polypropylene, polystyrene, acrylonitrile butadiene styrene, polycarbonate, nylon, polyamide, acrylic, polymethyl methacrylate, polyethylene terephthalate, polyvinyl chloride, polyether ether ketone, polyoxymethylene, thermoplastic polyurethane, thermoplastic elastomers, silicone rubber, liquid silicone rubber, polylactic acid, polyhydroxyalkanoates, glass fiber reinforced plastics, carbon fiber reinforced plastics, or metal powders. Each sample holder 304 may include individual lids that are ultrasonic welded onto the sample holder 304. This ultrasonic weld hermetically seals the individual lid to each sample holder 304.

[0066] Each sample holder 304 may also include a vent port 306. The vent port 306 allows for material to escape or be removed from the corresponding sample holder 304. In some embodiments, the sample holder 304 may also include a fill port 307 that allows for material to be injected or otherwise inserted into the sample holder 304.

[0067] In other embodiments, the lids of each sample holder 304 may be latch lids secured by a latch 308. The latch 308 is configured to mechanically secure the lid onto the4908-1847-6928 1 Page 14 of 53 068661-000100USPTcorresponding sample holder 304. When the latch 308 is engaged, the lid may be fixedly secured to the corresponding sample holder 304 due to a continuous engagement of the latch 308 to the corresponding sample holder 304 and / or a structure of the molded body 302. The latch 308 may be shaped to create a hermetic seal between the lid and the corresponding sample holder 304 when the latch 308 is closed. For example, the lid may include a gasket or other structure configured to hermetically seal the lid and the sample holder 304 when the lid is closed and the latch 308 engaged. A toolless force exerted onto the latch 308 may cause the lid to disengage from the corresponding sample holder 304 and / or the molded body 302. This may expose at least a portion of the sample holder 304. When the lid is open, cell samples may be injected into or removed from the sample holder 304.

[0068] In one example embodiment, the lid is configured to slide linearly within a track defined by the structure of the sample holder 304. The latch 308 may fixedly secure the lid within the track at a fixed position when the latch 308 is closed, covering the opening of the sample holder 304 and protecting the sample within. When the latch 308 is disengaged, the lid may be slid within the track to expose at least a portion of the opening of the sample holder 304. This may allow cell samples to be inserted or removed from the sample holder 304.

[0069] FIG. 4 a side view of the latch -lid cartridge 300, according to certain aspects of the present disclosure. As shown in FIG. 4, the molded body 302 is shaped to define one or more sample holders 304. Each sample holder 304 includes a lid substrate 402. The lid substrate 402 is shaped to cover at least a portion of the sample holder 304. The lid substrate 402 may be made of a plastic, polymer, polycarbonate, polyethylene, glass, metallic, or other material. In some embodiments, at least a portion of the lid substrate 402 may be transparent or translucent to electromagnetic radiation. For example, the lid substrate 402 may be made of a clear plastic that is transparent or translucent to optical wavelengths. This allows the sample inside of the sample holder 304 to be imaged by the imaging apparatus 106. The lid substrate may be disposed within a track defined by a structure of the lid such as the constraint frame 404. The constraint frame 404 may provide a structural basis for the lid and structures to fixedly secure the lid substrate 402 to the molded body 302. The constraint frame 404 may include one or more rails or tracks that are shaped to accept corresponding flanges or structures of the lid substrate 402. The lid substrate 402 may slide linearly within this track. In other embodiments, the constraint frame 404 is shaped to allow the lid substrate 402 to flip upwards or pivot to expose at least portion of the opening of the sample holder 304.

[0070] The lid also includes a seal 406. The seal 406 is configured to create a hermetic seal4908-1847-6928 1 Page 15 of 53 068661-000100USPTbetween the lid substrate 402 and the interior of the opening of the sample holder 304 that is defined by the molded body 302. The seal 406 may be a silicone seal, which may be overmolded. The seal 406 may also be made of rubber, latex, a flexible polymer, neoprene, or another material that is capable of creating a seal between the lid substrate 402 and the sample holder 304. The seal 406 may be shaped to expose at least a portion of the opening of the sample holder 304 when the lid substrate 402 is opened. The seal 406 may also be fixedly secured with the lid substrate 402, and may be removed alongside the lid substrate 402 when the lid substrate 402 is opened.

[0071] In some embodiments, the lid substrate 402 of the sample holders 304 is manufactured from materials whose intrinsic optical properties are configured to enable dark field microscopy imaging of the cells settled or adhered thereon, allowing for contrast for automated image analysis. In other embodiments, a transparent bottom surface of the sample holders 304 is manufactured from materials whose intrinsic optical properties are configured to enable dark field microscopy imaging of the cells settled or adhered thereon.

[0072] In some embodiments, the disposable cell culture cartridge may be provided with at least one sample holder 304 pre-loaded with a dried or lyophilized reagent, such as growth factors, cytokines, nutrients, or other culture medium supplements, intended to rapidly dissolve upon injection of the cell sample and culture medium.

[0073] In some embodiments, the exterior surface of the cartridge body further comprises printed indicia, such as identification markings, orientation alignment guides for insertion into the automated instrument, and machine-readable codes (e.g., bar codes or QR codes) for tracking and data management by the automated electronic instrument or a computing system. Also provided herein are methods for manufacturing a disposable cell culture cartridge.

[0074] In some embodiments, the method of manufacturing a disposable cell culture cartridge includes the steps of forming the molded body 302 by injection molding, forming the transparent bottom surface, incorporating the inlet port 307 and vent ports 306, attaching the lid substrate 402 configured to create a hermetic seal using seal 406 (e.g., via ultrasonic welding), and subsequently sterilizing the completed disposable cell culture cartridge.

[0075] In some example embodiments, suspensions of harvested stock culture cells are injected directly into the disclosed sample holders 206, and 304 (e.g., see FIGs. 1-3). Each disposable cell culture holder, as shown in FIGs. 1 and 2, accepts three independent cell injection samples to provide triplicate cell cultures for the analysis. Other numbers of injection samples may be used based on the design and structure of the cell culture holders 104. The4908-1847-6928 1 Page 16 of 53 068661-000100USPTholders require a small number of cells (<10,000) and a small amount of cell culture medium (<2 mLs per cell holder culture). Other populations of cells may also be used as sample. Nonadherent or adherent cells can be injected. Adherent cells attach to the bottom surface of the holders’ cell culture wells; and non-adherent cells settle onto the same bottom surface without adhering. Because the holder cultures are completely filled with cell culture medium and closed, they do not require a 5% carbon dioxide atmosphere, even when using sodium bicarbonate pH buffer systems.

[0076] Cell culture holders such as the cell culture holders 104 prepared with cell samples may be loaded into the system for incubation and direct cell counting. Each holder rests in a slot constructed in an array with slots for multiple cell culture holders, as shown in FIG. 1, allowing simultaneous analyses for multiple stock cell cultures. The automated electronic instrument 100 has a heater such as heating block 102 that maintains each respective inserted cell culture holder at 37 °C. If needed, other temperature settings in the range of 32 °C to 39 °C are available. Because of its use of closed cultures and its self-heating capability, the disclosed system operates on a lab bench in the ambient lab atmosphere. It does not require placement in a cell culture incubator to operate. Instead, the heating block 102 may be used to incubate the sample holders at the desired temperature.

[0077] Once cell culture holders prepared with cells are inserted into heater slots and the system is closed (e.g., see FIG. 1), incubation and cell counting begin. An X-Y motorized microscope objective lens, such as those shown in imaging apparatus 106 with connected digital camera 108, is located in one position underneath the cell culture holder slots. Illumination for cell imaging is provided above the inserted cell culture holders in the same position. Individual cell holders are rotated into position above the objective lens location for counting. The motorized objective lens is able to scan and directly image cells either adhering to or settled onto the transparent bottom surfaces of all three culture wells in inserted cell culture holders. Cell imaging software is used to convert the cell images into quantitative cell number data. Software for distinguishing live cells from dead cells is also present. The imaging system performs periodic counts of the total cells in cultures, at a frequency set by the user (e.g., daily, hourly). The disclosed system reports to the user the mean cell count data and the corresponding mean population doubling time (PDT) calculations at each of multiple designated cell counting time.

[0078] The disclosed system inherently provides more accurate and more precise quantification of stock cell cultures’ cell proliferation rate and their health status.4908-1847-6928 1 Page 17 of 53 068661-000100USPT

[0079] The disclosed system can also determine a mitotic index (MI) of the cells cultured in the closed cell culture holders 104. The mitotic index can also be determined by counting the number of cells that divide during a 1-hour interval of culture and dividing it by the total number of cells present. The mitosis phase, which ends with cells undergoing a division into two cells, takes on average 1 hour. So, the number of cells that divide during a 1-hour period are equivalent to the mitotic cells present during that period.

[0080] The automated electronic instrument 100 may also determine various other characteristics of the tested cell samples cultured in the closed cell culture holders 104. The imaging apparatus 106 may capture an image, a timeseries of images, or video data associated with the tested cell samples in the closed cell culture holders. Based on these data, the automated electronic instrument 100 may process the data and determine one or more metrics characterizing the tested cell sample. For example, the automated electronic instrument 100 may determine a fraction of the cells in the cell sample that are in a mitotic phase. In some example configurations, the fraction of cells in the tested cell sample that are in a mitotic phase is determined based on flow cytometry. In other example configurations, the automated electronic instrument 100 determines the fraction of cells in the tested cell sample that are in a mitotic phase based on cell biomarkers. The automated electronic instrument 100 may also use immunohistochemistry to determine a fraction of cells in the tested cell sample that are in a mitotic phase. For example, anti-phospho-histone H3 solution may be injected or otherwise inserted into the tested cell sample to determine the fraction of cells in the tested cell sample that are in a mitotic phase. Other methods of determining the fraction of cells in a mitotic phase may also be used by the automated electronic instrument 100.

[0081] The automated electronic instrument 100 may also determine effects of various contacting agent or other chemicals on the cells contained in the tested cell sample in the closed cell culture holders 104. For example, contacting agents such as growth factors, cytokines, nutrients, metabolites, toxins, and drug candidates may be injected or otherwise placed into the closed cell culture holders 104 so that they contact the cells in the tested sample being analyzed by the automated electronic instrument 100. The automated electronic instrument 100 may use the imaging apparatus 106 to determine an effect of the contacting agents or other chemicals on the cells. For example, the automated electronic instrument 100 may determine a cell proliferation rate of the cells prior to the introduction of the contacting agent, and a cell proliferation rate after the introduction of the contacting agent. Other metrics and effects may be determined by the automated electronic instrument 100.4908-1847-6928 1 Page 18 of 53 068661 -000100USPT

[0082] In some example embodiments, a rotary structure may be used by the automated electronic instrument 100 to analyze multiple cell culture holders 104. FIG. 5 is a perspective view illustrating a rotary array 500 of slots 504 for inserting, heating, imaging, and counting prepared triplicate cell culture holders, according to an aspect of the present disclosure. The rotary array 500 includes a main body 502 that defines a plurality of slots 504. The main body 502 may be made of plastic, polymer, polycarbonate, polystyrene, polyethylene, metal, rubber, or another material. The main body 502 may be manufactured by an injection molding process, a machining process, or a 3D printing process. In some example embodiments, each slot 504 is shaped to accept a corresponding cell culture holder 104. The slot 504 may include structures or features that hold the cell culture holder 104 in place in the slot 504 when the cell culture holder 104 is inserted into the slot 504. In other embodiments, the slot 504 includes latches, screws, adhesives, fasteners, or other devices to fixedly secure each cell culture holder 104 to each slot 504.

[0083] The rotary array 500 is configured to rotate around a point at its center. This causes each cell culture holder 104 inserted into a corresponding slot 504 of the rotary array 500 to move. When the rotary array 500 is installed into the automated electronic instrument 100, this may cause each cell culture holder 104 to move relative to the imaging apparatus 106 of the automated electronic instrument 100.

[0084] In some embodiments, the main body 502 of the rotary array 500 is composed of a thermally conductive material configured to reliably transfer heat from an external heating element to the cell culture contained within the slots 504. In some embodiments, this configuration ensures temperature uniformity of ±1°C across all individual sample holders throughout the culture period.

[0085] Each cell culture holder slot 504 has an independent heating element such as the heating block 102. In some example embodiments, each slot 504 has its own heating block 102. In other example embodiments, the automated electronic instrument 100 may have one or more heating blocks 102 that are thermally coupled by heat pipes or other mechanisms to the slots 504. Using the heating block 102, the automated electronic instrument 100 may heat the inserted cell culture holders 104. The cells can be counted over this 1-hour period by the imaging apparatus 106.

[0086] FIG. 6 is a set of graphs 600 depicting mitotic index moving average values over time during experimental configurations of certain aspects of the present disclosure. As shown in FIG. 6, the automated electronic instrument 100 may be used to determine a number of new4908-1847-6928 1 Page 19 of 53 068661-000100USPTcells produced in a tested sample in one of the sample holders 206 or 304 of the cell culture holder 104. The automated electronic instrument 100 may determine these values using the imaging apparatus 106. For example, the imaging apparatus 106 may capture an image of the tested sample at the start of a one-hour period of time. The computing system operably coupled to the automated electronic instrument 100 may then determine, by one or more processors, the number of cells present in the captured image. This may be done by analyzing the captured image to determine a number of cells present in the captured image via image processing algorithms, a trained machine learning or artificial intelligence process, or by another computer-implemented method. In some example embodiments, the imaging apparatus 106 may capture multiple images of the tested sample at the same time. The total number of cells present across the multiple images may be processed by the one or more processors to determine a total number of cells present in the tested sample at a certain time. The imaging apparatus 106 may capture one or more images of the tested sample at an end time of the one- hour interval, subsequent to the start time and separated by a period of one hour. The number of cells present in the end time image may be compared to the number of cells present in the start time image to determine the number of new cells produced during 1-hour culture intervals.

[0087] In some example embodiments, the automated electronic instrument 100 may associate each cell with identification information stored in a memory or storage of the computing system to distinguish it from the other cells. When processing the images captured by the imaging apparatus 106, the identification information may be used to distinguish new cells, which have not previously been captured in an image taken by the imaging apparatus 106, from cells that have already been identified, or cells that had been identified that have since died at the time of the image capture. Based on these data, the automated electronic instrument 100 may determine the number of new cells produced in the tested sample over the one hour period.

[0088] Other intervals of time may be used. The computing system operably coupled with the automated electronic instrument 100 may determine any period of time for the imaging apparatus 106 to capture images of the tested sample.

[0089] In the graphs 600, the MI values for the tested samples were determined. To obtain the MI values, the number of new cells produced over the one hour intervals was divided by the total number of cells present at the start of each one hour interval. This may determine an MI value for a tested cell sample. The MI values may be used to compute a moving average across a certain number of intervals. In graphs 600, the automated electronic instrument 1004908-1847-6928 1 Page 20 of 53 068661-000100USPTdetermined moving averages for 12-hour intervals and plotted these values versus the number of days of serial passage culture. Other moving averages and statistical values may be determined for any amount of time.

[0090] FIG. 7 is a set of graphs 700 depicting a relationship between mitotic index values and stem cell counts over time during experimental configuration of certain aspects of the present disclosure. FIG. 7 shows how the respective MI values and SCF values during the serial cell culture of the three distinct stem cell sources were interrogated for statistically-confident mathematical relationships.

[0091] Linear relationships were detected between a mathematical function relating MI to SCF versus days of serial culture for all three tissue stem cell sources. The statistical confidence of the individual linear relationships was high (p < 0.0001).

[0092] The three linear relationships differed significantly from each other, indicating that the relationships are specific for different types of tissue stem cells. The samples for the two types of HSCs were cultured in the same type of culture medium. Therefore, the quantitative difference in linear relationships for the two different HSC sources was not attributable to a difference in the type of cell culture medium.

[0093] All three linear regressions have a quantitatively similar y-intercept value. This indicates a universal conversion factor for converting the MI of primary, uncultured cell samples to their differential stem cell count. The SCF determined at Days = 0 (z.e., the y- intercept) provides an estimate of the SCF in the source organ or tissue from which the cell samples were isolated. The automated electronic instrument depicted in FIGs. 1-4 may use this universal conversion factor to determine a differential tissue stem cell count based on a measured mitotic index of the tested cell culture.

[0094] The automated electronic instrument 100 may therefore be able to estimate a fraction of stem cells in a source of a tested cell sample. For example, the source of a tested cell sample may be a human organ or tissue such as bone marrow. The tested cell sample may contain stem cells from the bone marrow. The automated electronic instrument 100 may perform any of the operations of the present disclosure to determine cell counts, mitotic indices, and other data characterizing the tested cell sample. Based on the mathematical relationships detailed with respect to FIG. 7, the MI value of the tested cell sample may be used to determine a fraction of stem cells in the bone marrow that the sample was taken from.

[0095] FIG. 8 depicts example images captured by an optical assembly of the automated electronic instrument, according to certain aspects of the present disclosure. The images 8004908-1847-6928 1 Page 21 of 53 068661-000100USPTdepict multiple images collected by the automated electronic instrument 100. These images may be captured by the imaging apparatus 106, for example using the digital camera 108. The images can be filtered and process to compute the percentage of the image that is colored white. The filtering steps can include edge detection, morphing, binary image processing, statistical analysis, and processing by a trained artificial intelligence or machine learning process. Based on the filtered image and the comparison of filtered images collected over a given time interval, cell growth, and therefore the mitotic index, of the tested cell culture can be determined.

[0096] Other methods of determining the mitotic index, such as flow cytometry with antibodies for mitotic cell-specific biomarkers; immunohistochemistry with antibodies for mitotic cell-specific biomarkers; morphological imaging by microscopy, can also be used by the automated electronic instrument 100.

[0097] In some example configurations, the automated electronic instrument 100 includes a processor configured to generate a graphical user interface. The graphical user interface can also be generated by a computing system communicatively coupled to the automated electronic instrument 100. The processor and / or the computing system can perform operations that cause the graphical user interface to be presented to a user via a display.

[0098] FIG. 9 is an example graphical user interface 900 of the automated electronic instrument 100, according to certain aspects of the present disclosure. The automated electronic instrument 100 may cause presentation of the graphical user interface 900 on a display communicatively coupled with the automated electronic instrument 100 and / or the computing system operably coupled with the automated electronic instrument 100. The graphical user interface 900 can be used to control the motion and poisoning of the imaging apparatus 106. For example, the graphical user interface 900 can include one or more control elements 902 that allow a user to specify a focus, a linear motion in an axis, or a rotary motion. Other control elements may be included in the control elements 902.

[0099] The visual display 904 of the graphical user interface 900 shows a live feed of images captured by the digital camera 108 of the imaging apparatus 106. For example, the digital camera 108 may capture a video data characterizing the view of the tested cell sample being analyzed by the automated electronic instrument 100. The digital camera and the imaging apparatus 106 may transmit the video data to the automated electronic instrument 100 and / or the computing system operably coupled with the automated electronic instrument 100. The automated electronic instrument 100 may cause presentation of the visual display 904 on the display communicatively coupled with the automated electronic instrument 100 and / or the4908-1847-6928 1 Page 22 of 53 068661-000100USPTcomputing system operably coupled with the automated electronic instrument 100.

[0100] In some example embodiments, the visual display 904 may display images, or video captured of the tested cell sample that have been processed. For example, the visual display 904 may show brightened, higher-contrast, false color, or edge-detected representations of the video data or images captured by the imaging apparatus 106. In some example embodiments, the visual display 904 also includes an indication including information associated with the visual transformations or processing that have been performed on the images or video data captured of the tested cell sample.

[0101] In other example embodiments, the visual display 904 may indicate which lens of the imaging apparatus 106 or the microscope 107 the images or video data captured of the tested cell sample were captured through. For example, the visual display 904 may indicate the lens selected during the capturing of the images or video data. The visual display 904 may also include graphical elements that allow a user to select a lens for the imaging apparatus 106 or the microscope 107 to use. Based on this selection, the automated electronic instrument 100 may perform operations that change the lens used by the imaging apparatus 106 or the microscope 107 to the lens selected by the user.

[0102] In some example configurations, the graphical user interface 900 of FIG. 9 includes a visual element 906 configured to indicate which sample holder of the closed cell culture holder 104 inserted into the automated electronic instrument 100 is being viewed by the imaging apparatus 106. As shown, the visual element may indicate to a user of the automated electronic instrument 100 which closed cell culture holder 104 is being viewed. In some example embodiments, the visual element 906 may also allow for a selection of the sample holder to be analyzed and / or viewed. For example, a user may indicate which sample holder should be viewed using the visual element 906. The automated electronic instrument 100 may then execute instructions that cause the selected sample holder to be moved into the view of the imaging apparatus 106. For example, the automated electronic instrument 100 may rotate the selected sample holder into the view of the imaging apparatus 106 using a rotary device such as the rotary array 500. The automated electronic instrument may also move the imaging apparatus 106 to view the selected sample holder.

[0103] FIG. 10 is a diagram depicting operations between the automated electronic instrument, a computing system, and a database, according to certain aspects of the present disclosure. The example operations depicted in FIG. 10 include operations amongst the components of FIG. 10. FIG. 10 depicts operations at stages A-C. The stages are examples and4908-1847-6928 1 Page 23 of 53 068661-000100USPTare not necessarily discrete occurrences over time (e.g., the operations of different stages may overlap). Additionally, FIG. 10 is an overview of example operations.

[0104] At stage A, the automated electronic instrument 100 captures an image of the cell sample. The automated electronic instrument 100, for example, can use an imaging apparatus 106 that includes a digital camera 108 to capture an image of a tested cell sample cultured in a receptacle such as closed cell culture holder 104. The image may be captured through the use of a microscope and the digital camera 108. In some example embodiments, the image may be a video feed captured by the digital camera 108

[0105] At stage B, the tested cell sample is analyzed by a computing system based on the captured image. For example, a computing system such as computing system 1002 operably coupled to the automated electronic instrument 100 can measure the number of cells in the sample based on the image. The computing system 1002 may also determine a proliferate rate of the cells in the tested cell sample. For example, the automated electronic instrument 100 may capture a first series of images over time, and the images in the first series of images may be compared to determine a proliferation rate for the cells in the tested cell sample. Computing system 1002 may be one or more processors, a memory, and storage included in the automated electronic instrument 100, a personal computer, a tablet computer, a laboratory instrument control system, or a computing server operably coupled to the automated electronic instrument 100. Other types of computing systems may also be operably coupled to the automated electronic instrument 100. The computing system may also be part of the automated electronic instrument 100.

[0106] At stage C, the mitotic index of the tested cell sample is determined based on the analysis and the captured image. Measuring the mitotic index can be done by filtering and analyzing the captured image by a processor of the computing system 702. In some configurations, the mitotic index can be determined by the application of a trained artificial intelligence or machine learning process by the computing system 702.

[0107] At stage D, the stem cell count is determined based on the mitotic index. The stem cell count may be a differential tissue stem cell count. The stem cell count can be calculated based on the computing system 702 based on the mitotic index and a linear relationship, such as the relationship shown in FIG. 4. In some example configurations, the linear relationship between the mitotic index and the stem cell count can be used to determine a universal conversion factor or equation that is used by the computing system 702 or the automated electronic instrument 100 to determine the stem cell count based on the mitotic index.4908-1847-6928 1 Page 24 of 53 068661-000100USPT

[0108] Exemplary Embodiment 2 - Method of Cell Proliferation Rate Determination. The automated electronic instrument 100 may be used to determine a cell proliferation rate for a tested sample cultured in a sample holder such as sample holder 206 or sample holder 304. The disclosed system can be readily deployed to make common cell proliferation research studies easier and less expensive to conduct. Examples of evaluated agents include cell growth factors, cell cytokines, environmental toxicants, and pharmaceutical drug candidates.

[0109] FIG. 11 is a flowchart of an example method 1100 of determining a cell proliferation rate of a tested cell culture, according to certain aspects of the present disclosure. The tested cell sample may be placed and / or grown in one of the sample holders 206 or 304, depending on the type of cell culture holder 104 used by the automated electronic instrument 100. The flow begins at block 1102.

[0110] At block 1102, a first time series of images of the cell sample is captured. The first time series of images may be captured across a first period of time by the imaging apparatus 106. The imaging apparatus 106 may perform periodic counts of the total cells in cultures, at a frequency set by the user (e.g., daily, hourly). In other embodiments, the frequency is determined by the computing system operably coupled to the automated electronic instrument 100. Counting is performed by taking periodic images of the cell culture sample using the digital camera 108. Each image may be analyzed by various algorithms to determine a cell count for the captured image. For example, each image captured by the digital camera 108 may be filtered by the one or more processors of the computing system. The filtering steps can include edge detection, morphing, binary image processing, statistical analysis, and processing by a trained artificial intelligence or machine learning process. In some example embodiments, multiple time series of images may be captured by the imaging apparatus 106.

[0111] At block 1104, a cell count is measured for each image. After each image is captured by the imaging apparatus 106, a cell count of the cells in the image is determined based on the filtered image. For example, the one or more processors of the computing system coupled to the automated electronic instrument 100 may count the number of spots in the image that depict a cell. This can be done with simple pixel comparison, edge detection, binary image processing, statistical analysis, and processing by a trained artificial intelligence or machine learning process. Each cell count value for each image may be stored in a memory or storage of the computing system.

[0112] At block 1106, a cell proliferation rate is determined for the sample based on the first time series of images. This may be done by determining a mean initiating cell number and4908-1847-6928 1 Page 25 of 53 068661-000100USPTmean final cell number for the first time series of images. In some examples, the mean initiating cell number and mean final cell number are determined based on a plurality of time series of images. Based on the initiating cell number and final cell number, a mean population doubling time (PDT) is determined. In some example embodiments, this PDT is the cell proliferation rate.

[0113] The disclosed system can perform the same evaluations conveniently at significantly reduced cost and with improved precision. By comparing PDT data from cell culture holders prepared with cells in control cell culture medium to PDT data from cell culture holders prepared with cells in cell culture medium supplemented with varied concentrations of a tested agent, the system can continuously evaluate for test agent-dependent differences in cell proliferation rate with increasing incubation time. The exemplary processes described above may be performed multiple times to determine multiple PDTs for a sample. Likewise, the processes described above may be performed multiple times across different samples of the cell culture holder 104. Mean PDTs may be determined from the plurality of PDTs determined by these processes. Other values for measuring the cell proliferation rate derived from the cell counts determined by the imaging apparatus 106 may be used and / or determined by the automated electronic instrument 100.

[0114] Exemplary Embodiment 3 - Method of Differential Tissue Stem Cell Counting. To perform a differential stem cell count for a tissue cell sample, the user introduces a suspension of the evaluated tissue cell sample into one of the disclosed system’s closed cell culture holders (FIGs. 2-3). The prepared cell culture holder is inserted into the automated electronic instrument; and the disclosed system is closed to begin incubation at 37°C, cell imaging, and cell counting.

[0115] To determine an algorithm for determining a differential tissue stem cell count without lengthy incubation times, kinetic stem cell (KSC) counting data for three different sources of human tissue stem cells were interrogated for mathematical relationships between the mitotic index (MI) of serial cultures of the stem cell samples and their stem cell-specific fractions (SCF). For reference, the SCF is a differential tissue stem cell count. It is the fraction of tissue stem cells in a sample and does not include the fraction of committed progenitor cells.

[0116] In one example embodiment, the human stem cell sources used were adipose tissue- derived mesenchymal stem cells (Ad-MSCs), CD34-selected umbilical cord blood hematopoietic stem cells (34CB-HSCs), and CD34-selected mobilized peripheral blood cell HSCs (34MPB-HSCs. Other stem cell or animal cell types may be used to derive the algorithms4908-1847-6928 1 Page 26 of 53 068661 -000100USPTdisclosed herein.

[0117] The algorithms to calculate the differential tissue stem cell count may take in a mitotic index (MI) as input. MI is the fraction of cells that divided during any 1-hour time interval while the tissue sample was cultured in the sample cartridges shown in FIGs. 2 and 3.

[0118] FIG. 12 is a flowchart of an example method of determining a differential stem cell count of a tested cell culture, according to certain aspects of the present disclosure. FIG. 12 may be performed by one or more processors of the automated electronic instrument 100 and / or the computing system, such as computing system 1002, operably coupled to the automated electronic instrument 100. The flow of FIG. 12 begins at block 1202.

[0119] At block 1202, an image of a cell sample is captured. For example, an automated electronic instrument 100 that includes a digital camera 108 can capture an image of the cell sample. The digital camera 108 can be optically coupled to a microscope to capture the image of the cell sample. For example, the digital camera 108 may be optically coupled to an imaging apparatus 106 that includes a microscope 107. The imaging apparatus 106 and / or the microscope 107 may also include one or more lenses for imaging the tested cell sample using a lens.

[0120] At block 1204, a mitotic index of the cell sample is determined. The mitotic index can be determined based on the captured image of the cell sample. For example the image can be filtered and / or analyzed to identify a number of cells in the sample, a growth rate of the cells, or other metrics of the cell sample. In some example configurations, a plurality of images of the cell sample can be captured over a time interval, and based on the plurality of images, a mitotic index of the cell sample is determined. The mitotic index may be determined by the application of a trained machine learning or artificial intelligence process to the one or more images captured by the automated electronic instrument 100.

[0121] At block 1206, a stem cell count of the cell sample is determined. This stem cell count is determined based on the mitotic index of the cell sample. For example, the stem cell count may be a differential tissue stem cell count, and the differential tissue stem cell count may be determined based on a mathematical relationship between the mitotic index and the differential tissue stem cell count.

[0122] Also provided herein are kits for use with the automated electronic instrument 100.

[0123] In some embodiments, a kit suitable for automated cell culture analysis may include a plurality of disposable cell culture cartridges. The kit comprises the disposable cell culture cartridges (e.g., at least 10 cartridges) packaged in sterile individual wrappers, instructions for4908-1847-6928 1 Page 27 of 53 068661 -000100USPTinjecting cell samples, and a sterile cell culture medium.

[0124] Although the disclosed embodiments have been illustrated and described with respect to one or more implementations, equivalent alterations and modifications will occur or be known to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In addition, while a particular feature of the invention may have been disclosed with respect to only one of several implementations, such features may be combined with one or more other features of the other implementations as may be desired and advantageous for any given or particular application.Exemplary Embodiment 3 - Method of Determining a Fraction of Tissue Stem Cells in a Tissue Sample

[0125] In one aspect, provided herein is a method of determining the fraction of tissue stem cells present in a tissue sample cultured in the automated electronic instrument. The method comprises:(a) culturing a heterogeneous population of cells, for example, in one of the one or more closed culture cell holders, from a specific tissue comprising tissue stem cells, transiently amplifying committed progenitor cells and terminally differentiated non-dividing cells;(b) serially passaging said population of cells wherein the total number of live and dead cells in the population of cells are counted via the automated electronic instrument detailed above;(c) processing the total cell count data by one or more processors of the automated electronic instrument to determine the stem cell fraction (SCF) of the population of cells throughout the periods of the serial passaging;(d) calculating, by the one or more processors of the automated electronic instrument, the population doubling times (PDT) for said tissue cells during non-saturated or non-confluent periods of the serial passaging; and(e) quantifying the number of tissue stem cells in the subsequent tissue samples of the same type based on (d), without serial passaging.

[0126] In another aspect, provided herein is a method of treating a subject in need thereof with a population of stem cells, the method comprising:4908-1847-6928 1 Page 28 of 53 068661-000100USPT(a) determining the stem cell fraction present in a tissue sample according to the method provided herein; and(b) administering to the subject in need thereof a dosage of stem cells based on the stem cell fraction (SCF).

[0127] In one embodiment of any of the aspects, the PDT is determined by the following Formula A: (Formula A),= initial starting time, t2= final time, N = the initial cell number, and N2= the final cell number. Operations based on Formula A may be performed by the one or more processors of the automated electronic instrument.

[0128] In another embodiment of any of the aspects, the SCF is determined by one of the following derivative Formulae B-D:SCF = PDT (mean quotient of SCF / PDT) (Formula B);SCF =e-(mPDT +b) (Formula C); orSCF = PDTe(at 3 + bt 2 + ct + d)(Formula D), where t = time. Operations based on Formulae B-D may be performed by the one or more processors of the automated electronic instrument.

[0129] It is noted that CPD is a routine measure of the proliferative capacity of tissue cell preparations

[0130] In another embodiment of any of the aspects, the cells are passaged every 12 hours or more, 24 hours or more, 48 hours or more, 72 hours or more, or 96 hours or more in the closed cell culture holders.

[0131] In another embodiment of any of the aspects, the cells are passaged on an irregular schedule based on when they achieve saturation density or confluence.4908-1847-6928 1 Page 29 of 53 068661 -000100USPT

[0132] In another embodiment of any of the aspects, a constant fraction of cells is serially passaged.

[0133] In another embodiment of any of the aspects, a constant number of cells are serially passaged.

[0134] In another embodiment of any of the aspects, the PDT is determined in vitro or by in vivo imaging techniques.

[0135] In another embodiment of any of the aspects, the PDT is determined by using a counting method selected from the group consisting of: counting cells, an absorbance assay, a turbidity assay, weighing cells, a fluorescent assay, and combinations thereof.

[0136] In another embodiment of any of the aspects, the cells are serially passaged until the number of total cells after two consecutive passages does not increase.

[0137] In another embodiment of any of the aspects, the processing time is determined by a computer processing system such as the automated electronic instrument.

[0138] In another embodiment of any of the aspects, the cells are processed until a predetermined number of tissue stem cells are present in the sample.

[0139] In another embodiment of any of the aspects, the cells are a vertebrate population of cells.

[0140] In another embodiment of any of the aspects, the cells are a mammalian population of cells.

[0141] In another embodiment of any of the aspects, the cells are a human population of cells.

[0142] In another embodiment of any of the aspects, the culturing is selected from the group consisting of: 3-dimensional cell culture; suspension cell culture; adherent cell culture; microcarrier cell culture; and any combination thereof.

[0143] In another embodiment of any of the aspects, wherein the cells are cultured in normoxic conditions. In another embodiment of any of the aspects, wherein the cells are cultured in hypoxic conditions.

[0144] In another embodiment of any of the aspects, the method further comprises contacting the cells with an agent.

[0145] In another embodiment of any of the aspects, the tissue stem cells are separated from initial transiently amplifying committed progenitor cells and terminally differentiated non-dividing cells.4908-1847-6928 1 Page 30 of 53 068661 -000100USPT

[0146] In another embodiment of any of the aspects, the tissue stem cells are used to treat an individual.

[0147] In another embodiment of any of the aspects, the method further comprises administering to a subject in need thereof an appropriate amount of stem cells based on the stem cell fraction (SCF).

[0148] In another embodiment of any of the aspects, the subject in need thereof has or is suspected of having a disease, disorder, or injury.

[0149] In another embodiment of any of the aspects, the disease, disorder, or injury is selected from the group consisting of: effects on organs and tissues such as, but not limited to: lungs, heart, blood vessels, blood, liver, pancreas, muscle, bones, joints, eyes, central and peripheral nervous systems.

[0150] In some embodiments of any of the aspects, the administered stem cells have been genetically modified, including gene editing. In some embodiments of any of the aspects, the gene modifications may include, but are not limited to, deleting gene sequences, inserting gene sequences, or editing gene sequences in the nuclear genome or the mitochondrial genomes. In some embodiments of any of the aspects, the genetically modified stem cells are administered as the therapeutic agent for reducing the signs and symptoms of diseases, disorders, and injuries or for providing cosmetic changes in an individual.

[0151] The methods provided herein enable the calculation of a tissue cell preparation’ s SCF (and equivalent stem cell-specific dosage) by inputting its PDT into a defined kinetic stem cell (KSC) counting algorithm. Interestingly, the method provided herein can be used to determine the exponential decay half-life of the SCF for a cultured tissue cell preparation containing tissue stem cells.

[0152] Without wishing to be bound by a theory, when the initial SCF of a primary tissue cell preparation is known, the SCF half-life (SCHE) makes it possible to calculate the SCF fraction of the cell preparation at any time in future cultures based on knowing the number of CPDs (cumulative population doubling). Accordingly, in another aspect provided herein is method for determining SCF at a future CPD (SCFCPD). The SCF at a future CPD is determined by the following Formula E:SCFCPD, SCF at any future CPD, = SCF0Xe-(ln2 / HL)CPD(Formula E), where SCF0= initial SCF and HL = half-life.4908-1847-6928 1 Page 31 of 53 068661-000100USPT

[0153] The methods provided herein comprise a step of processing the number of stem cells in the heterogeneous population of cells. The total cell count data can be processed using a model executed by one or more processors of the automated electronic instrument provided herein to determine the stem cell fraction (SCF) of the population of cells throughout the periods of the serial passaging. This can be achieved by calculating the population doubling times (PDT) for the tissue cells during non-saturated or non-confluent periods of the serial passaging, as explained in the embodiments detailed above; and quantifying the number of tissue stem cells in the subsequent tissue samples of the same type without serial passaging.

[0154] In some embodiments of any of the aspects, the cells are processed until a predetermined number of tissue stem cells are present in the sample.

[0155] In some embodiments of any of the aspects, the PDT is determined by the following formula:PDT = ^ ln 2 ln^ = initial starting time, t2= final time, N = the initial cell number, and N2= the final cell number.

[0156] In some embodiments of any of the aspects, the SCF is determined by the following derivative formulas:I. SCF = PDT (mean quotient of SCF / PDT)II. SCF = e'(mPDT +b)III. SCF = PDTe(at 3 + bt 2 + ct + d)where t = time

[0157] In some embodiments of any of the aspects, the PDT is determined by using a counting method selected from the group consisting of: counting cells, an absorbance assay, a turbidity assay, weighing cells, a fluorescent assay, and combinations thereof. Cell counts can be determined in vitro or by in vivo imaging techniques. For example, the cell count may be determined by the automated electronic instrument. The imaging assembly of the automated electronic instrument may determine a cell count for a cell sample cultured within a closed cell culture holder of the automated electronic instrument. For example, the imaging assembly may4908-1847-6928 1 Page 32 of 53 068661-000100USPTdetermine a cell count and / or a PDT for a cell culture using a trained machine learning algorithm applied to video or image data captured of the sample.

[0158] In some embodiments of any of the aspects, the method further comprises separating and / or isolating the tissue stem cells from initial transiently amplifying committed progenitor cells and terminally differentiated non-dividing cells. Methods of separating and isolating cells from a heterogeneous population of cells are known in the art, e.g., Fluorescence- activated cell sorting (FACS), Density Gradient Centrifugation, Immunodensity Cell Separation, Microfluidic Cell Separation, Immunomagnetic Cell Separation. Additional methods of isolating specific cell types are described, e.g., in Aijaz, A. etaL Biomanufacturing for clinically advanced cell therapies. Nat. Biomed. Eng. 2, 362-376 (2018); and Jiang Y. et aL Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Exp. Hematol. 2002; 30: 896-904; the contents of each of which are incorporated herein by reference in their entireties.

[0159] In some embodiments of any of the aspects, the isolated tissue stem cells are administered to a subject in need thereof.

[0160] In another aspect, provided herein is a method of treating a subject in need thereof with a population of stem cells, the method comprises: (a) determining the stem cell fraction (SCF) present in a tissue sample according to the method provided herein; and (b) administering to the subject in need thereof a dosage of stem cells based on the stem cell fraction (SCF).

[0161] In some embodiments of any of the aspects, the subject in need thereof has or is suspected of having a disease, disorder, or injury. Exemplary diseases, disorders, or injuries that may be treated in a subject with the cells provided herein include but are not limited to: lungs, heart, blood vessels, blood, liver, pancreas, muscle, bones, joints, eyes, central and peripheral nervous systems. Exemplary diseases and disorders include but are not limited to: neurodegenerative and neurological diseases, blood diseases, cardiovascular diseases, spinal cord injuries, diabetes, kidney disease, immunosuppressive diseases, cancer, liver diseases, lung diseases, muscular diseases, familial hypercholesterolemia, polycystic kidney disease, neurofibromatosis, hereditary spherocytosis, Marfan syndrome, Huntington's disease; ALS, Sickle cell anemia; cystic fibrosis; Tay-Sachs disease; phenylketonuria; mucopolysaccharidoses; mucopolysaccharidoses; lysosomal acid lipase deficiency; glycogen storage diseases; galactosemia; Duchenne muscular dystrophy; and hemophilia.4908-1847-6928 1 Page 33 of 53 068661-000100USPT

[0162] In some embodiments, the tissue stem cells are isolated and administered to a subject based on the SCF and / or PDT using the algorithms provided herein. The exact amount of cells required to treat a subject in need thereof will vary depending on factors such as the type of disease, disorder, or injury being treated.

[0163] The term “effective amount" or “appropriate amount” as used herein refers to the amount of a population of tissue stem cells or their differentiated progeny needed to alleviate at least one or more symptoms of a disease or disorder, including but not limited to an injury, disease, or disorder. An “effective amount” relates to a sufficient amount of a composition to provide the desired effect, e.g., treat a subject having an infarct zone following myocardial infarction, enhance vascularization of a graft, repair a spinal cord injury, restore muscle function, increase the levels or activity of blood cells, etc. The term "therapeutically effective amount" therefore refers to an amount of cells or a composition such cells that is sufficient to promote a particular effect when administered to a typical subject, such as one who has, or is at risk for, a disease or disorder. An effective amount as used herein would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a disease symptom (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. In some embodiments of any of the aspects, the appropriate amount of stems cells based on the SCF permits the engraftment of stems cells into the organ or tissue being treated. The administering can be done by direct injection (e.g., directly administered to a target cell or tissue) to the subject in need thereof. Administering can be transient, local, or systemic.

[0164] In some embodiments of any of the aspects, the stem cells are administered to the subject in a transplant composition. In some embodiments of any of the aspects, the transplant composition comprises a pharmaceutically acceptable carrier. In some embodiments of any of the aspects, the stem cells or the transplant composition are administered in combination with a therapeutic agent or a plurality of agents.

[0165] In general, the compositions comprising the cells provided herein are administered as liquid suspension formulations including the cells in combination with the pharmaceutically acceptable carrier. One of skill in the art will recognize that a pharmaceutically acceptable carrier to be used in a transplant composition will not include buffers, compounds, cryopreservation agents, preservatives, or other agents in amounts that substantially interfere with the viability of the cells to be delivered to the subject. A formulation comprising cells can include e.g., osmotic buffers that permit cell membrane integrity to be4908-1847-6928 1 Page 34 of 53 068661 -000100USPTmaintained, and optionally, nutrients to maintain cell viability or enhance engraftment upon administration. Such formulations and suspensions are known to those of skill in the art and / or can be adapted for use with the cells as described herein using routine experimentation.

[0166] Transplant compositions can optionally contain additional bioactive ingredients that further promote the survival, engraftment or function of the administered cells or, optionally, the tissue, organ or subject to which the composition is administered. Examples include, but are not limited to growth factors, nutrients, analgesics, anti-inflammatories and small molecule drugs, such as kinase activators, among others.

[0167] Physiologically tolerable carriers for the suspension of cells for a transplant composition include sterile aqueous physiological saline solutions that contain no additional materials other than the cells, or that contain a buffer such as sodium phosphate at physiological pH value, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.

[0168] The choice of formulation of the cells provided herein will depend upon the specific composition used and the number of cells to be administered; such formulations can be adjusted by the skilled practitioner. However, as an example, where the composition is tissue stem cells in a pharmaceutically acceptable carrier, the composition can be a suspension of the cells in an appropriate buffer (e.g., saline buffer) at an effective concentration of cells per mL of solution. The formulation can also include cell nutrients, a simple sugar (e.g., for osmotic pressure regulation) or other components to maintain the viability of the cells. Alternatively, the formulation can comprise a scaffold, such as a biodegradable scaffold.

[0169] In some embodiments of any of the aspects, the administered stem cells have been genetically modified, including gene editing. In some embodiments of any of the aspects, the gene modifications may include, but are not limited to, deleting gene sequences, inserting gene sequences, or editing gene sequences in the nuclear genome or the mitochondrial genomes. In some embodiments of any of the aspects, the genetically modified stem cells are administered as the therapeutic agent for reducing the signs and symptoms of diseases, disorders, and injuries or for providing cosmetic changes in an individual. Methods of genetically modifying stem cells and their differentiated progeny are known in the art. See, e.g., in WO2015 / 013583A2; US Pat No. 10,640,789 B2; US Pg. No. US2019 / 0367948 Al; Rees et al. Nature Rev Genet. 19(12); 770-788 (2018) and Kopmor et al. Nature 533, 420-424 (2016), the contents of each of which are incorporated herein by reference in their entirety. One4908-1847-6928 1 Page 35 of 53 068661 -000100USPTof ordinary skill in the art can design and test genetically modified adult stem cells described herein.

[0170] According to alternative embodiment Al of the present disclosure, an automated electronic instrument that incubates a tested cell sample injected into a closed cell culture holder is given. The closed cell culture holder is filled completely, without any dead space volume, with sterilized cell culture medium.

[0171] According to alternative embodiment A2 of the present disclosure, a further configuration of alternative embodiment Al, the closed cell culture holder is made of transparent materials, the transparent materials including plastics and glasses.

[0172] According to alternative embodiment A3 of the present disclosure, a further configuration of alternative embodiment A2, the automated electronic instrument is further configured to incubate tested cell samples that are injected into the closed cell culture holder. The cell culture surface area of the cell cultures in the closed cell culture holder are in the range of about 1 centimeters2(cm2) to about 3 cm2.

[0173] According to alternative embodiment A4 of the present disclosure, a further configuration of alternative embodiment Al, the automated electronic instrument is further configured to incubate a tested cell sample injected into the closed cell culture holder. The culture medium volume of the cell cultures in the closed cell culture holder is in the range of about 1 milliliter (mm) to about 3 mm.

[0174] According to alternative embodiment A5 of the present disclosure, a further configuration of alternative embodiment Al, the automated electronic instrument is further configured to use aluminum blocks and thermocouples, or other such heating devices, to regulate temperature of the sterilized cell culture medium in the closed cell culture holder.

[0175] According to alternative embodiment A6 of the present disclosure, a further configuration of alternative embodiment Al, the automated electronic instrument is further configured to maintain temperature of cultures in the closed cell culture holders in the range of room temperature of about 20 °C to about 45 °C.

[0176] According to alternative embodiment A7 of the present disclosure, a further configuration of alternative embodiment Al, the automated electronic instrument is configured to culture cells in the closed cell culture holder for periods of minutes, hours, days, or weeks.

[0177] According to alternative embodiment A8 of the present disclosure, a further configuration of alternative embodiment Al, the automated electronic instrument further comprises a light source that illuminates the closed cell culture holder from above when the4908-1847-6928 1 Page 36 of 53 068661-000100USPTinstrument is closed for operation.

[0178] According to alternative embodiment A9 of the present disclosure, a further configuration of alternative embodiment A8, the automated electronic instrument further comprises a microscope objective system and a digital camera for imaging cells in the closed cell culture holder from underneath.

[0179] According to alternative embodiment A10 of the present disclosure, a further configuration of alternative embodiment A9, the microscope objective system is of any of the types including light field microscopy, dark field microscopy, phase microscopy, Normarski optics, or differential interference contrast microscopy.

[0180] According to alternative embodiment Al l of the present disclosure, a further configuration of alternative embodiment A9, the microscope objective system and / or the closed cell culture holder are motorized for X-Y coordinate movement to allow imaging of different closed cell culture holders or different areas of the cell culture surfaces of cell cultures in individual closed cell culture holders.

[0181] According to alternative embodiment A12 of the present disclosure, a further configuration of alternative embodiment Al, the automated electronic instrument is further configured to convert captured cell images into quantitative metrics of cells that are in cultures in the closed cell culture holder. The quantitative metrics include one or more of a number of cells and a mass of total cells in the cultures.

[0182] According to alternative embodiment Al 3 of the present disclosure, a further configuration of alternative embodiment Al, the automated electronic instrument is further configured to convert captured cell images into quantitative metrics of the number of mitotic cells per total cells present, the mitotic index, in the cultures in the closed cell culture holder.

[0183] According to alternative embodiment A14 of the present disclosure, a further configuration of alternative embodiment Al, the automated electronic instrument is further configured to determine whether imaged cells are alive or dead and converts their images into quantitative metrics of the number of live cells and the number of dead cells.

[0184] According to alternative embodiment Al 5 of the present disclosure, a further configuration of alternative embodiment Al, the automated electronic instrument is further configured to serially collect time-lapse cell imaging data for addressed areas of cell cultures in the closed cell culture holder at scheduled intervals of minutes, of hours, or of days.

[0185] According to alternative embodiment Al 6 of the present disclosure, a further configuration of alternative embodiment Al, the automated electronic instrument is further4908-1847-6928 1 Page 37 of 53 068661 -000100USPTconfigured to quantify cell number and / or cell mass from adherent cells or non-adherent cells.

[0186] According to alternative embodiment Al 7 of the present disclosure, a further configuration of alternative embodiment Al, the automated electronic instrument is further configured to convert cell data collected over time into cell proliferation rate parameters of types like divided cells produced per unit time, cell population doublings, and cell population doubling time.

[0187] According to alternative embodiment Al 8 of the present disclosure, a further configuration of alternative embodiment Al, the automated electronic instrument is further configured to convert cell data collected over time into the differential tissue stem cell count of cell cultures in the closed cell culture holder.

[0188] According to alternative embodiment Al 9 of the present disclosure, a further configuration of alternative embodiment Al, the automated electronic instrument is further configured to convert collected mitotic index data into a differential tissue stem cell count of cell cultures in the closed cell culture holder.

[0189] According to alternative embodiment A20 of the present disclosure, a further configuration of alternative embodiment Al, the closed cell culture holder is configured to open for cell examinations, the cell examinations including secondary culturing, counting, staining, fixing, staining, or extracting.

[0190] According to alternative embodiment A21 of the present disclosure, a further configuration of alternative embodiment Al, the automated electronic instrument is further configured to quantify proliferation and differential tissue stem cell count of any vertebrate tissue cell preparation, including from companion animals, from agriculture animals, from human research subjects, from human cell donors, and from human patients.

[0191] According to alternative embodiment A22 of the present disclosure, a further configuration of alternative embodiment Al, the automated electronic instrument is further configured to quantify proliferation of any type of cell, including insect cells, microalgae, protozoa, microorganisms, and bacteria.

[0192] According to alternative embodiment A23 of the present disclosure, a further configuration of alternative embodiment Al, the automated electronic instrument is further configured to evaluate effects of contacting agents on the rate of cells’ proliferation, including growth factors, cytokines, nutrients, metabolites, toxins, and drug candidates.

[0193] While various embodiments of the present disclosure have been described above, it should be understood that they have been presented by way of example only, and not limitation.4908-1847-6928 1 Page 38 of 53 068661 -000100USPTNumerous changes to the disclosed embodiments can be made in accordance with the disclosure herein, without departing from the spirit or scope of the disclosure. Thus, the breadth and scope of the present disclosure should not be limited by any of the above described embodiments. Rather, the scope of the disclosure should be defined in accordance with the following claims and their equivalents.4908-1847-6928 1 Page 39 of 53 068661 -000100USPT

Claims

1. CLAIMSWhat is claimed is:

1. An automated electronic instrument comprising: a closed cell culture holder configured to receive a cell sample; a digital camera configured to image cells in the closed cell culture holder; a heating element configured to maintain the closed cell culture holder at a controlled temperature; and a processor communicatively coupled to the digital camera, the processor being configured to execute machine-readable instructions to cause the automated electronic instrument to: capture, by the digital camera, an image of a cell sample in the closed cell culture holder; and measure, by the processor, based on the image, a cell count of the cell sample.

2. The automated electronic instrument of claim 1, wherein the processor is further configured to: determine, by the processor based on analysis of the image and the cell count, a mitotic index of the cell sample, wherein the mitotic index represents a fraction of cells in mitotic phase; and determine, by the processor based on the mitotic index of the cell sample and a predetermined mathematical relationship between mitotic index and stem cell count, differential tissue stem cell count of the cell sample.

3. The automated electronic instrument of claim 1 , wherein the heating element comprises an aluminum heating block with a resistive film heater.

4. The automated electronic instrument of claim 1, wherein the closed cell culture holder comprises a transparent bottom surface, and wherein the digital camera is configured to be positioned to capture the image through the transparent bottom surface.4908-1847-6928 1 Page 40 of 53 068661-000100USPT5. The automated electronic instrument of claim 1, wherein the closed cell culture holder has a cell culture surface area of between 1 cm2to 3 cm2.

6. The automated electronic instrument of claim 1, wherein the closed cell culture holder has a culture medium volume capacity of between 1 mL to 3 mL.

7. The automated electronic instrument of claim 2, wherein determining differential tissue stem cell count includes calculating the differential tissue stem cell count based on a stem cell type-specific linear equation that accepts an input of a fraction of cells in the cell sample that are in a mitotic phase and a number of days that the cell sample has been cultured.

8. The automated electronic instrument of claim 2, wherein the processor is further configured to: receive, by the processor and via a communications interface, information characterizing a source of the cell sample; and determine, based on at least the information and the mitotic index, an estimated fraction of stem cells in the source of the cell sample.

9. The automated electronic instrument of claim 1, wherein the processor is further configured to: capture, by the digital camera, a first timeseries of images of the cell sample; measure, by the processor, for each image of the first timeseries of images, a plurality of cell counts of the cell sample based on each image; and determine, based on at least a portion of the plurality of cell counts, a cell proliferation rate.

10. The automated electronic instrument of claim 1, wherein the cell sample includes (i) human tissue cells, (ii) non-human animal tissue cells, or any combination of (i) and (ii).

11. The automated electronic instrument of claim 1, further configured to incubate cell samples that are injected into the closed cell culture holder, and wherein a cell culture surface area of the cell cultures in the closed cell culture holder being in a range of about 1 centimeters2(cm2) to about 3 cm2.4908-1847-6928 1 Page 41 of 53 068661-000100USPT12. The automated electronic instrument of claim 1, further comprising a microscope objective system.

13. The automated electronic instrument of claim 12, wherein the microscope objective system comprises (i) light field microscopy, (ii) dark field microscopy, (iii) phase microscopy, (iv) Normarski optics, or (v) differential interference contrast microscopy, or any combination of (i)-(v).

14. The automated electronic instrument of claim 12, wherein the digital camera uses the microscope objective system to capture a microscopy image of the cell sample, and wherein the processor determines a fraction of cells in the cell sample that are in a mitotic phase based on the microscopy image.

15. The automated electronic instrument of claim 1, further being analyze captured cell images to determined, based on the analysis of the captured cell images into one or more quantitative metrics of the number of mitotic cells per total cells present in the cell sample in the closed cell culture holder.

16. The automated electronic instrument of claim 1, further being configured to culture cells in the closed cell culture holder for periods of (i) minutes, (ii) hours, (iii) days, (iv) weeks, or (v) any combination of (i)-(iv).

17. A method for analyzing a cell sample, the method comprising: capturing, by a digital camera, an image of the cell sample, wherein the cell sample is injected into a closed cell culture holder; and measuring, by a processor communicatively coupled with the digital camera, and based on the image, a cell count of the cell sample.

18. The method of claim 17, wherein the processor is further configured to: determine, by the processor, a mitotic index of the cell sample based on the image and the cell count; and4908-1847-6928 1 Page 42 of 53 068661-000100USPTbased on the mitotic index of the cell sample, determine, by the processor, a differential tissue stem cell count of the cell sample.

19. The method of claim 18, wherein determining the differential tissue stem cell count includes calculating the differential tissue stem cell count based on a stem cell type-specific linear equation that accepts an input of a fraction of cells in the cell sample that are in a mitotic phase and a number of days that the cell sample has been cultured.

20. The method of claim 17, wherein the digital camera uses a microscope obj ective system to capture a microscopy image of the cell sample, and wherein the processor determines a fraction of cells in the cell sample that are in a mitotic phase based on the microscopy image.

21. The method of claim 17, further comprising converting captured cell images into one or more metrics of the number of mitotic cells per total cells present in the cell culture.

22. The method of claim 17, wherein the cell sample comprises cells obtained from an organ or tissue of an organism.

23. An automated electronic instrument comprising: a plurality of closed cell culture holder within a rotary holder; a cell sample injected into at least one of the closed cell culture holders; a heating block configured to heat the closed cell culture holder; a digital camera for imaging cells in the closed cell culture holder; and a processor communicatively coupled to the digital camera, the processor being configured to execute machine-readable instructions to cause the automated electronic instrument to: capture, by the digital camera, an image of the cell sample; and measure, by the processor, and based on the image, a cell count of the cell sample.

24. A method of determining a fraction of stem cells present in a tissue sample cultured in a closed cell culture holder of an automated electronic instrument, the method comprising:4908-1847-6928 1 Page 43 of 53 068661-000100USPTculturing, in the closed cell culture holder, a heterogeneous population of cells from a specific tissue comprising (i) tissue stem cells, (ii) transiently amplifying cells, wherein the transiently amplifying cells are transiently amplified by committed progenitor cells, (iii) terminally differentiated non-dividing cells, or (iv) any combination of (i)-(iv). determining, by one or more processors of the automated electronic instrument, a total cell count of the population of cells, wherein a total number of live and dead cells in the population of cells are counted based on an image of the population of cells captured by a digital camera of the automated electronic instrument; processing, by the one or more processors, the total cell count to determine a stem cell fraction (SCF) of the population of cells; calculating, by the one or more processors, one or more population doubling times (PDTs) for said tissue cells; and quantifying, by the one or more processors, the number of tissue stem cells in subsequent tissue samples of a tissue type of the tissue sample based on the calculated PDTs.

25. A system for automated tissue stem cell analysis comprising: the automated electronic instrument of claim 1; a computing system communicatively coupled to the automated electronic instrument; and a non-transitory computer-readable medium storing instructions that, when executed by the computing system, cause the system to: receive image data from the digital camera; process the image data to determine cell counts over time; calculate a mitotic index (MI) from the cell counts; and determine a differential stem cell count based on the mitotic index.

26. A method of manufacturing an automated electronic instrument according to claim 1.

27. Use of the automated electronic instrument of claim 1 for determining a differential tissue stem cell count.4908-1847-6928 1 Page 44 of 53 068661-000100USPT28. A disposable cell culture cartridge for use with an automated electronic instrument, the cartridge comprising: a body defining a plurality of sample holders, each sample holder configured to contain a cell culture; wherein each sample holder comprises: a transparent bottom surface configured to permit optical imaging of cells within the sample holder; an inlet port configured to receive injection of a cell sample and culture medium; and a vent port configured to permit gas exchange or fluid removal; wherein the body is configured to be heated by a heating element of the automated electronic instrument; and wherein each sample holder is configured to be hermetically sealed during cell culture.

29. The disposable cell culture cartridge of claim 28, wherein the body is manufactured from (i) polycarbonate, (ii) polystyrene, (iii) polyethylene, (iv) polypropylene, (iv) glass, or (v) any combination of (i)-(v).

30. The disposable cell culture cartridge of claim 28, wherein the transparent bottom surface comprises a material that is transparent to optical wave lengths and is thermally conductive.

31. The disposable cell culture cartridge of claim 28, wherein each sample holder has: a cell culture surface area of between 1 cm2to 3 cm2; and a culture medium volume capacity of between 1 mL to 3mL.

32. The disposable cell culture cartridge of claim 28, wherein the cartridge further comprises a sliding lid configured to move linearly with respect to the body to selectively expose or cover the inlet ports and vent ports.4908-1847-6928 1 Page 45 of 53 068661-000100USPT33. The disposable cell culture cartridge of claim 32, wherein the sliding lid is configured to create a hermetic seal over the sample holders when in a closed position.

34. The disposable cell culture cartridge of claim 32, wherein the body is machined from polycarbonate and defines a track for receiving the sliding lid.

35. The disposable cell culture cartridge of claim 28, wherein the body comprises thermally conductive material configured to transfer heat from an external heating element to cell cultures within the sample holders with a temperature uniformity of ±1°C across all sample holders.

36. The disposable cell culture cartridge of claim 28, wherein the transparent bottom surface has optical properties configured to enable dark field microscopy imaging of cells settled or adhered thereon.

37. The disposable cell culture cartridge of claim 28 for use in determining a differential tissue stem cell count.

38. The disposable cell culture cartridge of claim 28 for use in culturing human hematopoietic stem cells for transplantation therapy.

39. The disposable cell culture cartridge of claim 28, wherein at least one sample holder is pre-loaded with a dried or lyophilized reagent selected from the group consisting of: growth factors, cytokines, nutrients, and culture medium supplements.4908-1847-6928 1 Page 46 of 53 068661-000100USPT40. The disposable cell culture cartridge of claim 28, further comprising printed indicia on the body, the indicia comprising: identification markings for each sample holder; orientation markings for alignment with the automated electronic instrument; and machine-readable codes for tracking and data management.

41. The disposable cell culture cartridge of claim 28, wherein each sample holder comprises: a lid substrate configured to cover an opening of the sample holder; a seal disposed between the lid substrate and the body, the seal configured to create a hermetic seal when the lid substrate is in a closed position; and a latch mechanism configured to secure the lid substrate to the body in the closed position.

42. The disposable cell culture cartridge of claim 41, wherein the lid substrate comprises a transparent material configured to permit optical imaging of cells within the sample holder when the lid substrate is in the closed position.

43. The disposable cell culture cartridge of claim 41, wherein the seal comprises an overmolded silicone seal.

44. The disposable cell culture cartridge of claim 41, wherein the body is formed by injection molding.

45. The disposable cell culture cartridge of claim 41, wherein the cartridge further comprises a film lid configured to cover the inlet ports and vent ports, wherein the film lid is secured to the body by an adhesive or magnetic mechanism and is configured to be peeled off to provide access to the inlet ports and vent ports.4908-1847-6928 1 Page 47 of 53 068661-000100USPT46. The disposable cell culture cartridge of claim 45, wherein the film lid is reusable and comprises a reactivatable adhesive or magnetic attachment mechanism.

47. The disposable cell culture cartridge of claim 41, wherein each sample holder is configured to be completely filled with cell culture medium without dead space volume.

48. The disposable cell culture cartridge of claim 47, wherein the hermetic seal allows the cell culture to be maintained without a 5% carbon dioxide atmosphere.

49. The disposable cell culture cartridge of claim 41, wherein each sample holder is configured to culture adherent cells that attach to the transparent bottom surface or nonadherent cells that settle onto the transparent bottom surface.

50. The disposable cell culture cartridge of claim 41, wherein the cartridge comprises three sample holders configured to provide triplicate cell cultures for analysis.

51. A cell analysis system comprising: the disposable cell culture cartridge of claim 28; and an automated electronic instrument comprising: a cartridge holder configured to receive and secure the disposable cell culture cartridge; a heating element position to heat the disposable cell culture cartridge when received in the cartridge holder; a digital camera position to image cells through the transparent bottom surface of the disposable cell culture cartridge; and a processor configured to analyze images captured by the digital camera.4908-1847-6928 1 Page 48 of 53 068661-000100USPT52. The cell analysis system of claim 51, wherein the automated electronic instrument further comprises an illumination source configured to be positioned above the cell culture holder to illuminate the disposable cell culture cartridge from above during an imaging process of the disposable cell culture cartridge.

53. The cell analysis system of claim 51, wherein the cartridge holder is part of a rotary array configured to rotate multiple disposable cell culture cartridges into position for sequential imaging.

54. The cell analysis system of claim 51, wherein the heating element comprises an aluminum heating block with a resistive film heater configured to maintain the sample holders at a temperature of between 32°C to 39 °C.

55. A kit for automated cell culture analysis comprising; a plurality of disposable cell culture cartridges according to claim 24; instructions for injecting cell samples into the sample holders; and a sterile cell culture medium.

56. The kit of claim 55, wherein the plurality of disposable cell culture cartridges comprises at least 10 cartridges packaged in sterile individual wrappers.

57. A method of preparing a cell sample for automated analysis, the method comprising; providing a disposable cell culture cartridge according to claim 28; injecting a suspension of cells and cell culture medium into at least one sample holder through the inlet port; completely filling the sample holder with the cell culture medium such that no dead space volume remains; hermetically sealing the sample holder; and4908-1847-6928 1 Page 49 of 53 068661-000100USPTinserting the disposable cell culture cartridge into an automated electronic instrument configured to execute instructions stored in a memory of the automated electronic instrument, by one or more processors of the automated electronic instrument, that cause the automated electronic instrument to heat and image the cells.

58. The method of claim 57, wherein the injecting step comprises injecting fewer than 10,000 cells into the sample holder.

54. The method of claim 57, further comprising: after a period of cell culture, opening the sample holder by disengaging the hermetic seal; and performing secondary analysis of the cells in the sample holder by (i) secondary culturing, (ii) counting, (iii) staining, (iv) fixing, (v) extracting, or (vi) any combination of (i)-(v).

55. A method of manufacturing a disposable cell culture cartridge comprising: forming, by injection molding, a body that defines a plurality of sample holders; forming a transparent bottom surface for each sample holder; forming an inlet port and a vent port for each sample holder; attaching a lid structure to each sample holder, wherein the lid structure is configured to create a hermetic seal; and sterilizing the disposable cell culture cartridge.

56. The method of claim 55, wherein attaching the lid structure comprises ultrasonically welding individual lids onto each sample holder.

57. An automated electronic instrument comprising: a closed cell culture holder configured to receive a cell sample; a digital camera configured to image cells in the closed cell culture holder; a heating element configured to maintain the closed cell culture holder at a controlled temperature; and4908-1847-6928 1 Page 50 of 53 068661 -000100USPTa processor communicatively coupled to the digital camera, the processor being configured to execute machine-readable instructions to cause the automated electronic instrument to: capture, by the digital camera, an image of a cell sample in the closed cell culture holder over a time period of at least one hour; measure, by the processor, based on the image, a cell count of the cell sample; and determine, by the processor based on analysis of the image and the cell count, a one-hour cell production number.

58. The automated electronic instrument of claim 57, wherein the one-hour cell production number is indicative of a mitotic index of the cell sample.

59. An automated electronic instrument comprising: a closed cell culture holder configured to receive a cell sample; a digital camera configured to image cells in the closed cell culture holder; a heating element configured to maintain the closed cell culture holder at a controlled temperature; and a processor communicatively coupled to the digital camera, the processor being configured to execute machine-readable instructions to cause the automated electronic instrument to: capture, by the digital camera, an image of a cell sample in the closed cell culture holder over a time period of at least one hour; measure, by the processor, based on the image, a cell count of the cell sample; and determine, by the processor based on analysis of the image and the cell count, a mitotic index of the cell sample; determine, by the processor based on the analysis of the image and the cell count, a stem cell fraction of the cell sample; and determine, by the processor, a mathematical relationship between the mitotic index and the stem cell fraction.4908-1847-6928 1 Page 51 of 53 068661 -000100USPT60. The automated electronic instrument of claim 59, wherein the mathematical relationship is indicative of a mathematical relationship between the mitotic index and the stem cell fraction in a tissue of an organism.4908-1847-6928 1 Page 52 of 53 068661 -000100USPT

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