Incubator with spectrally controlled light source

Abstract

The present invention relates to a cell or tissue culture incubator with a spectrally controlled light source positioned in the incubator and a control unit configured to communicate with the light source. The control unit is configured to regulate the spectral characteristics of the light emitted by the light source to replicate, within the incubator, spectral characteristics of light conditions previously measured outside the incubator. In preferred embodiments, the control unit can also regulate other conditions in the incubator such as gas flow and composition, temperature, humidity, and pressure. The incubator may further include light sensors and weight sensors.

Description

INCUBATOR WITH SPECTRALLY CONTROLLED LIGHT SOURCE

[0001] This application claims priority to our co-pending US Provisional patent application with the serial number 63 / 536,930 filed 09 / 06 / 2023, incorporated by reference herein.Field of the Invention

[0002] The present disclosure is directed to devices, systems, and methods of cell or tissue culture incubators, particularly as it relates to illumination of the internal container of the incubator with a spectrally controlled light source to allow replication of light conditions previously measured outside the incubator.Background of the Invention

[0003] The background description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0005] Earth’s atmosphere is essential for life as we know it. It provides oxygen for breathing, protects life from harmful radiation, and creates the pressure needed for liquid water to exist. Due to the importance of atmospheric conditions, many have studied cells in environments where desired atmospheric conditions can be replicated, i.e., in an incubator. For example, in EP2906680A1, the inventors contemplated a chamber for cultivating embryos and a gas control system for maintaining a constant atmosphere in the incubator chamber. The contemplated system was configured to have pressure regulators for controlling the pressure, allows adjustment of nitrogen, carbon dioxide, and the gas pressure.

[0006] Like atmospheric conditions, light conditions also play a large role in life on Earth and cell function. Light sensitivity in living organisms is common in nature, with many cells and1SUBSTITUTE SHEET (RULE 26)tissues across various organisms exhibiting differential responses when exposed to light. Certain biological processes depend on such responses to light. For example, circadian rhythms in organisms such as mammals, birds, and insects are possible via specialized cells, such as cells in the suprachi asmatic nucleus in the brain, which are highly sensitive to light. Further, many unicellcular organisms, such as Euglena and Chlamydomonas, exhibit phototactic behavior in which they move either toward or away from light. Still further, light radiation such as ultraviolet (UV) radiation can cause damage to cells and their genetic material. In fact, “ [a]ll visible wavelengths of light can be toxic to cells in vitro, dependent upon the dose and the wavelength, with the more energetic shorter wavelengths of light below 500 nm being particularly detrimental.” Stockley, John H et al. “Surpassing light-induced cell damage in vitro with novel cell culture media.” Scientific Reports vol. 7,1 849. 12 Apr. 2017.

[0007] However, unlike replication of atmospheric conditions, it has not yet been contemplated how light conditions of a particular environment can be replicated within an incubator for purposes of cultivating and studying cells and / or tissue. Traditionally, scientists often utilize methodologies “such as optogenetics, super-resolution imaging, ion and voltage sensitive imaging, live cell imaging, and light triggered drug delivery.” Stockley, et al. (supra). Unfortunately, such methodologies often encounter challenges due to the necessity of customizing and consistently maintaining precise light conditions for cells under study, especially when investigating light-induced effects, while also maintaining desired atmospheric conditions.

[0008] Thus, even though various methods of studying the effects of light on cells are known in the art, there remains a need for new devices and methods that can address these limitations and provide solutions for investigating light-induced effects on cellular processes.Summary of The Invention

[0009] The inventive subject matter is directed to various compositions and methods of using incubators, preferably with controlled atmosphere, particularly as it relates to illumination of the internal container of the incubator with a spectrally controlled light source. Various objects, features, aspects, and advantages of the inventive subject matter will become more apparent from the following description of preferred embodiments.

[0010] In one aspect of the inventive subject matter, the inventors contemplate A method of cultivating cells and / or tissue in the presence of light that involves acquiring a spectral profile2SUBSTITUTE SHEET (RULE 26)in an environment outside of an incubator, wherein the incubator comprises a housing that at least partially encloses an internal container, and a lighting system comprising a spectrally controlled light source positioned within the internal container, using the spectral profile to instruct a control unit to adjust the spectral characteristics of the incubator to thereby create in vivo light conditions that replicate a spectral environment outside of the incubator, and cultivating the cells and / or tissue in the spectral environment replicated in the incubator.

[0011] In some embodiments, the in vivo light conditions increase and / or accelerate cell growth. In other embodiments, the in vivo light conditions reduce and / or slow cell growth. Moreover, it is generally contemplated that acquiring a spectral profile comprises collecting measurements of spectral characteristics that have an effect on metabolism, epigenetics, opsin dependency, optogenetics, immunology, gene expression, proteomics, bacterial defense, hormones, or pigmentation. However, acquiring spectral profiles may also include collecting measurements of spectral characteristics that influence vitamin D dependent processes or that influence UV dependent processes.

[0012] Viewed from another perspective, the inventors contemplate an incubator that includes a housing that at least partially encloses an internal container, a lighting system comprising a spectrally controlled light source positioned within the internal container, and a control unit in communication with the lighting system, wherein the control unit is configured to regulate spectral characteristics of light emitted by the spectrally controlled light source to recreate, within the incubator, spectral characteristics of light conditions previously measured outside the incubator.

[0013] Most typically, the contemplated incubator further comprises a weight transducer in the internal container that is configured to measure the weight of cultures placed in the internal container. In some embodiments, the contemplated incubator may also include a camera in the internal container, wherein the camera is configured to capture images of cell cultures in the internal container.

[0014] Viewed from yet another perspective, the inventors additionally contemplate a method of spectrally controlling light emitted within an incubator that includes providing an incubator that comprises a housing that at least partially encloses an internal container, and a lighting system comprising a spectrally controlled light source positioned within the internal container, coupling the spectrally controlled light source to a control unit, wherein the control unit is3SUBSTITUTE SHEET (RULE 26)configured to regulate spectral characteristics of light emitted by the spectrally controlled light source to recreate, within the incubator, spectral characteristics of light conditions previously measured outside the incubator.

[0015] Preferably, in all relevant embodiments considered above, the lighting system generally further includes one or more light sensors positioned in the internal container and in communication with the control unit, that are configured to measure the spectral characteristics of the light emitted by the spectrally controlled light source, and optionally, wherein the control unit utilizes the measurements obtained from the one or more light sensors to provide real-time feedback and adjust the spectral characteristics of the light emitted by the spectrally controlled light source to maintain desired light conditions. Furthermore, it is generally contemplated that the lighting system comprises at least 20 distinct light sources in a grid formation. In the embodiments considered above, the lighting system is also operable to emit ultraviolet (UV) light and / or infrared light, and wherein the control unit is optionally configured to selectively activate the UV and / or infrared light source.

[0016] In all relevant embodiments considered above, the previously measure light conditions typically comprise measurements taken subcutaneously or from a particular outdoor environment. Moreover, previously measure light conditions are contemplated to be transmitted over internet to provide real-time light control within the incubator.

[0017] In all relevant embodiments considered above, the spectral characteristics are regulated by the control unit to thereby replicate a diurnal light cycle in the internal container. In many embodiments, especially those contemplated above, the spectral characteristics comprise a wavelength range of between 365 and 900 nm.

[0018] In all relevant embodiments considered above, the control unit is configured to allow manual adjustment of the lighting system. In addition, the control unit is configured to allow selection of present spectral characteristics from a library of illumination profiles. In the embodiments considered above, it is also contemplated that the control unit is further configured to regulate flow and / or composition of a gas stream flowing into the incubator from an external gas source by controlling a flow control valve. Most typically the control unit is further configured to regulate temperature, humidity, and / or pressure in the incubator by activating a heater, humidifier, and / or pressure regulator. The control unit may also be configured to regulate atmospheric oxygen, nitrogen, and CO2 levels within the incubator.4SUBSTITUTE SHEET (RULE 26)

[0019] In all relevant embodiments considered above, an air flow control device is included that is coupled to the housing and / or the internal container that directs an air veil along or parallel to a hypothetical plane covering a surface of the incubator. A data logging system is also included that is configured to record and store the light conditions provided during cell culture experiments for future analysis and / or quality control. Lastly, an interface is included that is configured to allow an operator to customize the spectral characteristics of the light emitted from the light source.

[0020] Various objects, features, aspects, and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.Detailed Description

[0021] The inventors have now discovered that cell and tissue culture incubators can be manufactured and operated in a manner that provides superior spectrally controlled light emission. Indeed, the incubators presented herein have demonstrated an unprecedented capacity to customize and sustain spectral conditions of light emitted to cell cultures that replicate, within an incubator, a spectral environment outside of the incubator. Such capacity is achieved by use of a lighting system comprising at least one spectrally controlled light source located in the internal container of the contemplated incubator. Such lighting system can be configured to communicate with a control unit, and, where desired, the control unit can regulate spectral characteristics of light emitted by the spectrally controlled light source to create desired light conditions in the internal container.

[0022] Beneficially, the devices and methods presented herein solve a long-recognized problem and need. For example, Tahkamb, et al. teaches that “systematic reviews on other light-induced health concerns are needed, as well as meta-analyses of any negative health impacts, including circadian rhythm disruption, to further clarify the scientific evidence on impacts of light on human health.” Tahkamb, Leena et al. In addition to a “[systematic review of light exposure impact on human circadian rhythm.” Chronobiology International vol. 36,2 (2019). However, the present disclosure is not limited to replicating or disrupting circadian / diurnal rhythms. As described below, the present disclosure may also allow a researcher to change the intensity and spectral wavelengths on an arbitrary timing specified by the researcher for purposes of growth and / or development of cells and / or tissue.5SUBSTITUTE SHEET (RULE 26)

[0023] As used herein, the term “spectrally controlled light sourcefs]” should be interpreted as referring to a light source or sources operable to emit, simultaneously or sequentially, light at multiple prespecified wavelengths and (relative) intensities for prespecified periods of time. Therefore, and in a more general aspect of the inventive subject matter, a spectrally controlled light source can be operated as a monochromatic light source capable of emitting different wavelengths at different times, or as a polychromatic light source capable of emitting colored or ‘white’ light at the same time. For example, a spectrally controlled light source can be operated to replicate a spectral light composition that is characteristic for a specific geographic location and date / time, or a spectral light composition that is characteristic for a specific in vivo location of a cell within an organism.

[0024] In order to achieve such specific spectral conditions, contemplated light sources are contemplated to include sets or an array of individual lights where each individual light has a specific spectral range that is blended to create the ideal spectral profile (wavelength & intensity per wavelength) for a given experiment. For example, the lighting system may comprise at least 20, at least 25, at least 30, at least 40, at least 50, or at least 100 light sources that are blended to create a desired spectral profile. The contemplated light sources may be organized into a grid or a matrix formation. Moreover, the light sources may individually have a wavelength range of between 365 and 900 nm. For example, the light sources may have a wavelength of between 100 and 200 nm, between 200 and 400 nm, between 300 and 500 nm, between 250 and 450 nm, between 400 and 600 nm, between 500 and 700 nm, between 600 and 800 nm, between 700 and 900 nm, or between 800 and 1 mm. Due to each of the individual light sources having a wavelength range between 365 and 900 nm, the overall wavelength range of the lighting system, as a whole, may also be between 365 and 900 nm. For example, the overall wavelength of the lighting system may be 300 nm, 500 nm, 700 nm, 900 nm, or more. In still other examples, a spectrally controlled light source can be operated to emit light with different spectral characteristics according to any arbitrary timing sequence selected by a researcher, including but not limited to a diurnal or other circadian rhythm. Other timing sequences may include ultradian rhythms or infradian rhythms, or the researcher may configure the light source to replicate any 24-hour light cycle experienced by a specific cell type.

[0025] In especially preferred aspects, the incubator control unit includes a microprocessor and memory storing instructions executable on the microprocessor, wherein the instructions cause the control unit to cause the spectrally controlled light source to set or adjust the spectral6SUBSTITUTE SHEET (RULE 26)characteristics of the light emitted by the spectrally controlled light source. Most typically, the control unit is configured to allow manual adjustment of the lighting system such that a specific wavelength can be chosen. In various embodiments, the control unit is also configured to allow automatic adjustment of the lighting system, where the system has a library of illumination profiles that allow selection for and subsequent adjustment to a certain illumination. For example, the control unit may have an option for “rainforest” lighting conditions, which upon selection, adjust the spectral conditions of an incubator to match those of a rainforest. Likewise, the control unit is optionally configured to selectively activate and emit an ultraviolet (UV) and / or infrared light source as desired.

[0026] As will be readily appreciated, the control unit may further be coupled to or in communication with one or more flow control valves, wherein the control unit is configured to regulate flow and / or composition of a gas stream flowing into the incubator from an external gas source by controlling a flow control valve. In other embodiments, the control unit may be further electronically coupled to a temperature sensor, a gas sensor, an atmospheric pressure sensor, and / or a humidity sensor, and the instructions may cause the control unit to activate a heater, open a gas valve to allow entry of a gas into the incubator, and / or activate a humidifier. Additionally, or alternatively, the instructions may also cause the control unit to activate the heater, to open the gas valve to allow entry of the gas into the incubator, and / or activate the humidifier while a door is in an open or closed position. Most typically, the gas sensor is an 02 sensor and / or a CO2 sensor.

[0027] In some embodiments, the control unit is further electronically coupled to an access control device that is programmed to receive a user command and / or validate an authorized user of the incubator. Most typically, the instructions cause the control unit to move a door movable between a first position allowing access to the internal container from an outside position of the incubator and a second position preventing access to the internal container from the outside position of the incubator, from the first to the second position upon receiving the user command and / or validation of the authorized user. While not limiting to the inventive subject matter, the user command is a voice command or visual / gesture command. Therefore, in some embodiments the authorized user is validated by face recognition. Additionally, the control unit may also be electronically coupled to a sterilization unit, and the instructions cause the control unit to activate the sterilization unit.7SUBSTITUTE SHEET (RULE 26)

[0028] In preferred aspects of the inventive subject matter, one or more light sensors may be positioned in the internal container and in communication with the control unit, configured to measure the spectral characteristics of the light emitted by the spectrally controlled light source. These light sensors may be photodiodes, photoresistors, phototransistors, photovoltaic light sensors, or other types of light sensors. Alternatively, or additionally, suitable light sensors will include CMOS, EMCCD, or CCD chips, or other sensors with spectral resolution capacity. In some embodiments of the claimed invention, the light sensor(s) convert light in the incubator into an electrical current or other signal that can be then communicated to the control unit. Upon receiving such current or other signal, the control unit may regulate spectral characteristics of light emitted by the spectrally controlled light source to create desired light conditions in the internal container.

[0029] In further suitable embodiments, the inventors contemplate incubators or methods that may be used to study the effects of light exposure on cells, where the light exposure can be customized to replicate various light conditions for desired locations for specified periods of time. For example, the incubator may be set to replicate the average light conditions in Santa Monica, California, at an altitude of 45 meters in the month of August, for a full 24-hour cycle. In another example, the incubator may replicate the average light conditions underneath the surface of the Pacific Ocean, West of Santa Monica, at a depth of 50 fathoms (91.5 meters) in the month of February from 6:00AM to 6:00PM. In some embodiments, each factor (location, altitude, etc.) may be manually configured by a user to precisely select the desired conditions, while in other embodiments the user can select pre-configured settings. For instance, the incubator may offer a user a choice between “Santa Monica in Autumn Full Cycle” or “Pacific Ocean Subaquatic Half Cycle in Winter.”

[0030] To replicate the average light conditions in Santa Monica, for example, within an incubator, the light conditions of Santa Monica may be measured from the outdoor environment and transmitted over internet to provide real-time light control within the incubator. In other words, light conditions of a particular environment may be measured on a phone through the camera then stored and / or added to the contemplated illumination library for later use in the incubator. In some embodiments, measurements taken on a phone camera may be transmitted to the incubator over internet to provide real-time light measurements and allow subsequent adjustment of spectral conditions within the incubator to match those measured in the external environment.8SUBSTITUTE SHEET (RULE 26)

[0031] In addition to taking measurements of light conditions in an outdoor environment, it is also contemplated that a spectral profile can be created based on subcutaneous measurements. For example, a sensor may be placed underneath the skin or at a certain location in one’s body to measure light conditions of a particular organ. For example, if one wanted to study the differences in light sensitivity between kidney and dermal cells, a subcutaneous measurement of both locations could be taken and added to the illumination library of the incubator for use in cultivating kidney and dermal cells in the most realistic environment, within an incubator. A sensor may be implantable or an optical fiber that can remain in a body for at least 24 hours. In some embodiments, the sensor may also remain in a patient’s body for at least 36 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 168 hours. Alternatively, or additionally, measurements may be taken endoscopically in a more temporary manner, i.e., less than two hours. Electronic skin sensors may also be used for measurements of light conditions / effects on more superficial cells. Superficial sensors of this sort may be removed and replaced as long as necessary. For example, a skin sensor may be worn for seven days, replaced with another sensor for seven days, then repeated as needed. Measurements taken from body -placed sensors, either superficial or subcutaneous, may be either stored in the sensor and collected upon removal of the sensor or measurements may be transferred over the internet in real-time. The contemplated placements of a sensor may also be used in studying animals and effects of light conditions on their cells and / or tissue.

[0032] Consequently, a method of cultivating cells and / or tissue in the presence of specific light conditions is also herein contemplated. This method typically includes acquiring a spectral profile in an environment outside of an incubator, in ways disclosed above, wherein the incubator typically comprises both a housing that at least partially encloses an internal container and a lighting system that comprises a spectrally controlled light source positioned within the internal container. The method may also include using the spectral profile or a realtime sensor to instruct a control unit to adjust the spectral characteristics of the incubator to thereby create in vivo light conditions that replicate a spectral environment outside of the incubator and cultivating the cells and / or tissue in the spectral environment replicated in the incubator.

[0033] As will be readily appreciated, the in vivo light conditions may increase and / or accelerate cell growth. However, the in vivo light conditions may also reduce and / or slow cell growth. It is generally contemplated that the rate at which cells and / or tissue grow in response9SUBSTITUTE SHEET (RULE 26)to light conditions will depend on the light sensitivity of the particular cell and / or tissue being studied. For example, a kidney cell may grow rapidly under darker light conditions, but when exposed to light conditions that dermal cells typically experience, the kidney cells may begin to slow in growth or stagnate. Nonetheless, acquiring a spectral profile and comparing spectral profiles in such a context may be done through the techniques mentioned above. Most typically, the goal of acquiring and / or developing a spectral profile would be to collect measurements of spectral characteristics that influence metabolism, epigenetics, opsin dependency, optogenetics, immunology, gene expression, proteomics, bacterial defense, hormones, or pigmentation. Acquisition of a spectral profile may also include collecting measurements of spectral characteristics that influence vitamin D dependent processes, UV dependent processes, infrared dependent processes, or temperature dependent processes.

[0034] To simplify the user experience, it is contemplated that the instant invention may comprise an interface configured to allow an operator to customize the spectral characteristics of the light emitted from the light source. Such interface may include a computer mouse, a keyboard, a touch pad, and / or a screen which displays interactable information. In some embodiments, the interface may include a touchscreen displaying interactable information. In other embodiments, the interface is a command line interface, or a graphical user interface. In still other embodiments, the interface may include a voice user interface. Preferred aspects of the inventive subject matter include interfaces that allow a user to be recognized, such as via password or voice-recognition, which allows the user’s previous interaction with the interface to be remembered and re-displayed by the interface.

[0035] Upon growth of desired cells, light conditions could also be altered to study the light sensitivity and subsequent effects on a given cell and / or tissue. Despite the kind of environment being measured, whether outdoor or subcutaneous, it is generally contemplated that the incubator may comprise a data logging system that is configured to record and store the light conditions provided during cell culture experiments for future analysis and quality control.

[0036] In some embodiments of the instant invention, a camera may be positioned within the incubator, wherein the camera is configured to capture images of cell cultures in the internal container. The camera may utilize microscopy techniques to capture images of cell cultures, including brightfield microscopy, darkfield, digital imaging, and telepathology. In further embodiments of the claimed invention, the camera can be configured for fluorescent microscopy. For example, the camera may use a high-intensity light source that excites a10SUBSTITUTE SHEET (RULE 26)fluorescent molecule, such as a fluorophore in the cell culture being observed, and then separate the resulting emitted light from surrounding radiation to capture an image only displaying the fluorescing material. Ideally, the camera will be capable of analyzing the medium, cell shape, and cell density. Additionally, the camera (or associated software) in some embodiments may be capable of quantifying the number of cells in a culture, by counting the number of colonies or Colony Forming Units (CFUs) according to the ASTM D5465 standard. Alternatively, the camera may double as a spectral light sensor.

[0037] In still other embodiments, spectral photometers may be positioned in the incubator. Such photometers may measure certain qualities of cell cultures in the incubators by measuring the spectral characteristics of light emitted from the spectrally controlled light source through the cell cultures, and then measuring the spectral characteristics of the light that emerges on the opposite side of the cell cultures. The spectral photometers can analyze the differences between incident light and emitted or transmitted light to provide information about the cells and / or culture medium. For instance, a large difference in the intensity of the incident and transmitted light would indicate that the cell cultures absorb a significant portion of light in one or more spectral areas. This could be useful when studying recombinant expression of certain proteins, monitoring cell density, nutrient consumption in a culture medium, functioning of photoreceptor proteins in the cells, among other examples.

[0038] The inventors further contemplate that the devices presented herein include a weight transducer in the incubator. The weight transducer ideally is configured to convert an input mechanical force— such as the weight of a petri dish with cell culture being placed onto the transducer— into another physical variable such as an electrical output signal that can be measured, converted, and standardized. In this case, as the force applied to the sensor increases, the electrical signal changes proportionally. However, in other embodiments, the weight transducer may be a simple single point load cell weight sensor, commonly found in kitchen and bathroom weight scales. Such load cells may be helpful in logging events in cell or tissue cultures, detect evaporation of culture media, etc. In other embodiments of the present disclosure, the weight transducer is a strain gauge that converts varying weights into a change in electrical resistance which is then measured by associated circuitry of the strain gauge. As will be readily appreciated, contemplated weight transducers may house multiple strain gauges.

[0039] The inventors also contemplate that, in some aspects, the control unit of the incubator is further configured to regulate flow and / or composition of a gas stream flowing into the11SUBSTITUTE SHEET (RULE 26)incubator from an external gas source by controlling a flow control valve. In preferred embodiments, the gas in question is 02 and / or C02. As discussed in PatentNo. US 10,900,010, the gas compositions may be recirculated through a recirculation space that is formed between the housing and the internal container. In preferred embodiments, the recirculation space includes an 02 and a CO2 sensor, and, depending on the particular set points for the atmospheric parameters in the incubator, it should be noted that the sensors will provide signals to the control unit to regulate the flow of gas compositions through the flow control valve. In some aspects, the incubator may contain multiple flow control valves.

[0040] In another aspect of the inventive subject matter, it is contemplated that the incubator further comprises a door coupled to the housing and / or internal container such that the entire door is movable away from the hypothetical plane and such that the entire door is moveable in a horizontal or vertical direction. With respect to suitable doors, it is contemplated that any door that can at least temporarily close the opening is deemed suitable for use herein, and contemplated doors can provide access to the internal container by a rotating / pivoting motion (e.g., around a hinge), vertical or horizontal translating motion (e.g., using telescoping gear), or a compound motion (e.g., using trammel or compound pivot). However, it is generally preferred that the door is coupled to the housing and / or the internal container in a manner such that the door moves the door (first) away and (then) in an upward motion from the opening. Such manner of opening will advantageously reduce the severity of air motion forcing air from the inside of the incubator to the outside and / or the amount of turbulent air between the door and the inside of the incubator chamber. For example, the door will preferably be movable in a non-pivoting motion (not on a hinge or other pivoting mechanism extending along one edge of the door), typically in a first movement along a Y-axis (towards or away from the internal container) and a second movement parallel to a hypothetical plane extending across the opening (X- or Z-axis). Such movements are preferably sequential or may be performed in a single compound motion. Alternatively, the door may also be rotated about an axis that is near, at, or outside the perimeter of the door (typically after first moving the rotating door away from the opening). In further embodiments, the door may also be configured as a flexible or segmented cover with each segment coupled to the next via a flexible connector or film (thus being similar to a segmented garage door). Such flexible or segmented door can be moved in a sliding motion substantially parallel to the opening and towards a top or side wall of the internal container or housing.12SUBSTITUTE SHEET (RULE 26)

[0041] In addition, it should be noted that the door may also use a use magnetic (or mechanic) door seal to accommodate pressure differences between the inside and outside of the incubator. Where desired, it is further contemplated that the door may include a safety mechanism that is designed to prevent closing onto a shelf or an operator, and suitable safety mechanisms may be based on torque increase, optical sensors, and / or proximity sensors, all of which are well known in the art. Notably, it should be appreciated that contemplated incubators need not have a door at all so that the so modified incubators can be used as a glove box, a (biosafety level 2+) cell or tissue culture bench, as a fume hood, etc. Thus, it should be appreciated that the opening of the internal container is shielded by dual air curtains / veils. Advantageously, such shielding significantly reduces, or even entirely avoids contamination avoided due to a lack of contaminated air pushing or being sucked into the internal container as is common with heretofore known incubators. Viewed from a different perspective, the air veil(s) act as a virtual airlock that prevents contamination while preserving the environment of the internal container.

[0042] Viewed from a different perspective, the instant invention may further comprise means for performing gel electrophoresis within the incubator, which may be useful for studying the sizes and charges of biological samples under controlled conditions. For example, the incubator may store agarose, acrylamide, or one or more other suitable gel matrices. The incubator may further store buffer solution which can be mixed and heated with the gel. The incubator may contain an electrophoresis machine configured to load the cell samples into wells. The machine should be coupled to a power source that runs a voltage for a period of time that will achieve optimal separation. Alternatively, the incubator may further contain a vortex mixer configured to mix cell samples and / or other fluids rapidly. In another alternative embodiment, the incubator may contain a centrifuge configured to separate particles in a biological liquid mixture based on density.

[0043] A further embodiment of the inventive subject matter includes a culture exchange system wherein, rather than opening and closing a door each time a cell culture is replaced, removed, or added, a tray can be pulled out from a surface of the incubator, wherein the tray is configured to contain a cell culture. Alternatively, the culture exchange system may be configured to periodically replace cell cultures automatically with minimal disturbance to the cells.

[0044] It is contemplated herein that the incubator may contain one or more barcode scanners in the internal container configured to scan any barcode found on a cell culture petri dish,13SUBSTITUTE SHEET (RULE 26)wherein the barcode communicates the content of the cell culture, and wherein the barcode scanner is configured to communicate such content to the control unit. The control unit may be further configured to determine light conditions or other conditions (temperature, pressure, humidity, etc.) favorable to the content, and adjust the conditions accordingly. Alternatively, the control unit may determine the desirable conditions, and then suggest said conditions to the user, giving the user the option to either accept the control unit’s suggestion or reject it.

[0045] In interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a nonexclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C .... and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

[0046] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” As used herein, the terms "about" and "approximately", when referring to a specified, measurable value (such as a parameter, an amount, a temporal duration, and the like), is meant to encompass the specified value and variations of and from the specified value, such as variations of + / -10% or less, alternatively + / -5% or less, alternatively + / -1% or less, alternatively + / -0.1% or less of and from the specified value, insofar as such variations are appropriate to perform in the disclosed embodiments. Thus, the value to which the modifier "about" or "approximately" refers is itself also specifically disclosed. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0047] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope14SUBSTITUTE SHEET (RULE 26)of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0048] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise. As also used herein, and unless the context dictates otherwise, the term "coupled to" is intended to include both direct coupling (in which two elements that are coupled to each other contact each other) and indirect coupling (in which at least one additional element is located between the two elements). Therefore, the terms "coupled to" and "coupled with" are used synonymously.SUBSTITUTE SHEET (RULE 26)

Claims

AMENDED CLAIMS received by the International Bureau on 21 February 2025 (21.02.2025)What is claimed is:

1. An incubator, comprising: a housing that at least partially encloses an internal container; a lighting system comprising a spectrally controlled light source positioned within the internal container; and a control unit in communication with the lighting system, wherein the control unit is configured to regulate spectral characteristics of light emitted by the spectrally controlled light source to recreate, within the incubator, spectral characteristics of light conditions previously measured in vivo in a subcutaneous location.

2. The incubator of claim 1, wherein the lighting system further comprises one or more light sensors positioned in the internal container and in communication with the control unit, configured to measure the spectral characteristics of the light emitted by the spectrally controlled light source, and optionally, wherein the control unit utilizes the measurements obtained from the one or more light sensors to provide real-time feedback and adjust the spectral characteristics of the light emitted by the spectrally controlled light source to maintain desired light conditions.

3. The method of claim 1, wherein the previously measured light conditions previously measured in vivo in a subcutaneous location are based on measurements received by a sensor placed underneath a skin of a subject or at a certain organ in the subject’s body.

4. The method of claim 1, wherein the previously measured light conditions are transmitted over internet to provide real-time light control within the incubator.

5. The incubator of claim 1, wherein the spectral characteristics are regulated by the control unit to thereby replicate a diurnal light cycle in the internal container.

6. The incubator of claim 1, wherein the spectral characteristics comprise a wavelength range of between 365 and 900 nm.

7. The incubator of claim 1, wherein the lighting system comprises at least 20 distinct light sources in a grid formation.

8. The incubator of claim 1, wherein the control unit is configured to allow manual adjustment of the lighting system.

9. The incubator of claim 1, wherein the control unit is configured to allow selection of preset spectral characteristics from a library of illumination profiles.

10. The incubator of claim 1, further comprising an air flow control device coupled to the housing and / or the internal container that directs an air veil along or parallel to a hypothetical plane covering a surface of the incubator.

11. The incubator of claim 1, wherein the lighting system is operable to emit ultraviolet (UV) light and / or infrared light, and wherein the control unit is optionally configured to selectively activate the UV and / or infrared light source.

12. The incubator of claim 1, further comprising a data logging system configured to record and store the light conditions provided during cell culture experiments for future analysis and / or quality control.

13. The incubator of claim 1, wherein the control unit is further configured to regulate flow and / or composition of a gas stream flowing into the incubator from an external gas source by controlling a flow control valve.

14. The incubator of claim 1, wherein the control unit is further configured to regulate temperature, humidity, and / or pressure in the incubator by activating a heater, humidifier, and / or pressure regulator.

15. The incubator of claim 1, further comprising an interface configured to allow an operator to customize the spectral characteristics of the light emitted from the light source.

16. The incubator of claim 1, further comprising a weight transducer in the internal container configured to measure the weight of cultures placed in the internal container.

17. The incubator of claim 1, further comprising a camera in the internal container, wherein the camera is configured to capture images of cell cultures in the internal container.

18. A method of spectrally controlling light emitted within an incubator, comprising: providing an incubator that comprises a housing that at least partially encloses an internal container, and a lighting system comprising a spectrally controlled light source positioned within the internal container; coupling the spectrally controlled light source to a control unit, wherein the control unit is configured to regulate spectral characteristics of light emitted by the spectrally controlled light source to recreate, within the incubator, spectral characteristics of light conditions previously measured in vivo in a subcutaneous location.

19. The method of claim 18, further comprising positioning one or more light sensors in the internal container, wherein the one or more light sensors are configured to measure the spectral characteristics of the light emitted by the spectrally controlled light source, and coupling the one or more light sensors to the control unit, and optionally, wherein the control unit is configured to utilize the measurements obtained from the one or more light sensors to provide real-time feedback and adjust the spectral characteristics of the light emitted by the spectrally controlled light source to maintain desired light conditions.

20. The method of claim 18, wherein the light conditions previously measured in vivo in a subcutaneous location are based on measurements received by a sensor placed underneath a skin of a subject or at a certain organ in the subject’s body.

21. The method of claim 18, wherein the previously measured light conditions are transmitted over internet to provide real-time light control within the incubator.

22. The method of claim 18, wherein the control unit is configured to regulate the spectral characteristics to replicate a diurnal light cycle in the internal container.

23. The method of claim 18, wherein the spectral characteristics comprise a wavelength range of between 365 and 900 nm.

24. The method of claim 18, wherein the lighting system comprises at least 20 distinct light sources in a grid formation.

25. The method of claim 18, wherein the control unit is configured to allow manual adjustment of the lighting system.

26. The method of claim 18, wherein the control unit is configured to allow selection of preset spectral characteristics from a library of illumination profiles.

27. The method of claim 18, further comprising a step of providing an air flow control device coupled to the housing and / or the internal container that directs an air veil along or parallel to a hypothetical plane covering a surface of the incubator.

28. The method of claim 18, wherein the lighting system is operable to emit ultraviolet (UV) light and / or infrared light, and wherein the control unit is optionally configured to selectively activate the UV and / or infrared light source.

29. The method of claim 18, wherein the incubator further comprises a data logging system configured to record and store the light conditions provided during cell culture experiments for future analysis and quality control.

30. The method of claim 18, wherein the control unit is further configured to regulate flow and / or composition of a gas stream flowing into the incubator from an external gas source by controlling a flow control valve.

31. The method of claim 18, wherein the control unit is further configured to regulate temperature, humidity, and / or pressure in the incubator by activating a heater, humidifier, and / or pressure regulator.

32. The method of claim 18, further comprising a step of providing an interface configured to allow an operator to customize the spectral characteristics of the light emitted from the light source.

33. A method of cultivating cells and / or tissue in the presence of light, comprising: acquiring a spectral profile in an environment outside of an incubator; wherein the incubator comprises a housing that at least partially encloses an internal container, and a lighting system comprising a spectrally controlled light source positioned within the internal container; using the spectral profile to instruct a control unit to adjust the spectral characteristics of the incubator to thereby create in vivo light conditions that replicate a spectral environment outside of the incubator; and cultivating the cells and / or tissue in the spectral environment replicated in the incubator.

34. The method of claim 33, wherein the in vivo light conditions increase and / or accelerate cell growth.

35. The method of claim 33, wherein the in vivo light conditions reduce and / or slow cell growth.

36. The method of claim 33, wherein acquiring a spectral profile comprises collecting measurements of spectral characteristics that have an effect on metabolism, epigenetics, opsin dependency, optogenetics, immunology, gene expression, proteomics, bacterial defense, hormones, or pigmentation.

37. The method of claim 33, wherein acquiring a spectral profile comprises collecting measurements of spectral characteristics that influence vitamin D dependent processes.

38. The method of claim 33, wherein acquiring a spectral profile comprises collecting measurements of spectral characteristics that influence UV dependent processes.

39. The method of claim 33, further comprising positioning one or more light sensors in the internal container, wherein the one or more light sensors are configured to measure thespectral characteristics of the light emitted by the spectrally controlled light source, and coupling the one or more light sensors to the control unit, and optionally, wherein the control unit is configured to utilize the measurements obtained from the one or more light sensors to provide real-time feedback and adjust the spectral characteristics of the light emitted by the spectrally controlled light source to maintain desired light conditions.

40. The method of claim 33, wherein previously measured light conditions comprise measurements taken subcutaneously or from a particular outdoor environment.

41. The method of claim 33, wherein previously measured light conditions are transmitted over internet to provide real-time light control within the incubator.

42. The method of claim 33, wherein the control unit is configured to regulate the spectral characteristics to replicate a diurnal light cycle in the internal container.

43. The method of claim 33, wherein the spectral characteristics comprise a wavelength range of between 365 and 900 nm.

44. The method of claim 33, wherein the lighting system comprises at least 20 distinct light sources in a grid formation.

45. The method of claim 33, wherein the control unit is configured to allow manual adjustment of the lighting system.

46. The method of claim 33, wherein the control unit is configured to allow selection of preset spectral characteristics from a library of illumination profiles previously acquired.

47. The method of claim 33, further comprising a step of providing an air flow control device coupled to the housing and / or the internal container that directs an air veil along or parallel to a hypothetical plane covering a surface of the incubator.

48. The method of claim 33, wherein the lighting system is operable to emit ultraviolet (UV) light and / or infrared light, and wherein the control unit is optionally configured to selectively activate the UV and / or infrared light source.

49. The method of claim 33, wherein the incubator further comprises a data logging system configured to record and store the light conditions provided during cell culture experiments for future analysis and quality control.

50. The method of claim 33, wherein the control unit is further configured to regulate flow and / or composition of a gas stream flowing into the incubator from an external gas source by controlling a flow control valve.

51. The method of claim 33, wherein the control unit is further configured to regulate temperature, humidity, and / or pressure in the incubator by activating a heater, humidifier, and / or pressure regulator.

52. The method of claim 33, further comprising a step of providing an interface configured to allow an operator to customize the spectral characteristics of the light emitted from the light source.