Methods for handling biological materials in controlled and dynamic environments

Abstract

Disclosed is a set of methods relating to handling of biological material and liquids in which an instrument provide samples of biological material with several independent sets of controlled environmental conditions including temperature, humidity and gas composition; provide operational compatibility with standardized laboratory incubation vessel(s); operates with a pitch-roll orbital wave motion path to facilitate gravity assisted neutralization of diffusion gradients in liquids contained in such vessels; and enables observation, access and manipulation of internally kept biological material without causing exposure of such material to external atmospheric conditions.

Description

[0001] METHODS FOR HANDLING BIOLOGICAL MATERIALS IN CONTROLLED AND DYNAMIC ENVIRONMENTS

[0002] FIELD

[0003] The disclosure relates to methods, using key advantages of a described instrument, to the benefit of; eukaryotic cell cultivation, production of biological material, and biological assessment procedures.

[0004] INTRODUCTION

[0005] The handling of biological material such as (but not limited to) cells and microorganisms in a laboratory environment typically involves the use of equipment intended to provide environmental parameters that are supportive to the preservation of such biological materials, for example by providing controlled levels of temperature, pH, and availability of gases such as carbon dioxide (CO2) and oxygen (O2).

[0006] A frequently used conventional equipment for such handling of biological material is the common CO2incubator which features a openable cabinet in which biological materials, often kept in conventional models of incubation vessels including (but not limited to) petri dishes and multi-well plates, are set to rest on planar shelfs, with the incubator providing the internally kept materials with an temperature controlled and humidified environment while also regulating the amount of CO2. Typical daily work with CO2incubators, or incubators that in addition to CO2also feature the ability to regulate the concentration of O2(commonly referred to as hypoxic incubators), feature temporary removing the biological material from the incubator into the outer laboratory environment for subsequent handling or observation, before returning the biological materials to the incubator.

[0007] Another type of conventional equipment that may be used for handling of biological materials is the bioreactor, often employed for processes where handling of large amounts of biological materials is required, with examples including (but not limited to) industrial production of vaccines or hormones. While also providing a controlled environment in terms of temperature, pH, and concentration of gases, bioreactors differ from the above-mentioned incubators by operating in a manner that avoid exposing the handled biological material to the outer laboratory environment and being designed for large singular samples instead of accommodating conventional plasticware and multiple samples.

[0008] For handling of biological materials be it for, cultivation of cells and microorganisms, processes to produce biological materials, or using biological materials for biological assays, it is imperative to control and provide the biological material with the environmental conditions most suited to optimally support each application. Current methods using conventional equipment have inherent limitations in capability and features, makingthem ill-suited for optimally supporting handling of biological materials.

[0009] SUMMARY

[0010] The present invention seeks to overcome, eliminate and / or ameliorate one or more of the drawbacks and deficiencies of the prior art, for example those described in the above.

[0011] The disclosure provides methods of eukaryotic cell cultivation, biological material production, and biological assays, with an apparatus for facilitating and providing such biological material with contained and controlled delivery of temperature, humidity, and gases in a closed loop manner, with a solution to access the internal environment without exposure to the external environment, and with a solution to control the extent of exposure of the biological material to factors dissolved in liquids by means of a pitch-roll orbital wave movement to prevent the buildup of diffusion gradients, while also being operationally compatible with conventionally used models of incubation vessels and multi-sample approaches.

[0012] In an aspect, the disclosure relates to a method of eukaryotic cell cultivation, the method comprising i. Providing an instrument that can simultaneously cultivate multiple samples of cell culture with independent sets of controlled environmental conditions with respect to at least temperature, humidity and gas composition; ii. Position laboratory incubation vessel(s) used to house cell cultivation and cultivation medium in the instrument; iii. Operate the instrument with eukaryotic cells contained in a cultivation medium in at least one laboratory incubation vessel(s) so that the incubation vessel(s) follows a pitch-roll orbital wave motion path, resulting in gravity assisted flow and repositioning of the cell cultivation medium, thereby neutralizing diffusion gradients of any dissolved factors such as gases and nutritional components within the cell cultivation medium; iv. Wherein, during operation of the instrument, cell cultivation within the incubation vessel is observed, accessed and / or manipulated upon without exposing the cell cultivation medium to external atmospheric conditions.

[0013] The cell cultivation medium contained in the incubation vessel can preferably be of serum-free and defined composition (for example StemPro-34 SFM), or alternatively of undefined composition (for example DMEM-F12 + 10% Fetal Calf Serum). In addition, the cell cultivation medium may include an addition of phenol red to indicate pH in the medium.

[0014] The eukaryotic cells can be selected from pluripotent and multipotent stem cells (human and / or animal origin), cell derivates differentiated from pluripotent or multipotent stem cells (human and / or animal origin), male and female gametes / sex cells (human and / or animal origin), ex vivo cells and tissue retrieved from a living body (human and / or animal origin), cancerous cells and tumorigenic tissue (human and / or animal origin).

[0015] Observation of eukaryotic cells kept with cultivation medium within the incubation vessel can preferably be observed with an inverted microscope, with the air-tight containment solution being temporarily lifted from the instrument (without exposure to external atmospheric conditions) to facilitate placement upon the microscope.

[0016] Manipulation of eukaryotic cells kept within cultivation medium within the incubation vessel, such as replacement of the used medium to fresh medium, can preferably be carried out by use of the self-sealing access plugs of the containment solution through which medium can be transferred by use of a syringe (or similar) connected to a suitable perforator (such as a lance or needle) that temporary perforate the self-sealing access plug, thereby enabling medium exchange to the eukaryotic cells within the containment solution without causing exposure of the eukaryotic cells to external atmospheric conditions. If multiple samples of eukaryotic cells, each with separate medium compositions, are intended introduction of factors and / or differently composed medium to individual samples is similarly facilitated through the selfsealing access ports. If comparative use of distinct environmental conditions such as (but not limited to) differences in temperature or gas concentration is desired, multiple containment solutions housing eukaryotic cells, each with desired environmental parameters, can be operated simultaneously with the same instrument.

[0017] In a further aspect, the disclosure relates to a method of producing biological material, the method comprising i. Providing an instrument that can simultaneously cultivate multiple samples of biochemical processes with independent sets of controlled of environmental conditions with respect to at least temperature, humidity and gas composition; ii. Position laboratory incubation vessel(s) used to house biochemical processes and fluids supportive of such processes in the instrument; iii. Operate the instrument so that the incubation vessel(s) follow a pitch-roll orbital wave motion path, resulting in gravity assisted flow and repositioning of the fluid supportive of the biochemical process, thereby neutralizing diffusion gradients of any dissolved factors such as gases and biochemical factors within the fluid supportive of the biochemical process; iv. Wherein, during operation of the instrument, the biochemical process within the incubation vessel is observed, accessed and / or manipulated upon without exposing the fluid supportive of the biochemical process to external atmospheric conditions.

[0018] The fluid supportive of the biochemical process contained in the incubation vessel can preferably be of a chemically defined composition (for example Gibco High-Intensity Perfusion CHO Medium). In addition, the fluid supportive of the biochemical process may include an addition of phenol red to indicate pH in the medium.

[0019] Observation of production processes kept with fluid supportive of the biochemical process within the incubation vessel can preferably be observed with an inverted microscope, with the air-tight containment solution being temporarily lifted from the instrument (without exposure to external atmospheric conditions) to facilitate placement upon the microscope.

[0020] Manipulation of production processes kept within fluid supportive of the biochemical process within the incubation vessel, such as replacement of the used supportive fluid to fresh supportive fluid, can preferably be carried out by use of the self-sealing access plugs of the containment solution through which fluid supportive of the biochemical process can be transferred by use of a syringe (or similar) connected to a suitable perforator (such as a lance or needle) that temporary perforate the self-sealing access plug, thereby enabling exchange of fluid supportive of the biochemical process to the production process within the containment solution without causing exposure of the production process to external atmospheric conditions. If multiple samples of production processes, each with separate compositions of support fluid, are intended introduction of factors and / or differently composed support fluids to individual samples is similarly facilitated through the self-sealing access ports. If comparative use of distinct environmental conditions such as (but not limited to) differences in temperature or gas concentration is desired, multiple containment solutions housing production processes, each with desired environmental parameters, can be operated simultaneously with the same instrument. During the production process, one or more biochemical processes to produce biological material including organoids, viral particles, proteins, vectors, enzymes, antibodies and / or hormones can be performed.

[0021] It is possible that multiple separate biochemical processes be provided with controlled environmental conditions simultaneously by the same instrument.

[0022] During a production process, manipulation steps on biochemical processes in incubation vessels can comprise one of the following: enzymatic treatment, mechanical agitation, pH modulation, temperature modulation, catalytic reactions, fermentation, osmolarity modulation.

[0023] In a further aspect, the disclosure relates to a biological assay method, the method comprising i. Providing an instrument that can simultaneously perform multiple biological assays with independent sets of controlled environmental conditions with respect to at least temperature, humidity and gas composition; ii. Position laboratory incubation vessel(s) containing biological assays and fluids supportive of such biological assays in the instrument; iii. Operate the instrument so that the incubation vessel(s) follow a pitch-roll orbital wave motion path, resulting in gravity assisted flow and repositioning of the fluid supportive of the biological assay, thereby neutralizing diffusion gradients of any dissolved factors such as gases and biochemical factors within the fluid supportive of the biological assay; iv. Wherein, during operation of the instrument, the biological assay within the incubation vessel is observed, accessed and / or manipulated upon without exposing the fluid supportive of the biological assay to external atmospheric conditions.

[0024] The fluid supportive of the biological assay contained in the incubation vessel may be specific solutions intended for key steps of a chemically defined composition (for example Abeam Fluo- 8 AM, green fluorescent calcium binding dye).

[0025] Observation of biological assays kept with fluid supportive of the biological assay within the incubation vessel can preferably be observed with an inverted microscope, with the air-tight containment solution being temporarily lifted from the instrument (without exposure to external atmospheric conditions) to facilitate placement upon the microscope.

[0026] Manipulation of biological assay kept within fluid supportive of the biological assay within the incubation vessel, such as replacement of solutions intended for key steps of the biological assay, can preferably be carried out by use of the self-sealing access plugs of the containment solution through which fluid supportive of the biological assay can be transferred by use of a syringe (or similar) connected to a suitable perforator (such as a lance or needle) that temporary perforate the self-sealing access plug, thereby enabling exchange of fluid supportive of the biological assay to the containment solution without causing exposure of the biological assay to external atmospheric conditions. If multiple samples of biological assays are intended, introduction of factors and various fluids supportive of individual samples of biological assays is similarly facilitated through the self-sealing access ports. If comparative use of distinct environmental conditions such as (but not limited to) differences in temperature or gas concentration is desired, multiple containment solutions housing biological assays, each with desired environmental parameters, can be operated simultaneously with the same instrument.

[0027] The one or more assays can be performed as a measure of stability, functionality, viability, toxicity, biological factor resistance, chemical factor resistance, radiological factor resistance, pharmacokinetics and / or pharmacodynamics of one or more biological material. Multiple separate biological assays can be provided with controlled environmental conditions simultaneously by the same instrument.

[0028] During an assay, manipulation steps in incubation vessels can be applied, comprising one or more of the following: mechanical agitation, pH modulation, temperature modulation, osmolarity modulation.

[0029] BRIEF DESCRIPTION OFTHE DRAWINGS

[0030] FIG. 1 shows an illustration of pitch, roll and yaw motion.

[0031] FIG. 2 shows an exemplary containment solution.

[0032] FIG. 3 shows an exemplary heating plate.

[0033] FIG. 4 shows in (A) - (E) an illustration of various components of a motion control system.

[0034] FIG. 5 shows a mechanical construct wherein tilt of the motion control system can be varied.

[0035] FIG. 6 shows an exemplary circulation system for transporting humidified gas mixture.

[0036] FIG. 7 shows an exemplary system for preparing volumes of air with controlled concentrations of gas and humidity.

[0037] FIG. 8 shows a top view of an exemplary instrument. FIG. 9 shows pictures taken with brightfield microscopy of eukaryotic cells cultivated with the instrument including human pluripotent stem cells (A), human cancer cells (B), HEK cells (C), and cells / tissue from murine lung biopsy (D).

[0038] FIG. 10 shows pictures taken with brightfield microscopy of human hemogenic endothelium cultivated with a conventional hypoxic incubator (A) or cultivated with the instrument (B), with bar graphs indicating viability (C) and output of viable cells (D).

[0039] FIG. 11 shows pictures taken with brightfield microscopy of organoids produced with a conventional hypoxic incubator (A) or produced with the instrument (B), with bar graphs indicating organoid area (C) and organoid volume (D).

[0040] FIG. 12 shows administration of staining solution to biological material within cultivated with the instrument through a self-sealing port using a syringe and lance (A), with brightfield microscopic pictures confirming Trypan Blue staining of non-viable cells using the A549 cell line (B) and the H1975 cell line (C).

[0041] DESCRIPTION

[0042] The present disclosure relates to methods regarding handling of biological material, to which an instrument enables benefit and advantage by a set of features providing optimal and controlled environmental conditions. By combining compatibility with conventionally used plasticware and multi-sample approaches, with systems providing contained and controlled delivery of temperature and gases in a closed loop manner, with a solution to access the internal environment without exposure to the external environment, and with a solution to achieve exposure control to factors dissolved in liquids by means of a pitch-roll orbital wave movement to prevent the buildup of diffusion gradients in said liquids, the disclosed methods differentiates from prior art and conventional methods in technical nature and advantages.

[0043] Successful handling of biological material, such as cultivation of cells in the laboratory, is often not guaranteed. Cells may be difficult to successfully cultivate while maintaining their functional value, often resulting in failed experiments or undesired variability, while biological substrates may be hard to maintain due to sensitivity to unfavorable environmental factors. One major cause for these difficulties is arguably that if biological material becomes exposed to conditions that differ from conditions associated with the environment in which the biological material is naturally found, for example a cells natural niche in the body or the biological solutions in which proteins or hormones are found, exposure to such non-physiological conditions may directly cause undesired damage / alteration to the biological material. In the example of cultivated cells such undesired damage may result in cellular functional decline, which in turn limit the usefulness and application potential of cultivated cells in various life science applications, with examples including poor reliability of cells for analytical purposes, reduced yields when cells are used for production of drugs or biological substrates, or an inability to expand functional cells intended for therapeutic applications in healthcare. In the example of biological substrates including (but not limited to) proteins or hormones such undesired damage may result in altered chemical characteristics or 3-dimensional structure to potentially change the biological activity of the substrate, which in turn limit the scope of how effective the biological substrate may be across various life science applications, with examples including poorer production efficiency, shorter half-life, and less accurate understanding of the biological behavior of said substrates in the respective natural environment.

[0044] Be it a cell or a substrate, the environments in which biological material are naturally found are best described as dynamic environments where availability of relevant factors such as (but not limited to) nutrition, signaling substances, reactants, oxygen and antioxidants, are balanced to the optimal requirements of the biological material in question. For example, it is well documented that an overexposure of oxygen may cause increased amounts of oxidative damage to cellular components such as DNA, proteins, and lipids. However, oxygen is still a central component needed for cells to carry out metabolism, with cells absorbing dissolved oxygen present in their immediate environment, and situations where the minimal demands for oxygen cannot be provided is known to compromise cellular survival. Thus, for any given biological material the naturally intended exposure to oxygen is neither too much nor too little but corresponds to the amount found in the environment in which the biological material may be naturally observed.

[0045] Biological materials are often found as part of biological systems. Such biological systems, be it single cell organisms capable of self-propagation or large multicellular organisms equipped with circulatory systems, can be described as dynamic since the environment of the biological material is actively moved in relation to the biological material in question, which by extension help to control and maintain the local concentrations of factors and the environmental conditions. For systems that, instead of being dynamic, are passive in nature the only mechanism by which movement can happen will be through passive diffusion, which is a process by which something spontaneously move / diffuse from a region of higher concentration to a region of lower concentration until finally reaching equilibrium. This process follows the second law of thermodynamics and is described at the molecular level by Fick’s laws of diffusion. In practical terms, any system that relies on passive diffusion for movement will also face a physical limitation in terms of how quickly something can move from one point to another within that system. Using dissolved oxygen in a static volume of a liquid as an example, any oxygen that enters the solution at the surface interface (air to liquid) will diffuse towards the bottom of the solution at a limited rate, creating a diffusion gradient across the volume of the solution (with lower concentration at the bottom and higher concentration at the surface) until equilibrium has formed across the entire liquid volume. Imagine then the presence of a biological material (a cell) at the bottom of the solution that needs to consume a particular amount of dissolved oxygen per unit of time and area to stay alive, but simultaneously need to avoid becoming exposed to unnecessary high concentrations of dissolved oxygen since this would cause unwanted oxidative damage the cell. Depending on the concentration of oxygen gas in the air above the solution, and by extension the relative amount of oxygen dissolving into the solution and (by passive diffusion) reaching the cell per time and area, the cell will either get too much oxygen exposure, too little oxygen exposure, or (if the conditions are right) the desired optimal oxygen exposure. However, biological systems (like cells) are often in a state of continuous change. For instance, the metabolic activity of the cell(s) may suddenly accelerate, or the cell(s) may undergo division to suddenly double their numbers. These situations then change the balance between how much oxygen reach the location of the cell(s) (through passive diffusion) and how much oxygen the cell(s) optimally require, making it fundamentally challenging to use systems that are passive in nature to provide biological materials with controlled optimal environmental conditions, which is also why biological systems generally do not solely rely of passive diffusion but rather on dynamic movement. Using the above example of dissolved oxygen in a volume of a liquid, providing an active movement to the otherwise static liquid instead cause the liquid to dynamically mix with itself which result in facilitated and rapid equilibration of oxygen across the volume of the liquid. Since the delivery of dissolved oxygen to the cell(s) no longer is dependent on passive diffusion but rather on the oxygen concentration at equilibrium it now becomes possible to provide controlled amounts of oxygen to the cell(s) no matter if the cell(s) suddenly increase their consumption rate of oxygen. With these observations in mind, it can be argued that passive systems are fundamentally unsuitable for providing biological materials with controlled and stable environmental conditions, which is logical and explain why biological systems generally are dynamic in nature to better provide environmental control. By extension it could be argued that the ‘dynamic nature’ of a biological system, in itself, is a relevant feature if intending to provide biological material with ideal and controlled environmental conditions.

[0046] An important flaw with conventional methods is that the incubation vessels with biological materials are kept standing still (statically) when conventional incubators are used. This is fundamentally problematic since (as discussed above) reliance on passive diffusion in static scenarios result in that no actual control is provided over the biological materials relative exposure to factors dissolved in the solution in which the biological material is kept, resulting in suboptimal conditions where the material either becomes overexposed or underexposed to dissolved factors. Therefore, conventional incubators are not suitable for accurately providing biological materials with controlled environmental conditions. Alternative to common incubators are also conventional bioreactors, which do in fact provide a dynamic environment (typically achieved by impeller driven stirring of the volume of solution contained within the reactor vessel) to increase performance but are limited by the mechanical force exerted on the biological material through this mode of stirring as well as by an incompatibility to operate with multiple samples and with conventional models of plasticware on the same piece of equipment.

[0047] Another important flaw with conventional methods, where biological material is kept in conventional models of plasticware, is that the biological material becomes exposed to sudden changes in environment when they are retrieved from the common incubator for routine handling and work. Cells are sensitive to changes in their environment, and sudden changes in terms of oxygen exposure have been reported to directly impact cell signaling and behavior, also causing loss of viability. Daily handling of biological material in the laboratory typically includes the need to visually observe the material being handled as well as renewing the solution / medium in which the biological material is kept. The operator typically warms up fresh replacement media as well as preparing a sterile working station such as a laminar flow (LAF) cabinet, before opening the door of the incubator to access the biological material. This opening of the incubator allows the normal room environment / air to enter the incubator, and the gas-controlled environment of the incubator to spill into the room environment, causing a disturbance to the internal environmental conditions of the incubator and necessitating the addition of gas and heating to reestablish the internal target conditions. The operator then moves the biological material to the workbench and proceeds with the necessary operations typically taking between 3 and 20 minutes depending on what operations are required. During this time the biological material is unintentionally exposed to atmospheric air as well as experiencing a drop in temperature from the 37° C typically used in the incubator. Once all operations are completed the biological material is returned to the incubator where, once the disturbances of opening the incubator again have been compensated for, the biological material will again be kept in the internal environmental conditions. Since biological material (as mentioned above) is often sensitive to environmental changes, the inability of conventional incubators to prevent such disturbances during routine handling of biological material is another fundamental flaw with this type of equipment. Having already mentioned the incompatibility of bioreactors to operate with multiple samples and with conventional models of plasticware, it should be mentioned that bioreactors are incompatible with the typical routine operations (including microscopic observation and tracking) described above.

[0048] With these inherent flaws of conventional methods now apparent, a solution is presented herein that solves these flaws to provide distinct advantages over prior art solutions.

[0049] The disclosed solution relates to methods of handling biological material as dependent on an instrument having the following set of critical features: First, it allows for handling of multiple biological samples with the same instrument and is compatible with conventional models of lab plasticware including (but not limited to) petri dishes and multi-well plates. Second, it operates in a closed loop manner, relying on an airtight containment solution, a heating solution, a system for preparing volumes of air with controlled concentrations of gas and humidity, and a heated system for air circulation for providing desired environmental conditions to biological material without the need to expose the cells to sudden environmental changes or external conditions during operation. Third, by relying on a transparent barrier and integrated self-sealing plugs, it allows for observation of, and access to, biological material without the need to expose the biological material to sudden environmental changes or external conditions during operation. Fourth, it relies on a pitch-roll orbital wave motion to, by repositioning the lowest point for volumes of solution within conventional models of plasticware, facilitate gravitational displacement and mixing of the solution to the effect of achieving continuous equilibrium of dissolved factors across the volume of the solution, thereby preventing the buildup of diffusion gradients and the resulting undesired exposure levels (underexposure or overexposure) to dissolved factors associated with conventional methods. Be it a method of eukaryotic cell cultivation, a method of producing biological material, or a method relating to biological assay(s) these methods feature handling (including observation and manipulation) of s 1 sample(s) of biological material, kept in conventional models of plasticware, with an instrument providing controlled and dynamic environmental conditions in a closed loop manner.

[0050] An advantage of the disclosed method is that eukaryotic cells and biological assays can be maintained under controlled environmental conditions during the entire growth / reaction process, by virtue of the disclosed method including the controlled pitch-roll movement of the incubation vessel(s).

[0051] An instrument useful in the methods disclosed herein is an instrument for maintaining biological material under controlled environmental conditions.

[0052] Such an instrument is capable of; (i) simultaneously housing multiple samples of biological material with independent sets of controlled environmental conditions with respect to at least temperature, humidity and gas composition; (ii) position laboratory incubation vessel(s), used to house biological material and supportive liquid / medium, in the instrument; (iii) operate the instrument with biological material contained in supportive liquid / medium in at least one laboratory incubation vessel(s) so that the incubation vessel(s) follows a pitch-roll orbital wave motion path, resulting in gravity assisted flow and repositioning of the liquid / medium, thereby neutralizing diffusion gradients of any dissolved factors such as gases and nutritional components within the liquid / medium; (iv) wherein, during operation of the instrument, biological material within the incubation vessel is observed, accessed and / or manipulated upon without exposing the biological material and supportive liquid / medium to external atmospheric conditions. Furthermore, by comprising and air-tight containment solution, a dedicated solution to control temperature, a heated tank solution in which controlled amounts of water vapor and gas is prepared into an air mixture, and a solution for heated circulation of the airmixture, the instrument provides closed loop operational capability during handling of biological material with the instrument.

[0053] The instrument can generally comprise: (i) an air-tight containment solution having operational compatibility with conventional models of incubation vessels including (but not limited to) petri dishes and multi-well plates; (ii) the ability to run multiple (more than one) samples with the same instrument; (iii) a dedicated solution to control environmental temperature; (iv) a heated tank solution in which controlled amounts of water vapor and gas such as (but not limited to) carbon dioxide and nitrogen is prepared into an air-mixture; (v) a solution for a heated circulation system for the purpose of transporting heated air-mixture; and (vi) a solution providing motion following a pitch-roll orbital wave path to facilitate diffusion gradient neutralization, relating to factors dissolved in liquids within the containment solution, by gravity assisted flow and liquid repositioning. In addition, a solution allowing for observation, access to, and manipulation of biological material, without necessitating exposure of the biological material to external atmospheric conditions, is provided in the above-described instrument. Thus, the instrument provides biological material with closed loop handling (including observation, access to, and manipulation) and dynamic environmental parameter control.

[0054] The air-tight containment solution can be used together with conventional models of incubation vessels and plasticware such as multi-well plates or petri dishes commonly used when handling biological material in the laboratory. The air-tight containment solution can also feature a transparent barrier allowing for visual observation of the biological material kept within during operation. In addition, the air-tight containment solution can comprise at least one port (such as a self-sealing plug) to enable access to the biological material kept within without causing exposure of the biological material kept within to external atmospheric conditions. Such self-sealing ports can comprise or consist of any suitable self-sealing material. Selfsealing materials can automatically repair / close perforations to themselves without external intervention. Self-sealing materials include self-sealing polymers and elastomers such as natural or synthetic rubber materials. The air-tight containment solution can also comprise at least one air-tight sensor port for enabling the introduction of one or more sensors to measure one or more parameters within the air-tight containment solution. The parameters that are measured can for example be selected from gas concentration, temperature, pH, pressure and osmolarity.

[0055] By accommodating multiple incubation vessels for biological material, controlled environmental conditions can be simultaneously provided to multiple samples with the same instrument.

[0056] During incubation of biological material or cell growth, the instrument can be operated so that the pitch-roll movement is periodical, non-continuous in time or alternating in direction. For example, the instrument can be operated so that for a first period of time, the mechanism for providing the pitch-roll movement is turned on and for a second period of time that follows the first period, the movement mechanism is turned off so that the incubation vessels (conventional plasticware such as multi-well plates or the like) are stationary. This process can be repeated as necessary, generating on-off periods for the pitch-roll movement. Alternatively or additionally, the pitch-roll movement can be applied in alternating direction, i.e. in the clockwise direction for one period of time, followed by an counterclockwise direction for a second period of time.

[0057] Furthermore, incubation vessels with biological material can be individually provided with controlled amounts of environmental temperature with the same instrument.

[0058] The instrument can furthermore comprise heated tank solutions for the purpose of preparing air-mixtures of water vapor and gases such as (but not limited to) carbon dioxide and nitrogen. The heated tank solutions may for this purpose comprise one or more humidifiers, such as a heated humidifier. Such humidifier can comprise a volume of water that, when heater, provides the heated tank solution with humidified air. The heated tank solutions may for this purpose also comprise sensors to monitor the concentrations of relevant gases, ports for injection of pressurized gas, one or more fans to facilitate air mixing, and ports for exporting or importing prepared air mixture.

[0059] The gas and / or air mixture can be supplied at any suitable pressure, such pressure in the range of 0-300 kPa. By way of example the gas and / or air mixture can comprise carbon dioxide gas from a supply at 0-300 kPa and / or nitrogen from a supply that is at 0-300 kPa. The solution for heated circulation of air mixture can provide for sterile transportation of the prepared air mixture between the air-tight containment solution and the heated tank solution. Airflow rates can generally be in the range of 0-1000 mL / min, such as in the range of 0-10 mL / min, 0-100 mL / min and 0-500 mL / min.

[0060] Gas pressure within the air-tight containment solution may be maintained at any desired pressure which is typically in the range of 100-300kPa. For example, the gas pressure can be maintained at or near atmospheric pressure (101 .32 kPa + / - 30 kPa).

[0061] The solution for heated circulation of air-mixture can comprise one or more pumps, for moving air or gas mixture between the air-tight containment solution and the heated tank solution. The solution for heated circulation of air-mixture can furthermore comprise one or more filters, for filtering air or gas in the system.

[0062] The dedicated solution to control temperature, ensuring correct temperature to the air-tight containment vessel, can be provided through use of one or more heating plates. Thus, the dedicated solution to control temperature can comprise one or more heating plates adapted to meet the bottom of the air-tight containment solution, thereby providing heating to the containment solution and thereby to any biological material kept within the containment solution.

[0063] Alternatively, or additionally, controlled heating can be provided to the top of the air-tight containment solution. For example, controlled heating can further comprise heating by integration into the top of the air-tight containment solution, for providing the air-tight containment solution with controlled heating and preventing condensation on the inner top surface of the containment solution. Such controlled heating can further comprise heating means that is integrated into circulation filters and / or fittings for circulation filters in the system. Such controlled heating can further comprise heating means integrated into / onto the solution for heated circulation of air-mixture.

[0064] Controlled heating, comprising systems including the air-tight containment solution, the heated tank solution, and the solution for heated circulation of air-mixture, can be applied to any desired temperature that is in the range of about 5°C - 75°C, such as about 10°C - 65°C, 15°C - 50°C, 15°C - 40°C, 20°C - 37°C, 25°C - 37°C or 30°C - 37°C. In some cases, the desired temperature is ambient temperature. Cooling to temperatures below typical room temperature can be carried out, for example by placing the instrument within a cooled environment, e.g. a cooled room. Thereby, the instrument can be operated at the desired temperature using the internal heating mechanism(s).

[0065] An exemplary instrument for carrying out the methods described herein comprises: i. An airtight containment solution, the containment solution having at least one gas inlet and at least one gas outlet and at least one transparent surface for enabling observation of material within the system; ii. A solution for a heated circulation system that delivers gas / air or mixture thereof to and from the containment solution via the at least one gas inlet and the at least one gas outlet; iii. A motion control system that drives the containment solution, and any material contained therein along a pitch-roll orbital wave motion path that results in cyclical tilting movement; and iv. A heating solution that supplies heat to the containment solution and / or the circulation system in a controlled manner, thereby controlling temperature within the system.

[0066] A motion control system can comprise: i. At least one foundation having a lower surface and an upper surface for receiving one or more containment solutions for holding liquid for chemical reactants and / or biological materials; ii. A motorized component that is connected to the lower surface of the foundation, the motorized component having means to1 tilt the foundation with respect to a horizontal plane and drive the foundation in a pitch-roll fashion with respect to the plane of the foundation along a cyclical tilting path, while preventing a yaw motion of the foundation in the plane of the foundation. This way, the motorized component when in use drives the foundation along a pitch-roll orbital path so that any containment solution(s) positioned on the upper surface of the foundation follows the same motion.

[0067] A motion control system can alternatively comprise i. a base component, comprising a central shaft oriented in a z-direction perpendicular to a flat resting surface and a drive train for providing rotational movement around the central shaft; ii. A rotational component, the rotational component being operatively connected to the drive train so that the rotational component can rotate around the z-axis, the rotational component having an angled upper portion so as to form an angled wedge when viewed from the side, the upper portion following the rotational movement around the central shaft as the rotational component rotates; iii. A foundation, the foundation having a lower surface that meets the angled upper portion of the cylindrical rotational component and an upper surface, for receiving one or more containment solutions comprising chemical reactants and / or biological liquids; iv. An arrangement of bearings connecting the foundation to the rotational component so that the bearings form an interface between the rotational component and the lower surface of the foundation; and v. A linker component that is operatively connected to the platform, the linker component being at least partially deformable in the z-direction to facilitate pitch-roll motion of the foundation while preventing simultaneous yaw motion of the foundation.

[0068] When the apparatus is in operation the foundation follows the repositioning of the rotational component without itself rotating around the central shaft, thereby resulting in a pitch roll orbital wave motion of the foundation and any containment solution(s) placed on the foundation.

[0069] During the cultivation or biochemical process, motion following a pitch-roll orbital wave path to facilitate diffusion gradient neutralization is applied, relating to factors dissolved in liquids within the containment solution, by gravity assisted flow and liquid repositioning.

[0070] When in operation, the foundation follows the repositioning of the rotational component without itself rotation around the central shaft. This results in a pitch-roll orbital wave motion of the foundation and any air-tight containment solution placed upon the foundation. The linker component aids this functionality, by allowing movement in the z-direction (i.e., in the direction of the central shaft). At the same time, the linker component is minimally or not at all flexible in a direction perpendicular to the z-axis. For example, when the linker component is provided as comprising a generally flat elastic disc portion, the disc portion, when placed in the ground is partially flexible in the perpendicular direction while at the same time not being flexible in the plane of the disc.

[0071] The rotational component thus rotates around the central shaft of the base component when the instrument is operating and is driven by the drive train. The rotational component can be angled at its upper end, i.e. the upper end portion of the rotational component is angled when viewed from the side.

[0072] The upper surface of the foundation can be largely flat, to be able to accommodate dedicated solutions to control the environmental temperature of air-tight containment solutions placed upon them.

[0073] The cylindrical rotational component can have the bearings arranged on, at or near the upper end thereof, the bearings being aligned with the angled wedge of the upper end. Alternatively, the bearings can be arranged on, or attached to, the lower surface of the foundation. In either case, the bearings provide for the pitch-roll motion of the foundation as the rotational component rotates, without the foundation itself rotating.

[0074] The linker component can preferably contain, or consist of, elastic material that provides for the movement of the connected foundation in a pitch-roll fashion. The linker component can comprise an elastic disc with an aperture for connecting to the central shaft, thereby forming a connection between the central shaft and the foundation, the linker providing flexibility to movement of the foundation in terms of pitch and roll, while preventing yaw movement. Thus, the linker component can be connected to the central shaft so that the foundation is secured to the non-rotating central shaft. Through the flexibility of the linker component and the connection of the linker component to the rotational component via the bearings, the foundation is moved in a pitch-roll fashion as the rotational component rotates.

[0075] Turning now to FIG. 1 , the movement of pitch (tilting forward and backward), roll (tilting side to side), and yaw (tilting left and right) are illustrated. The methods and instrument described herein provides for a motion that includes a pitch and roll motion, i.e. tilting forward and backward, as well as tilting to the sides, while at the same time preventing yaw motion, i.e. motion dependent on movement around the z-axis. This way, an orbital wave motion is generated that results in favorable yet gentle mixing of liquid reagents within conventional models of plasticware housed with a containment solution of the instrument as described further herein.

[0076] In FIG.2, a side view of a containment solution 10 for use with an instrument for cell culturing and / or biological assaying is illustrated. The containment solution has a main body 11 , a lid portion 15 and a bottom portion 20. An import port 30 and an export port 35 are provided in the lid portion, for accommodating transport of humidified gas mixture to and from the containment solution. The lid portion and main body together serve to isolate the enclosed part(s) from the external environment, by forming a gas-tight seal / boundary to the upper part of the enclosed plasticware. This way, air / gas import and export ports can be used to control turnover of humidified gas mixture within the containment solution.

[0077] The lid portion of the containment solution has multiple access plugs 40, 41 , 42 located above the wells / vessels present on conventional models of plasticware, for delivery or removal of materials from within the containment solution. The access plugs can also be used for inserting sensors into the system. The access plugs are preferably self-sealing, allowing for delivery or removal from within the containment solution without exposing the interior of the containment solution to external environmental conditions. This way the containment solution can always remain air-tight during operation of the instrument. The lid portion of the containment solution has a heating wire 50 extending along the lid. The heating wire is shown to be embedded within the lid material, although it is possible to position the heating wire at or near the inner or outer surface of the lid.

[0078] Conventional models of plasticware 60 are kept within the main body of the containment solution, so that the plasticware rests on at least a part of the bottom portion 20. Liquid solutions, such as growth media for cultivated cells, are contained within wells 70 of the plasticware 60. During operation, the pitch-roll orbital wave motion provided by the instrument provides for the stirring of the liquid solutions kept within the containment solution to the effect of facilitating equilibrium of dissolved factors across the solution volume and to prevent the buildup of diffusion gradients.

[0079] The lid portion and / or the bottom portion of the containment solution can be comprised of transparent material, i.e. material that is transparent to visible light. This way, the interior of the containment solution can be viewed through the lid portion and / or bottom portion. Alternatively, the bottom portion can be at least partially open so that the conventional models of plasticware used together with the containment solution can be viewed directly from below. The containment solution may also be featured as a one-piece design, i.e. the containment solution features integrated vessels of non-conventional formats, for housing biological material.

[0080] Heating to the containment solution can additionally or alternatively be provided by means of a heating plate in which the containment solution rests. One such heating plate is shown in FIG. 3. The heating plate 80 has a main body in the form of a metal plate 82 that can be made from any suitable metal or material that conducts heat well, such as (but not limited to) aluminum or steel. The metal plate can have an upper surface that is designed to conform to the lower bottom of the containment solution, shown here by grooves 83 on the outer edges of the heating plate. Additional or other grooves or protrusions can be provided in the metal plate to accommodate containment solutions featuring differing dimensions and shapes.

[0081] The lower surface of the heating plate 80 has an electric heating pad 81 . This heating pad provides heat to the plate, driven by electricity. Any suitable heating pad or heating element can be used for this purpose. The heating pad can for example be an adhesive silicone heater mat, such as those supplied by http: / / rs-online.com.

[0082] In FIG. 4, an exemplary motion control system, designed to generate and provide a pitch-roll orbital wave motion to material placed thereon, is displayed. In (A), a base component 101 is shown in the form of a generally cylindrical housing with a central shaft 102 attached thereto. The base component furthermore has a drive train 104 designed to generate circular movement around the central shaft 102. In (B), a first rotational component 105 is shown, the rotational component having a generally cylindrical shape. The central shaft 102 extends through the rotational component. The first rotational component attaches to, and rests on, the base component 101 . Attachment of the first rotational component to the drive train 104 results in rotational movement of the first rotational component around the central shaft 102 during operation of the drive train 104. Rotation of the rotational component with respect to the base component is facilitated by bearings 103 on the base component that meet a lower end surface of the rotational component, allowing the rotational component to rotate with respect to the base component 101 . In (C), a second rotational component 110 is shown. This second rotational component is generally cylindrically shaped and is positioned and fastened on top of the first rotational component 105, with the central shaft 102 extending through the second rotational component 110. The upper end of the second rotational component 110 is slanted so that when viewed from the side, the upper end of the second rotational component 110 has a wedge-like shape. Bearings 111 , 112 are provided near the upper end of the second rotational component, to facilitate the rotational movement of the second rotational component with respect to the base component 101 . In (D), the underside of a foundation 120 is shown. This foundation has a generally flat upper surface (as shown in (E)), onto which one or more containment solutions may be accommodated. On the underside of the foundation shown in (D), a linker component 122 is shown. The linker component 122 has a disk-shaped portion 123 with a central opening. In this central opening an elastic disk 125 having a central aperture is attached. The central aperture of the linker component is designed to allow the central shaft 102 to extend therethrough. The disk-shaped portion 123 of the linker component 122 serves the purpose of meeting the bearings 111 , 112 of the second rotational component so that the second rotational component can freely rotate with respect to the non-rotating foundation 120. A circular protruding portion 124 on the linker component 122 is provided so that bearings 112 can meet an inner surface thereof, facing the central aperture, while bearings 111 meet the disk-shaped portion 123 of the linker component. The foundation 120 is secured to the central shaft 102 by a nut 126, as indicated in (E), where the assembled apparatus is shown from above. The nut can be replaced by other lock designs known in the art. The upper surface 121 of the foundation is generally flat, with protrusions 130 in the shape stoppers being provided to accommodate heating surfaces and containment solutions placed thereon. In the apparatus shown in (E), four sets of heating surfaces and containment solutions can be accommodated on the upper surface of the foundation. The elastic disk 125 of the linker component 122 can be seen through the central opening in the disk-shaped portion 123. It should be appreciated that the first and second rotational components 105, 110 can be provided as a single rotational component, through which the central shaft 102 extends. This single rotational component can preferably be generally cylindrically shaped with a wedge-like upper end, so that a foundation or platform attached thereto is tilted with respect to the surface on which the apparatus and instrument rest.

[0083] The foundation 120 is secured to the stationary central shaft 102. This has the consequence that as the rotational component(s) rotate, driven by the drive train 104, the foundation will be acted on to physically adjust its positioning to the rotational movement of the wedge-like upper portion of the rotational component 110. The elastic disk 125 if the linker component 122 allow for such adjustment, by being flexible in the z-direction (along the central shaft) so that the foundation can follow the rotation of the rotational component 110, facilitated by the bearings 111 , 112 on the second rotational component and bearings 103 in the base component 101 . The consequence is that the foundation, and any material placed thereon, will follow a movement path that results in motion back and forth and to the sides, i.e. a pitch-roll orbital wave motion. At the same time, there is no movement of the foundation that depends on rotation around the z-axis (yaw), i.e. side-to-side motion in the plane of the foundation since this is actively resisted by the linker component 122. It will be appreciated that bearings 111 , 112 shown here to be provided on the second rotational component, can alternatively be provided on the underside of the foundation 120. When so designed, the second rotational component will have a receiving surface so that the rotational component can move freely against the bearings located on the underside of the (non-rotating) foundation.

[0084] In FIG. 5, a side view of an alternative motion control system 222 is shown. The general mechanical construct is similar to that of the apparatus shown in FIG. 4. There is thus a base component 201 , a rotational component 210 and a platform-shaped foundation 220 resting on the rotational component 210. A central shaft 202 extends through the apparatus, being fixed to the base component and the foundation. There is further a tilting mechanism 230 shown, for varying the tilt of the foundation 220 with respect to the ground / plane upon which the apparatus and instrument is placed. The tilting mechanism can adjust the tilt of the foundation to any desired angle with respect to the ground, i.e. an angle in the range of 0° to 60°, such as 0° to 50°, 0° to 45°, 0° to 40° or 0° to 30°.

[0085] In FIG. 6, an exemplary circulation system for transporting humidified gas mixture is shown. The circulation system 300 can circulate humidified gas mixture through the overall systems of the instrument, driven by one or more pumps 302 having an entry point for humidified gas mixture 301 . The humidified gas mixture flows through tube 303, which is shown to have an (optional) air / gas filter holder 304, within which one or more filters 305 are placed. There is an exit point 306, from which humidified gas mixture exits the circulation system and into other systems of the instrument. An electrical heating wire 307 extends along the tube 303, with a second wire 308 providing heating to the air / gas filter holder 304, thereby allowing heating of the humidified gas mixture within the system and to prevent internal condensation of water vapor.

[0086] Alternatively, or additionally, the circulation system fortransporting humidified gas mixture may incorporate hollowed out sections on material (metal, plastics, etc.) through which the humidified gas mixture may travel. These sections of hollowed out material may also be heated by use of heating pads or similar.

[0087] An exemplary system for preparing volumes of air with controlled concentrations of gas and humidity 400 is shown in FIG. 7. The system consists of an enclosure 401 having a lid 404. The enclosure has a water import port 402 and a water drainage 403, for delivering water into and draining water from the system, respectively. During operation, the enclosure is partially filled with water 420. At the bottom of the enclosure there is a heating pad 411 , which serves the purpose of heating the water within the enclosure, thereby increasing the humidity within the enclosure. Heating of the water within the enclosure may also be facilitated by use of heat transfer from heated metal or similar.

[0088] The system for preparing volumes of air with controlled concentrations of gas and humidity has a port for exporting humidified gas mixture 413, as well as a port for importing humidified gas mixture 412, shown here to extend through the lid 404. Shown also is a port for the controlled injection of nitrogen (for the purpose of lowering the proportion of oxygen in the gas mixture) 405 and a port for the controlled injection of carbon dioxide (for the purpose of increasing the proportion of carbon dioxide in the gas mixture) 406. The system for preparing volumes of air with controlled concentrations of gas and humidity also feature a pressure escape port 407 to allow depressurization of the system as needed, a fan 408 used to continuously mix the internal volume of humidified gas mixture, and gas sensors 409, 410 used to determine the concentration of gases such as oxygen and carbon dioxide within the system.

[0089] When in operation the system for preparing volumes of air with controlled concentrations of gas and humidity can, dependent on what gases it can control, thus be used to supply the other systems of the instrument an air mixture of any desired composition of such gases, as well as having any desired humidity by means of controlling the heating of water within the system. There can also be a temperature sensor integral to the system to monitor the temperature of water within the system. Thereby, volumes of air with controlled gas composition and humidity can be regulated within the instrument.

[0090] In FIG. 8, an exemplary instrument is shown, referring also to FIG. 1-7. In the figure shown, one containment solution 10 seated on a heating plate 80 have been placed on top of a platform / foundation 120 that facilitates a pitch-roll orbital wave motion. The containment solution has ports 30, 35 for connecting the circulation system 300, themselves connecting to the ports 412, 413 on the system for preparing volumes of air with controlled concentrations of gas and humidity 400 (not shown), together facilitating circulation of humidified gas mixture to and from the containment solution 10. Two variations of self-sealing plugs 40, 41 are shown, for delivery or removal of materials from within the containment solution, or alternatively for inserting sensors into the system. Visible as part of the circulation system 300 is the air / gas filter holder 304, filter 305, and tubing 303. During operation of the system, up to four containment solutions may be supported on the platform / foundation 120, each containment solution either provided humidified gas mixture from the same system for preparing volumes of air with controlled concentrations of gas and humidity 400, or from individual such systems 400 allowing each containment solution on the instrument to operate with distinct environmental conditions. The containment solutions are all driven along the same pitch-roll orbital wave path provided by the platform / foundation 120.

[0091] Supporting the utility as a method of eukaryotic cell cultivation, FIG. 9 show pictures, captured during operation, of eukaryotic cell and tissue types cultured with an above-described instrument providing pitch-roll orbital wave motion at dynamic controlled conditions, with (A) showing a human pluripotent stem cell line (RB9-CB1 ) cultivated in the instrument for 24 hours at 37° C, 5% CO2, and 4% O2. In (B), cancer cells of a human glioblastoma line (U87) are seen at 72 hours of cultivation in the instrument at 37° C, 5% CO2, and 2,2% O2. In (C), HEK (Human Embryonic Kidney) cells are seen at 24 hours of cultivation in the instrument at 37° C, 5% CO2, and 2% O2. In (D), tissue and propagating cells from a murine lung biopsy is seen at 96 hours of cultivation in the instrument at 37° C, 5% CO2, and 3% O2.

[0092] Methods to cultivate cells with the instrument can be used for achieving superior output of more viable cells as compared to conventional incubators operating at similar temperature and gas conditions. FIG. 10 show the outcome of a trial where human pluripotent stem cells were used to generate hemogenic endothelium, with similar cells being kept at reduced oxygen level within a conventional incubator (24 hours at 37° C, 5% CO2, and 4% O2) or with the instrument operating at the same parameters but with pitch-roll orbital wave movement supporting equilibrium of dissolved factors in the media. Phenotypic observation of cells kept with conventional equipment (A) show cells with flatter and more dispersed morphology (indicating cell stress) while similar cells kept with the instrument (B) show an increased number of cells that also display s more 3-dimensional structure (as indicated by the white glow / halo associated with the cellular periphery). FACS based analysis using 7AAD staining demonstrated superior viability of cells cultivated with the instrument (C), and the total output of viable cells was also increased in the sample kept with the instrument (D).

[0093] Supporting the utility as a method of producing biological material, FIG. 11 show the recorded size of organoids (48 hours post formation) comparing the outcome using a conventional incubator (A) as compared to the instrument (B), both operating at similar parameters (37° C, 5% CO2, and 6% O2) but with the instrument using pitch-roll orbital wave movement to support equilibrium of dissolved factors in the media. Unbiased image analysis, and subsequent calculation of the organoids calculated area size (C) and volume size (D) was carried out showing superior organoid growth and generation with the method using the instrument.

[0094] Supporting the utility as a biological assay method, FIG. 12 show in situ staining of biological material with viability dye (Trypan Blue) carried out on cells kept with the instrument, operating at 37° C, 5% CO2, and 6% O2, without necessitating the exposure of the cells to external conditions, with (A) sequentially demonstrating the administration of staining solution into the containment solution through a self-sealing port using a syringe and lance. Microscopic analysis of the biological material kept within the containment solution confirmed successful staining of non-viable cells for cell line A549 (B) and H1975 (C) as indicated by retainment of Trypan Blue dye (see arrow heads).

[0095] As used herein, including in the claims, the terms “comprise”, “including”, “having”, and “contain” and their variations should be understood as meaning “including but not limited to” and are not intended to exclude other components or aspects of the invention.

[0096] Thos skilled in the art will readily appreciate that all parameters, dimensions, materials, compositions, and configurations described herein are meant to be exemplary ant that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present disclosure are used. The present invention also covers the exact terms, features, values and ranges etc. that are used in conjunction with terms such as about, around, generally, substantially, essentially, at least etc. (i.e., “about 3” shall also cover exactly 3 or “substantially constant” shall also cover exactly constant).

[0097] The term “at least one” should be understood as meaning “one or more”, and therefore includes both embodiments that include one or multiple components. Furthermore, dependent claims that refer to independent claims that describe features with “at least one” have the same meaning, both when the feature is referred to as “the” and “the at least one”.

[0098] Features disclosed in the specification, unless stated otherwise, can be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless stated otherwise, each feature disclosed represents one example of a generic series of equivalent or similar features.

[0099] Use of exemplary language, such as “for instance”, “such as”, “for example” and the like is merely intended to better illustrate the invention and does not indicate a limitation on the scope of the invention unless so claimed. Any steps described in the specification may be performed in any order or simultaneously, unless the context clearly indicates otherwise. All of the features and / or steps disclosed in the specification can be combined in any combination, except for combinations where at least some of the features and / or steps are mutually exclusive. In particular, preferred features of the invention are applicable to all aspects of the invention and may be used in any combination. The scope of the disclosure is only limited by the appended patent claims.

Claims

CLAIMS1 . A method of eukaryotic cell cultivation, the method comprising:

1. Providing an instrument that can simultaneously cultivate multiple samples of cell culture with independent sets of controlled environmental conditions with respect to at least temperature, humidity and gas composition; ii. Position laboratory incubation vessel(s) used to house cell cultivation and cultivation medium in the instrument; iii. Operate the instrument with eukaryotic cells contained in a cultivation medium in at least one laboratory incubation vessel(s) so that the incubation vessel(s) follows a pitch-roll orbital wave motion path, resulting in gravity assisted flow and repositioning of the cell cultivation medium, thereby neutralizing diffusion gradients of any dissolved factors such as gases and nutritional components within the cell cultivation medium; iv. Wherein, during operation of the instrument, cell cultivation within the incubation vessel is observed, accessed and / or manipulated upon without exposing the cell cultivation medium to external atmospheric conditions.

2. The method of claim 1 , wherein the eukaryotic cells are selected from pluripotent and multipotent stem cells (human and / or animal origin), cell derivates differentiated from pluripotent or multipotent stem cells (human and / or animal origin), male and female gametes / sex cells (human and / or animal origin), ex vivo cells and tissue retrieved from a living body (human and / or animal origin), cancerous cells and tumorigenic tissue (human and / or animal origin).

3. The method of claim 1 or claim 2, wherein the cultivation is done within a closed loop system by providing the instrument with a closed loop operational capability by the instrument comprising an air-tight containment solution, a dedicated solution to control temperature, a heated tank solution in which controlled amounts of water vapor and gas such as (but not limited to) carbon dioxide and nitrogen is prepared into an air mixture, and a solution for a heated circulation system for the purpose of transporting heated air-mixture.

4. The method of any one of the preceding claims, wherein multiple samples of cultivated cells are provided with controlled environmental conditions simultaneously by the instrument.

5. The method of any one of the preceding claims, wherein the movement following a pitch-roll orbital wave path is non-continuous or alternate in direction.

266. The method of any one of the preceding claims, wherein during the cultivation, at least one manipulation step on the cultivated cells in the incubation vessels is performed without exposing the cells to external air, the at least one manipulation step being selected from the following: enzymatic treatment, mechanical perturbation (of cells), pH modulation, modulation of cellular signaling pathways responses such as changing oxygenation or temperature, genetic manipulation, osmolarity modulation.

7. A method of producing biological material through a biochemical process, the method comprising: i. Providing an instrument that can simultaneously cultivate multiple samples of biochemical processes with independent sets of controlled of environmental conditions with respect to at least temperature, humidity and gas composition; ii. Position laboratory incubation vessel(s) used to house biochemical processes and fluids supportive of such processes in the instrument; iii. Operate the instrument so that the incubation vessel(s) follow a pitch-roll orbital wave motion path, resulting in gravity assisted flow and repositioning of the fluid supportive of the biochemical process, thereby neutralizing diffusion gradients of any dissolved factors such as gases and biochemical factors within the fluid supportive of the biochemical process; iv. Wherein, during operation of the instrument, the biochemical process within the incubation vessel is observed, accessed and / or manipulated upon without exposing the fluid supportive of the biochemical process to external atmospheric conditions.

8. The method of claim 7, wherein, during operation of the instrument, one or more biochemical processes to produce biological material including organoids, viral particles, proteins, vectors, enzymes, antibodies and / or hormones is performed.

9. The method of claim 7 or 8, the instrument facilitating closed loop operation by comprising an air-tight containment solution, a dedicated solution to control temperature, a heated tank solution in which controlled amounts of water vapor and gas such as (but not limited to) carbon dioxide and nitrogen is prepared into an air mixture, and a solution for a heated circulation system for the purpose of transporting heated air-mixture.

10. The method of claim 7-9, wherein multiple separate biochemical processes are provided with controlled environmental conditions simultaneously by the same instrument.11 . The method of any one of the claims 7-10, wherein the movement following a pitch-roll orbital wave path is non-continuous or alternate in direction.

12. The method of any one of the claims 7-11 , wherein manipulation steps on biochemical processes in incubation vessels comprises one of the following: enzymatic treatment, mechanical agitation, pH modulation, temperature modulation, catalytic reactions, fermentation, osmolarity modulation.

13. A biological assay method comprising the steps of: i. Providing an instrument that can simultaneously perform multiple biological assays with independent sets of controlled environmental conditions with respect to at least temperature, humidity and gas composition; ii. Position laboratory incubation vessel(s) containing biological assays and fluids supportive of such biological assays in the instrument; iii. Operate the instrument so that the incubation vessel(s) follow a pitch-roll orbital wave motion path, resulting in gravity assisted flow and repositioning of the fluid supportive of the biological assay, thereby neutralizing diffusion gradients of any dissolved factors such as gases and biochemical factors within the fluid supportive of the biological assay; iv. Wherein, during operation of the instrument, the biological assay within the incubation vessel is observed, accessed and / or manipulated upon without exposing the fluid supportive of the biological assay to external atmospheric conditions.

14. The biological assay method of claim 13, wherein, during operation of the instrument, the one or more assay is performed as a measure of stability, functionality, viability, toxicity, biological factor resistance, chemical factor resistance, radiological factor resistance, pharmacokinetics and / or pharmacodynamics of one or more biological material.

15. The biological assay method of claim 13 or 14, the instrument facilitating closed loop operation by comprising an air-tight containment solution, a dedicated solution to control temperature, a heated tank solution in which controlled amounts of water vapor and gas such as (but not limited to) carbon dioxide and nitrogen is prepared into an air mixture, and a solution for a heated circulation system for the purpose of transporting heated air-mixture.

16. The biological assay method of any one of claims 13-15, wherein multiple separate biological assays are provided with controlled environmental conditions simultaneously by the same instrument.

17. The biological assay method of any one of claims 13-16, wherein the movement following a pitch-roll orbital wave path is non-continuous or alternate in direction.

18. The biological assay method of any one of claims 13-17, wherein manipulation steps on biological assays in incubation vessels comprises one of the following: mechanical agitation, pH modulation, temperature modulation, osmolarity modulation.

19. The method of any one of the preceding claims, wherein the method is carried out using an instrument comprising: i. An air-tight containment solution, the containment solution having at least one gas inlet and at least one gas outlet and at least one transparent surface for enabling observation of material within the system; ii. A solution for a heated circulation system that delivers gas / air or mixture thereof to and from the containment solution via the at least one gas inlet and the at least one gas outlet; iii. A motion control system that drives the containment solution, and any material contained therein along a pitch-roll orbital wave motion path that results in cyclical tilting movement; iv. A heating solution that supplies heat to the containment solution and / or the circulation system in a controlled manner, thereby controlling temperature within the system.

20. The method of claim 19, wherein the motion control system comprises: i. At least one foundation having a lower surface and an upper surface for receiving one or more containment solutions for holding liquid for chemical reactants and / or biological materials; ii. A motorized component that is connected to the lower surface of the foundation, the motorized component having means to:1 . tilt the foundation with respect to a horizontal plane;2. drive the foundation in a pitch-roll fashion with respect to the plane of the foundation along a cyclical tilting path, while preventing a yaw motion of the foundation in the plane of the foundation, whereby the motorized component when in use drives the foundation along a pitch-roll orbital path so that any containment solution(s) positioned on the upper surface of the foundation follows the same motion.21 . The method of claim 19, wherein the motion control system comprises: i. A base component, comprising a central shaft oriented in a z-direction perpendicular to a flat resting surface and a drive train for providing rotational movement around the central shaft; ii. A rotational component, the rotational component being operatively connected to the drive train so that the rotational component can rotate around the z-axis, the rotational component having an angled upper portion so as to form an angled wedge when viewed from the side, the upper portion following the rotational movement around the central shaft as the rotationalcomponent rotates; iii. A foundation, the foundation having a lower surface that meets the angled upper portion of the cylindrical rotational component and an upper surface, for receiving one or more containment solutions comprising chemical reactants and / or biological liquids; iv. An arrangement of bearings connecting the foundation to the rotational component so that the bearings form an interface between the rotational component and the lower surface of the foundation; v. A linker component that is operatively connected to the platform, the linker component being at least partially deformable in the z-direction to facilitate pitch-roll motion of the foundation while preventing simultaneous yaw motion of the foundation, wherein when the apparatus is in operation the foundation follows the repositioning of the rotational component without itself rotating around the central shaft, thereby resulting in a pitch roll orbital wave motion of the foundation and any containment solution(s) placed on the foundation.30

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