Patent Application
20230085475
This application relates generally to cell isolation from tissue and more particularly, but without limitation, to methods and compositions for isolating lung cells from lung tissue, as well as cells produced from such methods. These cells may be used for research, cell therapies, tissue engineering, and other applications.
Methods to isolate different cell populations from cadaveric tissue are typically unique to each cell population, and often require different digestion enzymes, incubation times, and dissociation approaches. Due to the different isolation requirements for each cell type, tissue is often divided into separate pieces if multiple cell types are required from the same organ, thus reducing the overall possible yield for each cell type. In addition, these approaches are typically intensive and time-consuming, thereby further limiting the amount of tissue that can be processed while maintaining adequate cell health. Therefore, generating large numbers of different types of primary human cells from a single donor's tissue is a challenge. This challenge is of particular relevance to the field of tissue engineering for both autologous and allogeneic applications, where cell number requirements are high and primary cells have a limited expansion capacity. These isolation limitations could also impact personalized medicine and drug development and screening, whereby in vitro models may require generation of multicellular platforms from small pieces of donor tissue in order to achieve sufficient cellular complexity to accurately represent patient outcomes.
One specific example of a cell isolation challenge comes from processing lung tissue. The alveolar type 2 (AT2) cell is notoriously difficult to isolate. AT2 cells are often isolated from the right middle lung lobe. Isolating AT2 cells using existing methods requires multiple researchers and an entire day and yields only a few hundred million cells. This lengthy, hands-on process often limits isolation of other cell types that perform critical functions in the lung. Therefore, there is an unmet need for isolation methods that yield all critical cells of interest from donor lung tissue. Further, use of a single digestion method to isolate all cell types of interest would increase the cell yield of each cell type.
Described herein is a method to isolate different cell types using a single dissociation method from a donor organ. In some examples, disclosed is a method of isolating lung cells, such as one or more of alveolar type II cells (AT2), airway epithelial basal cells (AEP), stromal cells, and endothelial cells from human donor lung tissue. In some embodiments, the methods disclosed allow the isolation of a greater quantity of cells than other existing methods. Disclosed herein are a method of tissue dissociation and a method of cell purification, which may allow a greater number of cells to be isolated at once.
Obtaining sufficient numbers of a particular type of lung cell type, such as AT2 cells, from human donor lung tissue has been a long-standing challenge. These cells may be used to support diagnostic testing, drug discovery and development, cell therapy, or the construction of engineered organs. AT2 cells can be isolated using a method optimized in the Sannes lab at NC State (“Sannes method”, see Zhang, H., Newman, D. and Sannes, P. “HSULF-1 inhibits ERK and AKT signaling and decreases cell viability in vitro in human lung epithelial cells.” Respiratory Research. 2012; 13(1): 69), which is incorporated herein by reference in its entirety), originally developed by Leland Dobbs. AT2 isolation using the Sannes method typically yields a few hundred million AT2 cells from 1-2 lobes of human donor lung tissue. In contrast, the methods disclosed herein may enable the processing of all 5 lung lobes, which may produce as much as one billion AT2 cells without increasing staff or processing time.
Another major challenge is isolating a large number of specific types of lung cells, such as AT2 cells, with sufficient purity for downstream expansion using Sannes or other published methods. For instance, the Sannes method purification approach uses panning to remove white blood cells (differential adherence of cells to a plate), followed by a negative selection for fibroblasts. The panning approach does not easily scale and thus may be difficult with the increased number of cells generated by methods disclosed herein. Another existing purification method is use of a magnetic-activated cell sorting (MACS)- or fluorescence-activated cell sorting (FACS)-based positive selection approach based on the AT2 cell surface marker, HT2-280. However, this method is also not ideal as many fragile AT2 cells do not survive (average of 19% purification efficiency from MACS-based HT2-280 selection).
Disclosed herein is a method of purifying cells from lung tissue. The method may include removing white blood cells. The method may include removing one or more other types of cells. In some embodiments, antibodies bound to magnetic particles are used to select for and remove the white blood cells using magnetic-activated cell sorting techniques. Antibodies bound to magnetic particles may also be used to select for and remove one or more other type of cell. The remaining cells may be alveolar type II cells (AT2). The selected cells may be one or more of airway epithelial basal cells (AEP), stromal cells, endothelial cells, among others. The method may include purifying cell populations of interest from lung tissue isolate. The method may include removing white blood cells, stromal cells, and airway epithelial basal cells. The method may include selecting for endothelial cells. The method may include removing white blood cells and AT2 cells.
In some embodiments, cell surface proteins may be used to separate cells. The method may include selecting the less sensitive cells using antibodies for at least one marker chosen from CD45, CD16, CD32, CD90, CD144, CD31, CD140b, and CD271. The method may include removing the less sensitive cells using at least one marker selected from CD45, CD90, and CD271 markers. The CD45, CD90, and CD271 beads may be used to remove white blood cells, stromal cells, and airway epithelial basal cells. In some embodiments, antibodies bound to magnetic particles are used to select for and remove the white blood cells, stromal cells, and airway epithelial basal cells. In some embodiments, a 2-step selection may be performed whereby a CD45 selection is followed by a combined CD90 and CD271 selection.
Disclosed herein is a method of forming an engineered organ. The organ may be made from a synthetic or natural lung matrix. The method may include seeding a scaffold matrix with cells obtained from a method disclosed herein. In one embodiment, an engineered lung structure may be formed by seeding a lung scaffold with cells obtained from a method disclosed herein.
Disclosed herein is an engineered organ formed by seeding a scaffold with cells obtained from a method disclosed herein. Disclosed herein is an engineered lung structure formed by seeding a lung scaffold with cells obtained from a method disclosed herein. The cells may be purified by selecting white blood cells and at least one other types of cells using antibodies for one or more cluster of differentiation (CD) markers. The CD markers may be one or more of CD45, CD16, CD32, CD31, CD90, CD144, CD140b, and CD271. In some embodiments, white blood cells, stromal cells such as fibroblasts, endothelial cells, and airway basal cells may be selected. In some embodiments, the seeded cells may be one or more of alveolar type II cells, airway epithelial basal cells, lung stromal cells, and lung endothelial cells.
As used herein, “Lung Crush Method” or “LCM” refers to a method comprising application of mechanical force to crush tissue from which cells are to be isolated. The application of force can occur during or after enzymatic digestion of the tissue. The method may comprise additional steps, and the crushing force can be applied by any suitable means, e.g., mechanical grinding or the hands of a technician.
Cells isolated from human or animal organs may be used to support in vitro diagnostic and pharmaceutical testing, cell therapy development, and cellularization of scaffolds for regenerative medicine. These cellularized scaffolds may be used for transplantation into patients as clinical products. Obtaining sufficient numbers of a particular lung cell type, however, has been a long-standing challenge in the field. One such example is the isolation of AT2 cells from human donor lung tissue to use in the formation of an engineered lung tissue. The isolated AT2 cells may be banked, expanded, and used to cellularize porcine or 3D-printed lung scaffolds. These porcine or 3D-printed lung scaffolds may be transplanted into patients. The isolated AT2 cells may also be used to support research of AT2 cell identity and function, growth characteristics, disease states, and drug candidate screening in different platforms.
Isolations of AT2 cells have been performed using a method developed in the laboratory of Philip Sannes at NC State (herein described as the Sannes method). AT2 isolation using the Sannes method typically yields a few hundred million AT2 cells from 1-2 lobes of human donor lung tissue. Thus, the Sannes method must be repeated multiple times on different donors to secure a billion or more cells, leading to a high cost in time and material. Furthermore, pooling of cells from different donors for human cells, tissues, and cellular and tissue-based products is restricted by the FDA.
In contrast, the methods disclosed herein may enable the processing of all five lung lobes, which may produce as much as 1 billion AT2 cells, without increasing staff or processing time. Additionally, it may allow the processing of larger amounts of cells that may allow large-scale expansion in bioreactors. The methods disclosed herein allow for the production of an increased number of cells, which may decrease the need to isolate cells from additional donors. It may also allow for the build of donor-matched banks of multiple lung cell types and allow the repopulation of scaffolds with cells derived from a single donor. This is an important consideration for allogeneic tissue products, whereby using cells from a single donor with a close HLA match may be important to prevent organ rejection.
Additionally, it has been difficult to isolate a large number of AT2 cells or other specific lung cell types with sufficient purity for downstream expansion using Sannes or other published methods. Disclosed herein is a purification strategy that resolves this challenge. This purification method uses negative selection to remove non-AT2 cells that may overgrow in downstream cultures, leaving the sensitive AT2 cells unlabeled and in the negative population for downstream use. The positively selected non-AT2 cells can be seeded into culture to generate banks of other cell types of interest.
The Sannes Method (SM) may include removing the large, white airways and large chunks of undigested tissue. Small pieces of digested tissue may be transferred to a cup, minced using three pairs of surgical scissors taped together (referred to as triple-scissor) and collected. This process may be repeated several times until all of the digested tissue is minced.
The Lung Crush Method (LCM) may involve crushing the entirety of the digested tissue all at once using an object such as a hand or a mechanically automated crushing device, such as one using a rollers in series or parallel to apply force to crush the tissue. Crushing may include tearing open the pleura and allowing the digested tissue and cells to pour out and collect in a receptacle. Crushing may include squeezing the digested tissue. Crushing may include pulling apart the tissue. Crushing may include wringing out the tissue to collect additional cell suspension. The lung tissue may be crushed until only the airways and pleura remains. The airway tissue may be removed and the crushed tissue collected. The cell suspension following crushing has minimal undigested pieces of tissue remaining. In contrast, after cutting up the tissue using the Sannes method, pieces of tissue ranging from ˜1 to 5 mm are visible throughout the cell suspension. For example, tissue processed using the LCM may have no more than 20%, 10%, 5%, 2%, or 1% by weight of tissue pieces that are 1 mm, 2 mm, or 5 mm or more in diameter. In some embodiments, tissue processed using the LCM contains no more than 5% by weight of tissue pieces that are 5 mm or more in diameter. In some embodiments, tissue processed using the LCM contains no more than 5% by weight of tissue pieces that are 2 mm or more in diameter. In some embodiments, tissue processed using the LCM contains no more than 5% by weight of tissue pieces that are 1 mm or more in diameter. Determining the relative amount of pieces of tissue of a particular size can be accomplished using sieves, mesh, or the like of the appropriate size.
After being dissociated, the collected liquid may be filtered. The liquid may be filtered through surgical gauze or a mesh, silk, or nylon filter. The liquid may be filtered multiple times and through multiple filters. The liquid may also be centrifuged one or more times and the cell pellet resuspended.
Three head-to-head isolations were performed on donor-matched tissue to compare the LCM to the Sannes method prior to scaling up the LCM. For each donor in this comparison study, the tissue was divided into left and right lungs. One lung from each donor was processed using the Sannes method and one was processed using LCM. The lung that was assigned to each process was changed with each donor, as well as the operator performing the isolation. Data from this comparison study are included in
After filtration, the desired cells may be purified. The selection process may be a negative or positive selection process. In the Sannes purification method, undesired cells may be removed by differential adherence to non-tissue-culture Petri dishes with or without the use of antibodies. In some embodiments, a combination of differential adherence and magnetic removal may be used. The Sannes AT2 purification process may involve plating and panning to remove white blood cells and stromal cells, such as fibroblasts. The purification process may involve using an antibody, such as an AS02 antibody to selectively attach to the stromal cells. The antibody may be attached to a metal particle, allowing the stromal cells to be removed magnetically. Another commonly used method is to positively select the cells using an antibody for the AT2 cell surface marker, HT2-280 (Terrace Biotech, Mouse IgM monoclonal antibody), followed up by staining with an anti-mouse IgM magnetic bead. While positive selection via HT2-280 results in a high purity sample with low levels of contaminating cell types that may overgrow the culture, the purification efficiency is low with this selection method, thus leading to a low overall AT2 yield.
In the purification method described herein, other non-AT2 cells may also be removed. For instance, CD45, CD90, and CD271 antibodies may be added to bind to white blood cells, stromal cells, and airway basal cells. These antibodies may be bound to a metal particles. The metal particle, antibodies, and attached blood cells, stromal cells, and airway basal cells may be removed magnetically.
The antibodies bound to magnetic particles may also be used to select for and remove one or more types of cells to leave the most sensitive, desired cells behind. The desired cells left behind in the negative population may be banked for future use. The isolated cells may be alveolar type II cells (AT2). The isolated cells may be one or more of airway epithelial basal cells, stromal cells, endothelial cells, among others.
Depending on the identity of the desired cell, the method may include removing white blood cells, stromal cells, and/or airway epithelial basal cells. The method may include removing white blood cells and/or alveolar type II cells. Magnetic beads bound to antibodies for cell surface proteins may be used to selectively separate the cells that are not AT2 cells. The selected cells may be removed using at least one antibody for a cell surface protein selected from CD45, CD16, CD32, CD90, CD31, CD144, CD140b and CD271. The selected cells may be removed using at least one antibody for a cell surface protein selected from CD45, CD90, and CD271 markers. The CD45, CD90, and CD271 antibodies may be used to white blood cells, stromal cells, and airway basal cells from the AT2 population. Alternate markers may be used to remove all but the desired cell from the sample.
A negative selection approach using magnetic beads bound to antibodies for CD45, CD90, and CD271 was performed to purify AT2 cells from the head-to-head isolation tests of LCM and Sannes dissociation methods that were presented in
Following the head-to-head comparisons, the LCM was scaled up to process both the left and right lung tissue (bilateral lungs) from donors. The triple scissor dissociation step for the Sannes protocol is labor- and time-intensive, which limits the amount of tissue that can be processed at one time. The simplified dissociation with lung crush method allows the entirety of the lung tissue from one donor to be handled at once and decreases the overall processing time.
These purification methods are summarized in the following tables:
The following example describes specific aspects of some embodiments of this disclosure to illustrate and provide a description for those of ordinary skill in the art. The examples should not be construed as limiting this disclosure, as the example merely provides specific methodology useful in understanding and practicing some embodiments of this disclosure.
In this method, a lobe (usually the right middle), is dissected out for processing.
Cleaning: The lung lobe vasculature was perfused free of blood with Solution II (an aqueous solution of NaCl, Na2HPO4, HEPES, CaCl2), and MgSO4 7 H2O) at 37° C. Air was removed from the lung lobe, the lobe was cannulated, and washed with Solution I (an aqueous solution of NaCl, Na2HPO4, HEPES, glucose and EGTA). Lavage was repeated until draining solution ran clear.
Digestion: Elastase was dissolved in Solution II at 37° C. The lung lobe was incubated in a water bath set to 37° C. and filled with the warm elastase solution. The lung was allowed to digest until it became well-relaxed.
Tissue Dissociation (Sannes Method): Large, undigested chunks of tissue were excised. Large white airways were removed and discarded. Smaller lobe pieces were added to a chilled cup on ice containing 5 mL cold DNase solution (25 mg DNase in 50 mL of Solution II). These smaller lobe pieces were minced in batches with three pairs of surgical scissors “triple scissors” held or taped together in tandem. The minced cell solutions were collected into a 1 liter flask kept cold on ice. Once all of the tissue was processed, FBS was added to the cell suspension and the flask was shaken vigorously in a water bath (37° C.) for 3 minutes.
Filtration: The cell suspension was filtered through a layer of moistened surgical gauze up to 3 times. The cell suspension was filtered through 2 layers of moistened surgical gauze. This was repeated at least once to remove most of the large tissue pieces. The cell suspension was then filtered once or twice through moistened triple layer gauze. The cell suspension was filtered through a 165 μm silk or nylon mesh.
Centrifugation: The cell suspension was centrifuged at 200×g for 10 minutes at 4° C. The supernatant was discarded and the cell pellet resuspended in 5 mL DMEM media.
Plating: A petri dish was prepared with 500 μg/mL human IgG in Tris buffer at pH 9.5. In some cases, the dishes were incubated overnight at 4° C. About 5 mL of cell solution was delivered to the prepared IgG dishes.
Panning: The prepared cell dishes were panned for up to one hour in the incubator and until the white blood cells appeared well-adhered and gray but AT2 cells were still refractile and not attached. Fibroblasts also began to attach. The cell dishes were removed from the incubator and gently rocked to mobilize the AT2 cells. The unattached cell solution was collected and centrifuged at 200×g for 10 minutes at 4° C. The supernatant was discarded.
Fibroblast depletion option 1-Differential Adherence: The fibroblast population was depleted by differential adherence to non-tissue culture-treated Petri dishes for ˜1 hour.
Fibroblast depletion option 2-Magnetic Removal: The fibroblast population was depleted using an AS02 anti-fibroblast antibody negative selection step. The cell pellet was resuspended in DMEM. AS02 antibody was used to selectively attach to the fibroblasts. Tubes of cells and antibodies were gently rolled for a 10 minute incubation period at 4° C. DMEM/0.1% cell culture-grade BSA was added and the solution was centrifuged (10 min, 800 rpm, 4° C.). The supernatant was removed and the cells were resuspended in DMEM/0.1% BSA.
Dynabead prep: Pan-mouse IgG dynabeads were washed in 1 mL DMEM/0.1% BSA, collected magnetically and resuspended in DMEM/0.1% BSA. The Dynabeads were added to the cells and the solution incubated for 30 min at 4° C., rolling slowly end-over-end. The fibroblasts were removed magnetically for ˜2 minutes by a DynaMag-15 magnet. The unattached AT2 cells were poured off to collect and count. The cells were centrifuged to concentrate and to exchange medium for seeding. The pellets were resuspended in DMEM with 10% FBS and 2× antibiotic/antimycotic. The cells were counted and stored for future use.
Fibroblast depletion option 3: Fibroblasts were depleted using a combined method of option 1 and option 2.
In this method, bilateral lungs (all lung lobes) are used for processing.
Cleaning: The lung airway was cannulated and instilled with 1 L HBSS (−MgCl2, −CaCl2)). The HBSS was drained from the lung with gentle massage. Lavage was repeated 3 times. 2 final rinses were completed with HBSS (+MgCl2, +CaCl2)).
Digestion: Elastase, collagenase type IV, calcium chloride, and DNase was dissolved in HBSS (−MgCl2, −CaCl2) at 37° C. and instilled into the lungs (collagenase type IV and Dnase are not used in the Comparative Example 1). The lungs were placed into a Whirlpak bag and placed in a water bath set at 37° C. and the lungs were allowed to digest for approximately 45 minutes.
Tissue Dissociation (Lung Crush Method): Wearing sterile gloves, a human operator placed a hand inside bag. The pleura was torn open and the lung tissue was pulled apart and crushed by hand until only the airways remained. At this point, the remaining airway tissue was removed from the bag and disposed. The liquid contents of the Whirlpak bag were collected.
Filtration: The cell suspension was filtered through a series of mesh sheets with decreasing pore size (2000 μm, 1000 μm, 200 μm, 100 μm). After filtering, the cell suspension was brought up in DMEM/F12 media with DNase. 5% FBS was added to the cell suspension and mixed.
Centrifugation: The cell suspension was centrifuged at 300×g for 8 min. The supernatant was discarded and the cell pellet was resuspended in 5 mL DMEM/F12 with DNase.
Once a cell suspension was secured according to the above methods, AT2 cells, AEP cells, and stromal cells were purified according to the following method.
Magnetic Bead labeling of Stromal Cells, Airway Basal Cells, and White Blood cells: The cells were counted, for instance, with a K2 Cellometer (Nexcelom). The cell suspension was centrifuged, for instance at 300×g for 5 minutes at 4° C. The cell suspension was resuspended in media. In some cases, the media included DNase. CD45, CD90 and CD271 beads were added to bind with white blood cells, fibroblasts, and airway basal cells, respectively. In some embodiments, the beads were added in excess to the number of stromal cells, airway basal cells, and/or white blood cells expected in the sample. The sample was mixed well and incubated. In some embodiments, incubation occurred for 45 minutes at room temperature. The cells were washed, centrifuged, and resuspended in media.
Magnetic Separation of AT2 cells from Stromal Cells, Airway Epithelial Basal Cells, White Blood cells: The cells were placed into a container, such as a blood transfer bag, and attached to CliniMACS tubing set. A depletion program was selected, in this example specifically the Depletion program 3.1 program on the CliniMACS™ system (a cell purification system). The cells (AT2 cells) that were not selected using the Depletion program were counted via the K2 Cellometer (cell counter) and stored for future use.
The cells that were selected (CD45+/CD90+/CD271+) were split and seeded at 300,000-400,000 cells/cm2 into separate flasks in culture medium designed to support airway epithelial basal or stromal cells. These cultures generated purified populations of AEP and stromal cells over passage.
Lung cells were isolated from lung tissue according to the following method. The donor lung tissue was cleaned and digested according to the methods disclosed herein or known to those skilled in the art. The lung tissue was dissociated using a method such as the Lung Crush Method. The cell suspension was filtered through surgical gauze, nylon, mesh, or other porous material according to methods disclosed herein or other methods known in the art.
Once a cell suspension was secured according to the above methods, stromal cells were purified according to the following method.
A post-filtration sample was frozen down. A sample taken following isolation was evaluated for CD90 expression. Post-filtration cells were thawed and seeded in stromal cell medium at a concentration of 3,000 CD90+ cells/cm2. These cultures generated a purified population of stromal cells over passage. This purification method serves as an alternative to the use of selected cells to generate a stromal cell culture, and enables maximization of the selected population to be used for generation of airway epithelial basal cell cultures.
Endothelial cells were isolated from lung tissue according to the following method. The donor lung tissue was cleaned and digested according to the methods disclosed herein or known to those skilled in the art. The lung tissue was dissociated using a method such as the Lung Crush Method. The cell suspension was filtered through surgical gauze, nylon, mesh, or other porous material according to methods disclosed herein or other methods known in the art.
Once a cell suspension was secured according to the above method, endothelial cells were selected according to the following method.
Magnetic Bead labeling and Separation of Endothelial Cells: The cells were counted, for instance, with a K2 Cellometer (Nexcelom). The cell suspension was centrifuged, for instance at 300×g for 5 minutes. The cell suspension was resuspended in media. In some cases, the media contained DNase. CD45 beads were added to the cell suspension to bind with white blood cells. In some embodiments, incubation occurred for 15 minutes. The cells were placed into a container, such as a blood transfer bag, and attached to CliniMACS tubing set. A depletion program was utilized to select for the CD45 positive white blood cells. CD31 beads were then added to the negative fraction from the first purification step, in order to bind with endothelial cells. In some embodiments, the beads were added in excess to the number of endothelial cells expected in the sample. The sample was mixed well and incubated. In some embodiments, incubation occurred for 15 minutes at room temperature. The cells were washed, centrifuged, and resuspended in media. A MultiMACS instrument was used to then select for the endothelial cells. The endothelial cells that were selected were counted via the K2 Cellometer and seeded into culture in endothelial cell culture medium.
Endothelial cells can also be obtained by seeding cells directly into culture following digestion with a purification using CD31 selection following 1-2 passages of culture.
As used herein, the singular terms “a,” “an,” and “the” may include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an object may include multiple objects unless the context clearly dictates otherwise.
As used herein, the terms “substantially” and “about” are used to describe and account for small variations. When used in conjunction with an event or circumstance, the terms can refer to instances in which the event or circumstance occurs precisely as well as instances in which the event or circumstance occurs to a close approximation. When used in conjunction with a numerical value, the terms can refer to a range of variation of less than or equal to ±10% of that numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%. When referring to a first numerical value as “substantially” or “about” the same as a second numerical value, the terms can refer to the first numerical value being within a range of variation of less than or equal to ±10% of the second numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%.
Additionally, amounts, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.
While the disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure as defined by the appended claim(s). In addition, many modifications may be made to adapt a particular situation, material, composition of matter, method, operation or operations, to the objective, spirit and scope of the disclosure. All such modifications are intended to be within the scope of the claim(s) appended hereto. In particular, while certain methods may have been described with reference to particular operations performed in a particular order, it will be understood that these operations may be combined, sub-divided, or re-ordered to form an equivalent method without departing from the teachings of the disclosure. Accordingly, unless specifically indicated herein, the order and grouping of the operations is not a limitation of the disclosure.
This application claims priority to U.S. Provisional Application No. 63/237,003, filed Aug. 25, 2021, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63237003 | Aug 2021 | US |
