Methods for isolating, culturing, differentiating, and detaching myeloid cells from vessels

Abstract

A method for isolating, culturing, differentiating, and detaching myeloid cells, using untreated vessels made of plastic, thermoplastic, polycarbonate, or other similar materials, and detaching them using a dissociation agent applied at a temperature gradient.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 588,572, filed October 6, 2023, and U.S. Provisional Application No. 63 / 591,669, filed October 19, 2023, pursuant to 35 USC § 119(e). The contents of each of these applications, in their entirety, are incorporated herein by reference. Invention Field

[0003] This disclosure relates to methods for isolating, culturing, and differentiating myeloid cells (e.g., into macrophages) such that they can be readily detached and used for other downstream purposes while minimizing cytotoxicity and cell death. Specifically, embodiments of this disclosure involve using dishes untreated with hydrophilic agents or ultra-low cell adhesion agents to isolate, culture, and differentiate cells into other cell types, such as macrophages, and efficiently detaching these cells using dissociation agents at temperature gradients, thus eliminating the need for scrapers and other more destructive detachment methods. Background Technology

[0004] Peripheral blood mononuclear cells (“PBMCs”) are a mixed population of myeloid cells and lymphocytes. Myeloid cells are a type of blood cell that originates in the bone marrow. These heterogeneous cell populations are essential for the normal functioning of an organism's immune system and other systems because they can differentiate into neutrophils, eosinophils, basophils, macrophages, and other cell types. Because myeloid cells are important for the development and maintenance of the immune system and other systems, they are frequently used in research to explore and elucidate the mechanisms of disease and to test new therapeutic alternatives.

[0005] Macrophages are myeloid-derived cells found in various tissue types and exhibit a wide range of functions, including metabolism, bone remodeling, hematopoiesis, and immune surveillance. Macrophage functions are diverse and not static throughout their lives. For example, macrophages can be activated by antigens and antigen-activated immune cells, leading to a signaling cascade necessary for initiating, maintaining, and resolving inflammation. Due to the multiple roles and stages of macrophage activity, numerous biomarkers are used to differentiate them from other cell populations. Generally, macrophages can be classified based on the pro-inflammatory (M1) or anti-inflammatory (M2) characteristics produced by their precursor cells (homeostatic macrophages (M0)) after exposure to certain cytokines and other environmental factors. Different types of macrophages can be classified based on their cell surface markers. For example, common pan-macrophage markers include the expression of CD64, CD68, MerTK, CD115, etc. M1 macrophages can be identified based on increased expression of CD80, CD86, HLA-DR, CD40, etc. M2 macrophages can be identified by increased expression of CD206, CD163, CD200R, and CD209. Further references describing the expression patterns of various macrophage types include Murray et al. 2014, Moser 2003, and Mosser & Edwards 2008.

[0006] Although myeloid cells, macrophages, and other myeloid-derived cells are widely studied and used, barriers exist when culturing these cells, limiting downstream applications and experiments. For example, the strong attachment of macrophages to culture dishes may require the use of harsh desorption reagents and protocols, such as ethylenediaminetetraacetic acid (EDTA) and scrapers, which can be toxic or lethal to the cells. These effects can lead to loss of cell viability, reduced yields in downstream experiments and assays, artifacts that interfere with result analysis, and other undesirable consequences.

[0007] PBMCs and other myeloid-derived cells can originate from tissue donors in humans or other organisms, which can lead to donor-to-donor variability in cell populations. For example, PBMC donors can have a lymphocyte population ranging from 70% to 90%, while the monocyte population can range from 10% to 20%. These donor-to-donor population ratio differences can affect optimal culture conditions as well as downstream processes and applications. For instance, optimal culture of PBMCs and other myeloid-derived cells may include consideration of monocyte seeding density. Proper monocyte seeding density allows these cells to grow and differentiate into macrophages with healthy morphology, allowing for better yields after electroporation and other assays. Similarly, proper seeding density allows cells to adhere better to the plate, minimizing cell loss during washing and culture medium changes.

[0008] Cell culture and growth, such as myeloid cells, macrophages, and other myeloid-derived cells, can be performed in tissue culture-treated dishes. To convert these dishes into tissue culture-treated dishes, the dishes are exposed to reagents such as gas plasma treatment, where the surface of the dish (where cells can attach and grow) becomes more hydrophilic and negatively charged, making the dish more suitable for cell attachment. Other dishes that can be used to culture cells (such as myeloid cells) include ultra-low adhesion plates, which may contain a hydrogel layer or other materials covalently bound to the surface of the cell-attachable dish, thereby inhibiting cell attachment to the plate. However, for many cell types, such as macrophages, growing cells in tissue culture-treated dishes or ultra-low adhesion plates may not be ideal. For example, isolating, culturing, and growing macrophages in tissue culture-treated dishes can result in a reduced number of cells attaching to the dish. Additionally, macrophages grown in tissue culture-treated dishes may exhibit unhealthy and elongated morphologies and more immature phenotypes, making them unsuitable for many downstream applications such as electroporation and cell therapy. On the other hand, macrophages grown on ultra-low adhesion plates result in a phenotype with lower packing density and a higher proportion of elongated stress cells (which may indicate immune activation), which may be undesirable for many assays and other downstream applications.

[0009] Suboptimal isolation, culture, differentiation, and detachment of myeloid-derived cells using tissue culture-treated dishes or ultra-low adhesion plates also results in undesirable strong adhesion of these cells to the plate. Because of the stronger adhesion in tissue culture-treated dishes or ultra-low adhesion plates, when detachment is required, operators may have to use harsh detachment methods, such as a combination of ethylenediaminetetraacetic acid (EDTA) and mechanical detachment using tools (such as cell scrapers), which can damage cells and affect downstream applications.

[0010] The combined use of inappropriate glassware (as described above), inappropriate seeding densities, and harsh desorption methods leads to inefficient resource utilization, as cells become less healthy, more likely to die, may grow slower, and may be activated. These effects can be undesirable because they can result in lower yields for downstream applications or render cells insufficient for many downstream applications, such as electroporation and cell therapy. Furthermore, slower growth and reduced yields necessitate the use of additional resources, such as cell culture media, growth factors, and operator time and effort. In short, these inefficiencies can lead to unnecessary costs.

[0011] Therefore, there is a need to improve methods for isolating, culturing, growing, differentiating, and detaching myeloid cells. In some embodiments, improved methods utilize growth vessels, for example, those untreated by tissue culture and made of materials such as glass, plastic, thermoplastic, polyolefins, polycarbonate, and polystyrene, which allow the use of less stringent dissociation reagents, such as Accutase® and trypsin (e.g., Gibco). TM Trypsin-EDTA (0.25%) (phenol red) is used under a temperature gradient, so desorption leads to reduced toxicity and cell death. Invention Overview

[0013] According to a non-limiting aspect of this disclosure, a method for culturing myeloid cells is provided, the method comprising using a vessel untreated with a hydrophilic agent such as plasma gas. The vessel may be made of glass, plastic, thermoplastic, polyolefin, polycarbonate, polystyrene, or other materials. The vessel may be a culture dish having a surface area of, for example, 10 mm, 11 mm, 12.7 mm, 35 mm, 60 mm, 94 mm, 100 mm, and 145 mm. The vessel may have a surface area of ​​less than, greater than, or equal to 640 cm². 2 Cell culture bags with a culture area of ​​[missing information]. This vessel can be a substrate for culturing cells in a 3D environment, such as a gel matrix, and can have volumes of 1 mL, 5 mL, 20 mL, 50 mL, 100 mL, etc. This vessel can be a 6-well, 12-well, 24-well, 48-well, or 96-well plate. This vessel can have a volume including 25 ml (12.5 cm²). 2 ), 50ml (25cm) 2 ), 250ml (75cm) 2 ) and 600ml (182cm) 2 The method also includes using a flask or other medium suitable for cell culture. The method further includes using a cell culture medium supplemented or unsupplemented with serum, such as fetal bovine serum (FBS) or human serum albumin (HSA), in an amount of about 2-15%. The method also includes culturing cells under standard cell culture conditions. For example, cells can be cultured at a temperature of 32°C to 37°C, a CO2 concentration of 4% to 7%, and a humidity of about 80% to 95%. Cell culture conditions may vary depending on the cell type, downstream application, and conditions suitable for the situation as determined by a person skilled in the art, and may exceed the limitations described. Methods for culturing myeloid cells include using a 2 x 10⁻⁶ cell culture medium. 4 cells / cm 2 At most, for example, 8 x 10 5 cells / cm 2The methods for culturing cells described herein include inoculating cells at concentrations of M-CSF to promote their attachment by incubating them for a period of time, including, for example, 18 to 36 hours. The methods for culturing cells described herein include culturing cells to a desired confluence, for example, ranging from 75% to 100%. The methods for culturing cells described herein include culturing cells to differentiate them into other cell types by using, for example, macrophage colony-stimulating factor (M-CSF) at concentrations ranging from, for example, 10 ng / mL to 200 ng / mL. Other examples of factors that can be used to differentiate macrophages include granulocyte-macrophage colony-stimulating factor (GM-CSF) at concentrations ranging from, for example, 1 ng / mL to 200 ng / mL. The methods for culturing cells described herein include culturing cells with M-CSF under similar temperature, humidity, and CO2 conditions as described above to promote differentiation for a period of time, including, for example, 4 to 7 days.

[0014] Another non-limiting aspect of this disclosure includes a method for desorbing myeloid-derived cells, such as macrophages, from a culture dish. In some embodiments, the dish may be made of glass, plastic, thermoplastic, polyolefin, polycarbonate, polystyrene, or other materials. In some embodiments, the dish described in these methods need not be treated with a hydrophilic agent such as plasma gas. The dish may be a culture dish having a surface area of, for example, 10 mm, 11 mm, 12.7 mm, 35 mm, 60 mm, 94 mm, 100 mm, and 145 mm. The dish may have a surface area of ​​less than, greater than, or equal to 640 cm². 2 Cell culture bags with a culture area of ​​[missing information]. This vessel can be a substrate for culturing cells in a 3D environment, such as a gel matrix, and can have volumes of 1 mL, 5 mL, 20 mL, 50 mL, 100 mL, etc. The vessel can be a 6-well, 12-well, 24-well, 48-well, or 96-well plate. The vessels described in these methods can have volumes including 25 ml (12.5 cm²). 2 ), 50ml (25cm) 2 ), 250ml (75cm) 2 ) and 600ml (182cm) 2The method involves using a flask, or other container, for example, to promote cell desorption from the vessel using a dissociation agent, which may be enzymatic or non-enzymatic. In some embodiments, the method described herein includes using a dissociation agent, desorption agent, or enzyme-based cell dissociation medium or solution, such as Accutase® or trypsin. In some embodiments, the method described herein includes using a temperature gradient when using a dissociation agent to promote cell desorption at high cell viability. The method described herein includes using a temperature gradient that starts at a lower temperature and gradually increases to a higher temperature. For example, the temperature gradient described herein includes applying a dissociation agent at a temperature of 4°C to 20°C. For example, the dissociation agent is at a temperature of about 4°C. The dissociation agent used under the temperature gradient includes culturing cells with the dissociation agent at an initial temperature of 2°C to 6°C for a period of up to 20 minutes. The dissociation agent used under the temperature gradient includes culturing cells with the dissociation agent at a second temperature ranging from 19°C to 23°C for up to 20 minutes. The dissociation reagent used under a temperature gradient includes culturing cells with the dissociation reagent at a third temperature ranging from 32°C to 38°C for up to 60 minutes. In some embodiments, the incubation and culture periods described herein can be performed under the aforementioned CO2, humidity, and other condition parameters. In some embodiments, the methods described herein include transferring cells into suitable vessels for downstream applications such as electroporation, cell therapy, or others.

[0015] As used herein, “processing component” (PA) is intended to describe a unit or units that contain the mechanism of loading and electroporation of cells.

[0016] As used in this article, "room temperature" is intended to describe a temperature in the range of 20°C to 22°C.

[0017] As used herein, “macrophage” refers to a cell that possesses one or more of the following characteristics: large, adhesive, phagocytic, and derived from monocytes. Furthermore, as used herein, macrophages include those expressing pan-macrophage markers, including CD64, CD68, MerTK, CD115, etc. As used herein, macrophages include M1 macrophages, which can be identified by increased expression of CD80, CD86, HLA-DR, CD40, etc. As used herein, macrophages include M2 ​​macrophages, which can be identified by increased expression of CD206, CD163, CD200R, CD209, etc.

[0018] As used in this article, "myeloid cells" is intended to describe cells that express, for example, the following characteristics: CD11b, CD206, CD33, CD52, and other markers.

[0019] As used herein, “vessel,” “growth vessel,” and “culture vessel” are used interchangeably and include flasks, well plates, substrates, culture bags, culture dishes (e.g., culture dishes for culturing bacteria), and other similar vessels for isolating, growing, or culturing cells.

[0020] As used herein, “untreated tissue culture vessels” or “untreated tissue culture vessels” refers to those that have not been treated with a hydrophilic agent (e.g., with plasma gas) to enhance their hydrophilic properties, which typically helps adherent cells attach to the vessel.

[0021] As used in this article, an “ultra-low adhesion vessel” is a vessel containing a layer of compound (such as a hydrogel) on a cell-adherable surface that prevents cell adhesion.

[0022] As used in this article, “macrophage colony-stimulating factor” (“M-CSF”) is used to describe hematopoietic growth factors and related cofactors used to proliferate, differentiate and maintain monocytes, macrophages and other cell types.

[0023] As used in this article, “differentiation” refers to the process or outcome of cells or populations that includes cell division and modifications that can lead to functional or phenotypic characteristics of another cell type. For example, myeloid cells or cell populations differentiate into macrophages or cell populations.

[0024] As used herein, "cell dissociation reagent" describes a solution that causes cells to detach from a vessel via chemical, enzymatic, or other mechanisms. Examples of enzyme dissociation reagents or enzyme-mediated cell desorption media include TrypLE. TM Trypsin and Accutase®. Examples of chemical or non-enzymatic dissociation reagents include Non-Enzymatic Cell Dissociation Solution 30-2103™, as well as EDTA alone or in solution or buffered form such as PBS (e.g., PBS / EDTA). Attached Figure Description

[0025] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate some disclosed embodiments and, together with the specification, serve to explain the disclosed embodiments. The details shown are as examples and for illustrative purposes, embodiments of this disclosure are discussed. The description taken in conjunction with the drawings makes it apparent to those skilled in the art how embodiments of this disclosure can be practiced.

[0026] Figure 1 This is a collection of four images of macrophages grown on various dishes. The images include embodiments of disclosures 103 and 104, in which macrophages are grown on plastic dishes that have not been treated with a hydrophilic agent.

[0027] Figure 2 These are images of embodiments of the present invention, showing the ideal phenotype of macrophages grown on polystyrene culture dishes without tissue culture treatment.

[0028] Figures 3A-3E The method described above was used to culture and extract mRNA / DNA ( Figure 3A ), RNP ( Figure 3C Cell viability of macrophages subjected to electroporation, mRNA / DNA ratio ( Figure 3B ) and RNP ( Figure 3D The transfection efficiency and knockout efficiency of ) Figure 3E ).

[0029] Figure 4 The results show a microscopic comparison of the transfection efficiency of macrophages prepared using the methods described herein compared to conventional macrophage culture methods. Invention Details

[0031] The following is a detailed description with several non-limiting embodiments and figures, and the provided components and steps may be substituted, added or modified.

[0032] The embodiments disclosed herein relate to methods for isolating, culturing, and differentiating myeloid cells to optimize their health status and differentiation process, using, for example, 25 ml (12.5 cm) of polystyrene that has not been treated with hydrophilic agents such as plasma gas. 2 A flask. Furthermore, embodiments of this disclosure include placing the flask in a 2 x 10 inch container. 4 cells / cm 2 The seeding density of the cells is determined by plating them at a suitable density and culturing them for a sufficient time to allow cell attachment to the vessel, including culturing the cells for 18 hours at 37°C, 5% CO2, and 95% humidity. Furthermore, embodiments of the invention include washing the culture with phosphate-buffered saline (PBS) and adding cell culture medium supplemented with 100 ng / mL M-CSF to promote myeloid cell differentiation into macrophages. Additionally, embodiments of this disclosure include culturing the attached cells in M-CSF-supplemented medium at 37°C, 5% CO2, and 95% humidity for 5 days to allow the cells to differentiate into macrophages. Furthermore, embodiments described in this disclosure include removing 12.5 mL of M-CSF-supplemented medium from the flask and adding 12.5 mL of M-CSF-supplemented medium (100 ng / mL) to the flask, and culturing for an additional 2 days under the aforementioned temperature, CO2, and humidity conditions.

[0033] Embodiments of this disclosure include using methods such as flow cytometry to determine the ratio or percentage of monocytes, lymphocytes, or other cell types in the donor prior to seeding and culturing the cells. If the monocyte population is found to be below the optimal 50% to 55%, the seeding density of the seeded cells should be increased. For example, if the monocyte percentage is approximately 30%, the seeding density should be increased to approximately 2 x 10⁻⁶ cells / year. 8 cells / cm 2 PBMC, instead of approximately 2 x 10 4 cells / cm 2 Density inoculation of PBMCs.

[0034] Embodiments of the invention also include modifying the cell culture protocol to reduce cell loss or increase the yield of cultured and differentiated cells. For example, if the monocyte population in the PBMC donor is found to be below the optimal 50% to 55%, the cell culture protocol can be modified. For instance, if the monocyte percentage is about 30%, the PBMCs can be cultured for 36 to 48 hours at seed with, for example, 100 ng / mL M-CSF, instead of culturing the cells for 18 to 36 hours in the aforementioned M-CSF-free medium to allow cell attachment. Additionally, when the monocyte population is below optimal, for example, about 30% monocytes in the PBMC donor, other modifications to the cell culture conditions include not washing the cells with PBS before adding fresh medium or other fluids.

[0035] Figure 1 This is a collection of macrophage culture images grown in different containers, including embodiments of the present invention, wherein macrophages are grown in sterile polystyrene culture dishes that have not been treated with hydrophilic agents such as plasma gas 104 (without tissue culture treatment). Figure 1 As shown, macrophages grown in tissue culture dish 101 exhibited reduced confluence and a stressed and unhealthy morphology, characterized by a greater number of spindle-shaped cells and a reduced number of omelet-shaped cells. Similarly, Figure 1 Macrophages growing on ultra-low attachment plate 102 are shown, where macrophages grow to a lower level of confluence and based on flow cytometry data ( Figure 1 (Not shown in the image) exhibits unusual spiderweb morphology and macrophage markers. Figure 1Macrophages grown on a non-tissue culture-treated dish 103 are also shown, in which the cells exhibit healthy morphologies, including a greater frizzle phenotype (characterized by flattened and relatively rounded cytoplasm and small, relatively rounded nuclei; this morphology is also described as having a prominent perinuclear halo and a centrally located round nucleus) and some elongated or spindle-shaped macrophages (characterized by a wider middle section that gradually narrows at both ends away from the wider middle; other terms describing this morphology include conical, spindle-shaped, elliptical, and cylindrical). Finally, Figure 1 Cells grown on a polystyrene culture dish 104 without tissue culture treatment were also shown. Macrophages grown under the latter condition 104 exhibited higher confluence levels and a healthy mixed phenotype (e.g., Figure 2 As shown in the figure, it is ideal for easier detachment and subsequent measurement and treatment.

[0036] Figure 2 This is an image illustrating an embodiment of the invention, in which macrophages are cultured on polystyrene dishes without prior tissue culture treatment. The image shows cells growing to a healthier, denser level and the desired confluence, ideal for subsequent assays and treatments. The morphology of this population includes spindle-shaped morphology 201 (characterized by a wider middle section, narrower towards the ends; other terms describing this morphology include conical, spindle-shaped, elliptical, and cylindrical) and omelet-shaped morphology 202 (characterized by a flattened and relatively rounded cytoplasm and a smaller, relatively rounded nucleus; this morphology is also described as having a prominent perinuclear halo and a centrally located round nucleus). Such morphological variations may indicate that the macrophages are not yet activated and may be more suitable for subsequent applications.

[0037] Figure 3 shows the results of the experiment described in Example 5, which tested the cell viability of macrophages cultured and then electroporated according to the disclosed method. Post-electroporation evaluation included: loading mRNA / DNA ( Figure 3A ), RNP ( Figure 3C Macrophage activity after ( ); mRNA / DNA ( Figure 3B ) and RNP ( Figure 3D The transfection efficiency of RNP-transfected macrophages; and the knockout efficiency of RNP-transfected macrophages. Figure 3E Four days after electroporation, cell viability and gene knockout efficiency were assessed using flow cytometry.

[0038] Figure 4 This study compares the transfection efficiency of macrophages transfected using electroporation according to conventional methods of macrophage culture with that of the method described herein. Figure 4Further, it was shown that, compared with cells cultured using conventional methods (“control group”), cells cultured using the disclosed method (“experimental group”) exhibited higher viability and improved morphology even after electroporation. Figure 4 This further demonstrates the improved electroporation and transfection efficiency of macrophages cultured using the disclosed method.

[0039] Embodiments of this disclosure also relate to growing polystyrene in a vessel (e.g., 25 ml (12.5 cm²) without hydrophilic agents such as plasma gas treatment, under a temperature gradient, using a dissociation agent. 2 A method for desorbing macrophages from a flask. One embodiment of the method disclosed herein involves applying an enzymatic dissociation reagent or an enzyme cell desorption medium, such as Accutase®, to the cell culture at a temperature of 4°C, and applying a volume sufficient to cover the surface of the flask where the cells are growing, for example, in 25 ml (12.5 cm²) of water. 2 Apply 7 mL of Accutase® to the flask. Furthermore, embodiments of this disclosure include incubating cells with Accutase® at an initial temperature of 4°C for 20 minutes, followed by culturing the cells at 20°C for 20 minutes, and finally culturing the cells at 37°C for 60 minutes. Additionally, embodiments described herein include aspirating the flask with a 10 mL serum pipette and transferring it to a suitable dish or container (such as a 10 mL Falcon tube) for further processing or subsequent applications, such as electroporation or transfection.

[0040] It should be understood that the method claimed is scalable and not limited to any particular cell density range. It should also be further understood that the cells were incubated at standard incubation parameters (37°C, 5% CO2).

[0041] In one embodiment, the temperature of the incubator is selected from, but is not limited to, a temperature range of 30°C–40°C and a CO2 range of 1%–10%.

[0042] In one implementation, the initial concentration of myeloid cells in the plating can be 1 x 10⁻⁶. 4 Up to 1 x 10 6 cells / cm 2 between.

[0043] In one implementation, the growth vessel used is an untreated culture vessel or a sterile bacterial culture vessel.

[0044] In one embodiment, the growth vessel may be made of materials including, but not limited to, plastics, polycarbonate, thermoplastics, polyolefins, polystyrene, or other similar materials.

[0045] In one embodiment, any standard cell culture medium can be used with the claimed method, such as, but not limited to, Dulbecco Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco Modified Eagle Medium Nutrient Mixture F-12 (DMEM F12), and TexMACS. TM Minimum Essential Medium (MEM) and Iscove Modified Dulbecco Medium (IMDM).

[0046] In one implementation, agitating the culture medium to loosen non-adherent cell populations can be done using standard laboratory techniques, such as, but not limited to, pipetting, rotating, washing, and shaking.

[0047] In one implementation, the removal of the culture medium from the growth vessel can be accomplished by pipetting, vacuum treatment, or pouring out the culture medium.

[0048] In one implementation, the myeloid cell population is incubated for a period of time sufficient to allow the cell population to attach to the vessel, including incubation of the cells for up to 40 hours.

[0049] In one embodiment, differentiation medium may be added to the adherent myeloid cell population. The differentiation medium may contain M-CSF. The concentration of M-CSF in the differentiation medium may be in the range of 0.1 ng / mL to 500 ng / mL. In another embodiment, M-CSF is not added to the differentiation medium.

[0050] In one implementation, cells stimulated with differentiation medium are incubated for up to 10 days.

[0051] In one implementation, the desired confluence level of cells achieved after the addition of differentiation medium can be between 50% and 100%.

[0052] In one embodiment, macrophages are detached from the cell culture dish by adding a dissociation reagent, incubating the macrophages for a first incubation period of 10 minutes at 4°C, then for a second incubation period of 10 minutes at 20°C, and then for a third incubation period of 30 minutes at 37°C.

[0053] In one embodiment, the dissociation agent or cell detachment medium is any enzymatic dissociation agent, such as, but not limited to, Accutase®, trypsin, and PBS / EDTA.

[0054] In one embodiment, the dissociation reagent is added to a cell culture vessel at a temperature between 3°C and 20°C.

[0055] In one implementation, the first incubation period is carried out at a temperature between 1°C and 8°C for a period of up to 25 minutes.

[0056] In one embodiment, the second incubation period is carried out at room temperature or at a temperature range between 18°C ​​and 25°C for up to 25 minutes.

[0057] In one implementation, the third incubation period is carried out at a temperature between 30°C and 40°C for up to 60 minutes. Example

[0058] The following are a series of embodiments of the invention described in a non-limiting manner. Therefore, the following detailed description is not limited to the disclosed embodiments and examples. Rather, the appropriate scope is defined by the appended claims.

[0059] Example 1: Macrophage differentiation protocol. 3 x 10 8 1 x 10 peripheral blood mononuclear cells (3 bottles, [1 x 10]) 8 Cells / mL were thawed in a 37°C water bath to form a cell suspension. The cell suspension was slowly added dropwise to 47 mL of pre-warmed XVIVO15® medium. With the brake off, the cells were centrifuged at 150 xg for 10 min at 22°C. The medium was aspirated, and the cell pellet was gently resuspended in 3 mL of pre-warmed TexMACS™ medium. 1 mL of the cell suspension was transferred to 29 mL of pre-warmed TexMACS™ medium. The above steps were repeated with the remaining 2 mL of cell suspension. 30 mL of the cell suspension was transferred to a 150 mm polystyrene dish without hydrophilic treatment and incubated overnight at 37°C. The medium was then gently swirl to loosen the non-adherent cells. The medium was then aspirated, while the adherent cells were retained, and the mixture was washed with 25 mL of room temperature phosphate-buffered saline. To remove any remaining non-adherent cells, the mixture was gently swirl. The process was aspirated and repeated. 30 mL of TexMACS™ medium containing 50 ng / mL M-CSF was then added. Cells were incubated at 37°C for 5 to 7 days to differentiate monocytes into macrophages. On day 3 or 4, half of the culture medium (~15 mL; retain 15 mL in the culture dish) was removed, and 15 mL of fresh, preheated TexMACS™ medium containing 50 ng / mL M-CSF was added (considering the original 15 mL of medium). No additional M-CSF was added to promote detachment for at least 2 to 3 days prior to cell detachment.

[0060] Example 2: Macrophage differentiation protocol when monocytes >50% in the PBMC population. The percentage or ratio of monocytes in the PMBC population was determined using standard flow cytometry. Flow cytometry buffer was removed from the PBMC population using standard centrifugation, and the cells were resuspended in 1 mL of TexMACS™. An ​​appropriate amount of cell suspension was transferred to 29 mL of preheated TexMACS™ medium, resulting in approximately 2 x 10⁻⁶ cells / mL. 8 cells / cm 2 Cells were seeded into 150 mm polystyrene culture dishes without hydrophilic agent treatment. The cells were then incubated at 37°C for 36 to 48 hours with 50 ng / mL M-CSF added to the medium. The medium was then gently rotated to loosen non-adherent cells, and subsequently aspirated. 30 mL of TexMACS™ medium containing 50 ng / mL M-CSF was then added. Cells were incubated at 37°C for 5 to 7 days to differentiate monocytes into macrophages. On day 3 or 4, half of the medium (~15 mL; leaving 15 mL in the culture dish) was removed, and 15 mL of fresh, preheated TexMACS™ medium containing 50 ng / mL M-CSF was added (considering the original 15 mL of medium).

[0061] Example 3: Protocol for detaching cultured macrophages and preparing for transfection. On the day of electroporation, use a first culture dish untreated with a hydrophilic agent and include rotation to loosen any dead or floating cells, followed by aspiration of the medium. Wash cells with 25 mL of room temperature phosphate-buffered saline (PBS). Gently rotate the mixture to remove loosened cells and extracellular proteins, aspirate, and repeat. Add 15 mL of ice-cold Accutase® to the culture dish and incubate at 4°C for 10 min. Perform a 10-minute continuous incubation step at room temperature and a 30-minute continuous incubation step at 37°C. Pipette 10 mL of serum to detach cells and dilute the cell suspension with at least 30 mL of preheated TexMACS™ medium in a 50 mL Falcon tube. Loosely cap the tube and incubate at 37°C until ready for washing. Repeat the process with a second and third culture dish. If you want to increase yield, repeat the process at least once per plate. With the brake off, centrifuge the cells at 150 xg for 10 minutes at 22°C. Aspirate the culture medium and resuspend the cells in electroporation buffer. Combine the cells and add electroporation buffer to 1 mL. Use an acridine orange / propidium iodide counter or automated cell counter to remove 20 μL of suspension to check cell count / viability. Transfer the cells to a 15 mL Falcon tube. Rinse the original 50 mL Falcon tube with another 9 mL of electroporation buffer (sequential rinsing), then add 9 mL of electroporation buffer to the 1 mL cell suspension in the 15 mL tube. With the brake off, centrifuge the cells at 150 xg for 10 minutes at 22°C. Aspirate the electroporation buffer and resuspend the cells in fresh electroporation buffer to an appropriate concentration, such as 2 x 10⁻⁶. 7 cells / mL to 3 x 10 7 Cells / mL. Prepare premixes containing appropriate cargo (mRNA, DNA, siRNA, sgRNA, proteins such as Cas9 nuclease, transposons / transposases, etc.) as needed. Load the treatment kit (PA) (OC-25x3) with 25 μL of each sample. Fill the empty wells with electroporation buffer. Electroporate the cells using an appropriate electroporation protocol. Transfer 20 μL of electroporated cells to a 12-well non-tissue culture plate. Incubate the cells at 37°C for at least 20 min. Then add 1 mL of preheated TexMACS containing 50 ng / mL M-CSF. TM Culture medium. Examples of inoculation density after electroporation include 2 x 10⁻⁶. 5 Up to 6 x 10 5 cells / mL or, for example, 6 x 10⁻⁶ cells / mL. 4 cells / cm 2Up to 2 x 10 5 cells / cm 2 Incubate the cells at 37°C overnight or for 24 hours.

[0062] Example 4: Post-electroporation analysis protocol. For each sample, collect the culture medium containing any dead and / or floating cells into a 15 mL tube and wash with 1 mL of room temperature PBS. Gently rotate the mixture and aspirate with a pipette to remove loose cells, and combine with the corresponding culture medium in the 15 mL tube. Add at least 2 mL of pre-warmed TexMACS to each tube to aid in Accutase® dilution and washing. Gently cap the tubes and place them in a 37°C incubator until the remaining cells are detached. Add 1 mL of ice-cold Accutase® and incubate at 4°C for 10 min, followed by incubation at room temperature for 10 min and then at 37°C for 30 min. Aspirate the cells with a pipette to detach them and collect them as individual samples into their respective 15 mL tubes. Wash the cells by centrifuging them at 150 xg for 10 min with the brake off. Then aspirate the culture medium (leaving ~100 µL-500 μL to ensure no cell loss). Resuspend the cells and transfer them to 1.5 mL Eppendorf tubes. Add 1 mL of fluorescence-activated cell sorting (FACS) staining buffer along with the cells, then centrifuge at 300–500 xg for 5 minutes with the brake off. Decant the supernatant and resuspend the cells in the remaining staining buffer. Add an appropriate monoclonal antibody (mAb) for flow cytometry and stain for 20 minutes at room temperature. Add 1 mL of staining buffer and centrifuge at 300–500 xg for 5 minutes with the brake off. Gently decant the supernatant and resuspend the cells in the remaining buffer. Transfer the cells to FACS tubes and run flow cytometry to assess cell viability (7-AAD) and transfection efficiency based on green fluorescent protein light emission.

[0063] Example 5: Cultured macrophages transfected with mRNA, DNA, or RNP. As described in Examples 1 and 3, cultured macrophages dissociated from culture dishes were loaded using electroporation. For cells loaded with mRNA or DNA, prior to electroporation, the cells were washed and resuspended in electroporation buffer and combined with different sizes of mRNA-GFP or DNA plasmids (pSmall-GFP, 2 kb; pMedium-GFP, 4.2 kb; and pLarge-GFP, 9.2 kb). Cargo was added at low and high concentrations (100 µg / mL and 200 µg / mL, respectively). Cells were transfected using two electroporation protocols, each specific for DNA or mRNA. Twenty-four hours after electroporation, cell viability and transfection efficiency were assessed using flow cytometry as described in Example 4.

[0064] For macrophages loaded with ribonucleoprotein complexes (RNPs) to knock out the expression of specific genes, macrophages were cultured and detached from culture dishes as described in Examples 1 and 3 of this document with slight modifications. In this example, monocyte differentiation into macrophages took 3 days. While washing the cells, RNPs were prepared by conjugating recombinant Cas9 protein with guide RNA (sgRNA) targeting B2M or SIRPα. After RNP preparation, cells were resuspended in electroporation buffer and combined with B2M RNPs, SIRPα RNPs, or a combination of B2M and SIRPα RNPs, and then electroporated. Four days after electroporation, cell viability and gene knockout efficiency were assessed by flow cytometry as described in Example 4.

[0065] Cargo loaded with mRNA DNA Figure 3A ) and RNP ( Figure 3C The viability of macrophages after electroporation demonstrates that the culture and detachment methods described herein result in high cell viability after electroporation (high-energy and low-energy electroporation protocols, independent of cargo). This was used for loading mRNA / DNA ( Figure 3B ) and RNP ( Figure 3D Post-electroporation evaluation of transfection efficiency showed that macrophages cultured using the method described herein could be effectively transfected with cargo. Furthermore, Figure 3E This demonstrates that there is no significant difference in knockout efficiency when knocking out a single gene or two genes in macrophages cultured and detached as described in this paper.

[0066] Example 6: Comparison of macrophages electroporated using conventional culture methods with macrophages electroporated using the disclosed method. Cells in the control group were thawed at 1 x 10⁻⁶ cells / day. 8PBMCs were cultured. The PBMCs were resuspended in 10 mL of cell culture medium (RPMI 1640, 10% FBS, 2 mM GlutaMAX). TM Centrifuge the cells at 180 g for 7 minutes. Discard the supernatant, resuspend the remaining cell pellet in 30 mL of culture medium, and seed on T175 cm⁻¹. 2 In a flask, incubate for 2 hours. Replace the medium with cell culture medium supplemented with 10 ng / mL M-CSF to remove non-adherent cells and initiate differentiation. Incubate cells overnight and allow differentiation. After incubation, add 20 mL of medium supplemented with 50 ng / µL M-CSF. Return cells to the incubator and incubate for another 2 days. On day 5, remove 34 mL of medium, add 12 mL of fresh cell culture medium supplemented with 83 ng / mL M-CSF, and incubate cells for another 3 days. On day 7, dissociate cells by continuous washing with PBS / EDTA. The harvested cells are then divided into two groups. Group 1 was not electroporated, and Group 2 was electroporated but without additional electroporation with pDNA-GFP. Macrophages in the experimental groups were prepared using the protocols described in Examples 1, 2, and 4 and divided into the same two groups as the control. Figure 4 The results from the experiments presented showed that, in both groups, the disclosed method resulted in a higher population of viable macrophages. Importantly, Figure 4 The results showed that a higher percentage of macrophages cultured and differentiated using the disclosed method expressed higher levels of GFP compared to the same cells cultured and differentiated using conventional methods. Furthermore, significant macrophage reattachment was observed when cells were prepared using the claimed method.

Claims

1. A method for culturing myeloid cells, the method comprising: Myeloid cell populations were plated together with culture medium using growth dishes. The myeloid cell population is incubated at a time and temperature sufficient to allow the cell population to adhere to or attach to the vessel. Stir the culture medium to loosen the non-adherent cell population. The culture medium is removed from the growth vessel while retaining the adherent cells. Add differentiation medium containing macrophage colony-stimulating factor (M-CSF) to the vessel. The adherent cells were cultured at a time and temperature sufficient to allow them to differentiate into macrophages and to increase the cell population to the desired confluence level. After the incubation period, a certain amount of the culture medium containing M-CSF was removed, and Add culture medium containing M-CSF to the vessel.

2. The method of claim 1, wherein the density of the myeloid cell population after plating is 2 x 10⁻⁶. 4 cells / cm 2 Up to 8 x 10 5 cells / cm 2 between.

3. The method of claim 1, wherein the growth vessel is made of plastic, polycarbonate, thermoplastic, polyolefin, polystyrene or other similar materials.

4. The method of claim 1, wherein the growth vessel has not undergone tissue culture treatment.

5. The method of claim 1, wherein the culture medium is a cell culture medium.

6. The method of claim 5, wherein the culture medium further comprises serum.

7. The method of claim 1, wherein the concentration of M-CSF in the differentiation medium is in the range of about 10 ng / mL to 200 ng / mL.

8. The method of claim 1, wherein the incubation temperature is in the range of about 32°C to 37°C.

9. The method of claim 1, wherein the incubation is carried out at a CO2 concentration of 3% to 7%.

10. The method of claim 1, wherein the incubation time sufficient to allow the cell population to adhere to the vessel is about 18 to 36 hours.

11. The method of claim 1, wherein the time for culturing the adherent cells to a level sufficient to differentiate the adherent cells into macrophages and increase the cell population to a desired confluence level is about 4 to 7 days.

12. The method of claim 11, wherein the desired confluence level is between 75% and 100%.

13. The method of claim 1, wherein the amount of M-CSF-containing culture medium removed and added is equal to half the total volume of the M-CSF-containing culture medium in the growth vessel.

14. A method for detaching macrophages from a cell culture dish, the method comprising: Add dissociation reagent to macrophages cultured in culture medium and growth dishes. The macrophages were incubated at 2°C to 6°C for up to 20 minutes. The macrophages were then incubated at 19°C to 23°C for up to 20 minutes. The macrophages were then incubated at 32°C to 38°C for up to 60 minutes, and The detached macrophages were transferred to a vessel for further processing.

15. The method of claim 14, wherein the growth vessel is made of plastic, polycarbonate, thermoplastic, polyolefin, polystyrene and other similar materials.

16. The method of claim 14, wherein the growth vessel has not undergone tissue culture treatment.

17. The method of claim 14, wherein the culture medium is any culture medium suitable for cell culture.

18. The method of claim 14, wherein when applied to the cell culture, the temperature of the dissociation reagent is about 4°C.

19. The method of claim 14, wherein when applied to the cell culture, the temperature range of the dissociation reagent is about 4°C to 20°C.

20. The method of claim 14, wherein the dissociation reagent is an enzyme cell desorption medium; the method of claim 14, wherein the dissociation reagent is a non-enzymatic dissociation reagent.

21. A method for culturing myeloid cell populations, wherein the monocyte population is less than 55%, wherein: Myeloid cell populations were plated together with culture medium using growth dishes. The myeloid cell population was incubated with a culture medium containing 50 ng / mL M-CSF for up to 50 hours at a temperature sufficient to allow the cell population to adhere to or attach to the vessel. Stir the culture medium to loosen the non-adherent cell population. Remove the culture medium from the growth vessel while retaining the cell population that is adhering to or attached to the vessel. The adherent cells were cultured for up to 7 days in medium supplemented with 50 ng / mL M-CSF at a temperature sufficient to allow the adherent cells to differentiate into macrophages and to increase the cell population to the desired confluence level. After the incubation period, a certain amount of the culture medium containing M-CSF was removed, and Add culture medium containing M-CSF to the vessel.

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