Methods for isolating, culturing, differentiating, and detaching myeloid cells from a vessel

Abstract

A method of isolating culturing, differentiating, and detaching myeloid cells using vessels comprised of plastic, thermoplastic, polycarbonate, or other similar materials not treated with hydrophilic agents and detached using dissociation reagents administered in a temperature gradient.

Description

METHODS FOR ISOLATING, CULTURING, DIFFERENTIATING, AND DETACHING MYELOID CELLS FROM A VESSELCross-Reference to Related Applications

[0001] This application claims priority under 35 U.S.C. § 119(e) 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. The contents of each of these applications are incorporated herein by reference in their entirety.Technical Field

[0002] The present disclosure relates to a method of isolating, culturing, and differentiating myeloid cells into, for example, macrophages, such that they can be readily detached and used for other downstream purposes while minimizing cell toxicity and death. In particular, embodiments of the present disclosure relate to using vessels not treated with hydrophilic agents or ultra-low cell attachment agents to isolate, culture, and differentiate cells into other cell types, such as macrophages, and effectively detach these cells by using dissociation reagents in a temperature gradient, discarding the need for scrappers and other more harmful detachment methods.Background

[0003] Peripheral blood mononuclear cells (“PBMCs”) are a mixed population of myeloid and lymphoid cells. Myeloid cells are a type of blood cell that originate in the bone marrow. These heterogenous populations of cells are critical for the proper functioning of an organism’s immune system and other systems as they can differentiate into neutrophils, eosinophils, basophils, macrophages, and other types of cells. As myeloid cells are important for the development and maintenance of the immune system and other systems, they are often used for research and to study and elucidate mechanisms of disease and to test new treatment alternatives.

[0004] Macrophages are myeloid-derived cells that have been found in every type of tissue and exhibit a wide array of functions, including metabolism, bone remodeling, hematopoiesis, immune surveillance, and more. Macrophage functions are varied and not static throughout their life. For example, macrophages can become activated by antigens and antigen-activated immune cells, which result in a cascade of signals necessary for initiation, maintenance, and resolution of inflammation. Because of the multiple roles andstages of macrophage activity, there are numerous biomarkers used to identify them from other cell populations. Generally, macrophages can be classified based on their pro- inflammatory (Ml) or anti-inflammatory (M2) properties which arise after their progenitor cells, the steady-state macrophage (MO), are exposed 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 expression of CD64, CD68, MerTK, CD 115, among others. Ml macrophages can be identified based on their increased expression of CD80, CD86, HLA-DR, CD40, among others. M2 macrophages can be identified based on their increased expression of CD206, CD 163, CD200R, CD209, among others. Some references that further describe the expression pattern of various types of macrophages are Murray et al. 2014, Moser 2003, and Mosser & Edwards 2008.

[0005] Though myeloid cells, macrophages, and other myeloid-derived cells are studied and used extensively, there are obstacles when culturing these cells that limit the downstream applications and experiments that can be performed. For example, the powerful attachment of macrophages to culture vessels can necessitate the use of stringent detachment reagents and protocols, like ethylenediaminetetraacetic acid (EDTA) and scrapers, which can be toxic or lethal to cells. These effects can result in loss of cell viability, reduction of yield for downstream experiments and assays, artifacts that interfere with result analysis, and other undesirable effects.

[0006] PBMCs and other myeloid derived cells can originate from tissue donors of human or other organisms, which can lead to donor-to-donor differences in the population of cells. For example, PBMC donors may have a lymphocyte population in the range of 70 to 90%, while the monocyte population can range from 10 to 20%. These donor-to-donor differences in population ratios can affect optimal culture conditions and downstream processes and applications. For example, optimal culturing of PBMCs and other myeloid derived cells may include considerations of monocyte seeding densities. Correct monocyte seeding density allows for these cells to grow and differentiate into macrophages with a healthy morphology, allowing for better yields following electroporation and other assays. Similarly, correct seeding density allows better attachment of the cells to the plates, minimizing cell loss during washes and media change.

[0007] Culturing and growing cells, like myeloid cells, macrophages, and other myeloid derived cells, can occur in tissue culture treated vessels. To be converted to tissue culture treated vessels, the vessels are exposed to agents like gas plasma treatment where the surface of the vessel (where the cells would attach and grow) is made more hydrophilic and negatively charged, making the vessel more suitable for cells attaching to the surface. Other vessels that can be used for culturing cells, like myeloid cells, include ultra-low attachment plates, which are plates that can contain a layer of hydrogel, or other materials covalently bound to the surface of the vessel where cells would attach, inhibiting cell attachment to the plate. For numerous cell types, like macrophages, however, growing cells in tissue culture treated vessels or ultra-low attachment vessels may not be ideal. For example, isolating, culturing, and growing macrophages in tissue culture treated vessels can result in reduced number of cells attaching to the vessels. In addition, macrophages grown in tissue culture treated vessels can adopt an unhealthy and elongated morphology and a more immature phenotype, making them inappropriate for numerous downstream applications like electroporation, cell therapies, and more. On the other hand, macrophages grown on ultra-low attachment plates result in a less densely packed phenotype, along with a higher proportion of elongated stressed cells that may indicate immune activation, which may not be desirable for numerous assays and other downstream applications.

[0008] Isolating, culturing, differentiating, and detaching myeloid-derived cells sub- optimally by using tissue culture treated vessels or ultra-low attachment plates also results in an undesirably robust attachment of these cells to the plates. Because of the stronger attachment in tissue culture treated vessels or ultra-low attachment vessels, when detachment of the vessel is required, the operator may have to use stringent detachment methods, like ethylenediaminetetraacetic acid (EDTA) combined with mechanical detachment with tools like cell scrapers that can harm the cells, affecting downstream applications.

[0009] The combination of using an inadequate vessel, like the ones described above, improper seeding densities, and stringent detachment methods results in ineffective use of resources as the cells become less healthy, are more likely to die, possibly grow slower, and could become activated. These effects may be undesirable because they can lead to lower yield for downstream applications or make the cells inadequate for numerous downstream applications, like electroporation and cell therapies. In addition, the slowergrowth and reduced yield leads to the use of additional resources, like cell culture media, growth factors, as well as operator time and effort. Together, these inefficiencies can lead to unnecessary costs.

[0010] Therefore, there is a need for improved methods for isolating, culturing, growing, differentiating, and detaching myeloid cells. In some embodiments, the improved methods use a growth vessel that is, for example, not tissue culture treated, and made of materials like glass, plastic, thermoplastic, polyolefins, polycarbonate, polystyrene, and others, that allow the use of less stringent dissociation reagents, such as Accutase® and Trypsin (e.g., Gibco™ Trypsin-EDTA (0.25%), phenol red), used in a temperature gradient, so detachment results in reduced toxicity and cell death.Summary

[0011] According to one non-limiting aspect of the present disclosure, there is provided a method of culturing myeloid cells, the method comprising the use of a vessel not treated with hydrophilic agents, like plasma gas. The vessel may be made of glass, plastic, thermoplastic, polyolefins, polycarbonate, polystyrene or other materials. The vessel can be a petri dish with 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 vessels can be cell culture bags with a culture area of less than, more than, or equal to 640 cm2. The vessel can be a matrix for culturing cells in a 3D environment, like, for example, a gel matrix, and it can be of a volume of 1 mL, 5 mL, 20 mL, 50 mL, 100 mL, and more. The vessels can be 6-, 12-, 24- , 48 or 96- well plates. The vessel can be a flask of a volume including 25ml (12.5cm2), 50ml (25cm2), 250ml (75cm2) and 600ml (182cm2), or others. The method of culturing also comprises the use of cell culture media that can be any cell culture media fit for cell culture. The method of culturing also comprises the use of cell culture media with or without supplementation of serum, like fetal bovine serum (FBS) or human serum albumin (HSA), at an amount of about 2 to 15%. The method of culturing also includes culturing the cells under standard cell culture conditions. For example, cells can be cultured at a temperature of 32 °C to 37 °C, with a CO2 concentration of 4% to 7%, and a humidity of about 80% to 95%. The cell culture conditions can vary beyond the limitations described depending on the cell type, the downstream applications, and according to what a person of ordinary skill in the art would determine applicable under the circumstances. The methods of culturing the myeloid cells include seeding the cells at a concentration of 2 x 104cells / cm2up to for example 8 x 105cells / cm2. The methods ofculturing cells described herein include culturing the cells to promote their attachment by incubating them for a time period including, for example, 18 to 36 hours. The methods of culturing cells described herein include culturing the cells up to a desired confluency ranging from, for example, 75% to 100%. The methods of culturing cells described herein include culturing the cells to differentiate them into other cell types by using, for example, macrophage colony stimulating factor (M-CSF) at a concentrating 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 of culturing cells described herein include culturing the cells with M-CSF to promote differentiation for a time period including, for example, 4 to 7 days under similar temperature, humidity, and CO2 conditions described above.

[0012] Another non-limiting aspect of the present disclosure includes methods of detaching myeloid derived cells, such as macrophages, from a vessel. In some embodiments, the vessel may be made of glass, plastic, thermoplastic, polyolefins, polycarbonate, polystyrene, or other materials. In some embodiments, the vessel described in these methods do not have to be treated with hydrophilic agents, like plasma gas. The vessel can be a petri dish with 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 vessels can be cell culture bags with a culture area of less than, more than, or equal to 640 cm2. The vessel can be a matrix for culturing cells in a 3D environment, like, for example, a gel matrix, and it can be of a volume of 1 mL, 5 mL, 20 mL, 50 mL, 100 mL, and more. The vessels can be 6-, 12-, 24-, 48 or 96- well plates. The vessel described in these methods can be a flask of a volume including 25ml (12.5cm2), 50ml (25cm2), 250ml (75cm2) and 600ml (182cm2) or others. In some embodiments, the disclosed methods include, for example, the use of a dissociation reagent that can be enzymatic or non-enzymatic to promote cell detachment of the vessels. In some embodiments, the methods described herein include the use of a dissociation reagent, detachment reagent, or enzyme cell detachment medium or solution, like Accutase® or Trypsin. In some embodiments, the methods described herein include the use of a temperature gradient when using the dissociation reagent to promote cell detachment with a high cell viability. The methods describe herein include the use of a temperature gradient that starts with a lower temperature and gradually increases to a higher temperature. For example, the temperature gradient described herein includeadministering the dissociation reagent where the dissociation reagent is at a temperature of 4 °C to 20 °C. For example, the dissociation reagent is at a temperature of about 4 °C. The dissociation reagent used at a temperature gradient includes culturing the cells with the dissociation reagent for a period of up to twenty minutes at an initial temperature of 2 °C to 6 °C. The dissociation reagent used at a temperature gradient includes culturing the cells with the dissociation reagent at a second temperature ranging from 19 °C to 23 °C for up to 20 minutes. The dissociation reagent used at a temperature gradient includes culturing the 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 culturing periods described herein can occur in CO2, humidity, and other condition parameters described above. In some embodiments, the methods described herein include transferring the cells to an appropriate vessel for downstream applications like electroporation, cell therapy, or others.

[0013] As used herein, “processing assembly” (PA) is meant to describe the unit or units comprising the mechanisms to load and electroporate cells.

[0014] As used herein, “room temperature” is meant to describe temperatures ranging between 20 °C to 22 °C.

[0015] As used herein, “macrophages” are meant to describe cells including those with the one or more of the following characteristics: large, adherent, phagocytic, differentiated from monocytes. In addition, the macrophages as used herein include those expressing pan-macrophage markers, including expression of CD64, CD68, MerTK, CD115, among others. As used herein macrophages include Ml macrophages, which can be identified based on their increased expression of, for example, CD80, CD86, HLA- DR, CD40, among others. As used herein macrophages include M2 macrophages, which can be identified based on their increased expression of, for example, CD206, CD 163, CD200R, CD209, among others.

[0016] As used herein, “myeloid cells” are meant to describe cells containing, for example, the following characteristics: expression of CDl lb, CD206, CD33, CD52, among other markers.

[0017] As used herein, “vessel,” “growth vessel,” and “culture vessel” are used interchangeably and include flasks, well plates, matrices, culture bags, petri dishes (suchas petri dishes meant for culturing bacteria), and other similar vessels used to isolate, grow, or culture cells.

[0018] As used herein, “non-tissue culture treated vessels” or “not tissue culture treated vessels” are those not treated to enhance hydrophilic characteristics with hydrophilic agents, such as with plasma gas, that ordinarily helps adherent cells attach to the vessel.

[0019] As used herein, “ultra-low attachment vessels” are those that contain a layer of a compounds, like hydrogel, on the surface where cells would attach to prevent their attachment.

[0020] As used herein, “macrophage colony stimulating factor” (“M-CSF”) is used to describe the hematopoietic growth factors and associated cofactors used to proliferate, differentiate, and maintain monocytes, macrophages, and other cell types.

[0021] As used herein, “differentiation” refers to the process or result of a cell or population that includes cell division and a modification of functional or phenotypical characteristics that can lead to another cell type. For example, a myeloid cell or cell population differentiating into a macrophage cell or cell population.

[0022] As used herein “cell dissociation reagent” is used to describe solutions that result in the detachment of cells from a vessel under chemical, enzymatic, or other mechanisms. Examples of enzymatic dissociation reagents or enzyme cell detachment medium include TrypLE1M, Trypsin, and Accutase®. Examples of chemical or non- enzymatic dissociation reagents include Non-Enzymatic Cell Dissociation Solution SO- 2103™ and ethylenediaminetetraacetic acid (EDTA) alone or in a solution or buffer like PBS (e.g., PBS / EDTA).Brief Description of the Figure

[0023] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate some disclosed embodiments and, together with the description, serve to explain the disclosed embodiments. The particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the present disclosure. The description taken with the drawings makes apparent to those skilled in the art how embodiments of the present disclosure may be practiced.

[0024] FIG. 1 is a collection of 4 pictures of macrophages growing on various vessels. The pictures include an embodiment of the present disclosure 103 and 104, wherein the macrophages are grown on vessels comprised of plastic that is not treated with hydrophilic agents.

[0025] FIG. 2 is a picture of an embodiment of the present invention showing the ideal phenotype of macrophages growing on a petri dish comprised of polystyrene that is not tissue culture treated.

[0026] FIG. 3A-3E show cell viability for macrophages cultured using the described method and electroporated with mRNA / DNA (FIG. 3A), RNPs (FIG. 3C), transfection efficiency for mRNA / DNA (FIG. 3B) and RNPs (FIG. 3D), and knock-out efficiency (FIG. 3E).

[0027] FIG. 4 shows a comparison of transfection efficiency, demonstrated by microscopy, in macrophages prepared using the methods described herein relative to traditional macrophage culturing methodologies.Detailed Description

[0028] The following is a detailed description with several non-limiting embodiments and accompanying figures, and substitutions, additions, or modifications may be made to the components and steps provided.

[0029] Embodiments of the present disclosure are directed to methods of isolating, culturing, differentiating myeloid cells to optimize their health, differentiation, using, for example, a 25ml (12.5cm2) flask comprised of polystyrene and not treated with hydrophilic agents like plasma gas. Furthermore, embodiments of the present disclosure include plating and culturing the cells, at a seeding density of 2 x 104cells / cm2, in the vessel described for sufficient time to allow the cells to attach to the vessel described, which include culturing the cells for 18 hours at 37 °C, with 5% CO2 and a 95% humidity. Moreover, embodiments of the present invention include washing the culture with phosphate-buffered saline (PBS) and adding cell culture media supplemented with 100 ng / mL of M-CSF to promote differentiation of the myeloid cells into macrophages. Furthermore, embodiments of this disclosure include culturing the attached cells with media supplemented with M-CSF for 5 days at 37 °C, with 5% CO2 and a 95% humidity for the cells to differentiate into macrophages. In addition, the embodiments described inthis disclosure include removing 12.5 mL of media supplemented with M-CSF from the flask and adding 12.5 mL of media supplemented with M-CSF (100 ng / mL) to the flask and culture for 2 additional days under the temperature, CO2 and humidity conditions described above.

[0030] Embodiments of the present disclosure include determining the ratio or percentage of monocytes, lymphocytes, or other cell types in a donor prior to seeding and culturing the cells using methods like flow cytometry. If the population of monocytes is found to be below the optimal 50% to 55%, then the seeding density of the cells seeded should be increased. For example, if the monocyte percentage is about 30%, instead of seeding at an approximate density of 2 x 104cells / cm2of PBMCs, the seeding density should be increased to about 2 x 108cells / cm2PBMCs.

[0031] Embodiments of the present invention also include reducing the cell loss or increasing the yield of cultured and differentiated cells by modifying cell culture protocols. For example, if the population of monocytes is found to be below the optimal 50% to 55% in a PBMC donor, then cell culture protocols can be modified. For example, if the monocyte percentage is about 30%, then the PBMCs can be cultured for 36 to 48 hours with M-CSF at a concentration of, for example, 100 ng / mL when seeded, instead of culturing the cells in the media described above without M-CSF for 18 to 36 hours for the cells to attach. In addition, other modifications to the cell culture conditions when the population of monocytes is below optimal, such as about 30% monocyte in the PBMC donor, include not washing the cells with PBS prior to adding fresh media or other fluids.

[0032] FIG. 1 is a collection of macrophage culture pictures grown on different vessels, including embodiments of the present invention, wherein the macrophages are grown in sterile polystyrene petri dishes not treated with hydrophilic agents (not tissue culture treated) like plasma gas 104. As illustrated in FIG. 1, macrophages grown in tissue culture treated vessels 101 demonstrate reduced confluency along with a stressed and unhealthy morphology, characterized by more cells in a spindle-like shapes and a reduced number of cells displaying a fried egg morphology. Similarly, FIG. 1 illustrates macrophages grown on ultra-low attachment plates 102 wherein the macrophages grow to a lower level of confluency and exhibit an unusual spider web morphology and macrophage markers based on flow cytometry data (not shown in FIG. 1). FIG. 1 also illustrates macrophages grown on non-tissue culture treated vessels 103, wherein the cellsexhibit a healthy morphology that includes more fried egg phenotype (characterized by a flattened and relatively rounded cytoplasmic shape with a smaller and relatively round nucleus; this morphology is also described as having prominent perinuclear halo and a centrally located and round nuclei) and some elongated or spindle-shaped macrophages as well (characterized by a wider middle with two ends that become less wide as they distance themselves from the wider middle; other words to describe this morphology include conical, fusiform, oblong, cylindroid). Lastly, FIG. 1 also illustrates cell grown on petri dishes comprised of polystyrene that are not tissue culture treated 104. Macrophages grown in these latter conditions 104 display a higher level of confluency and a healthy mixed phenotype (further shown in FIG. 2) ideal for easier detachment and subsequent assays and processing.

[0033] FIG. 2 is a picture of an embodiment of the present invention, wherein the macrophages are cultured on a petri dish comprised of polystyrene that is not tissue culture treated. The picture shows the cells growing to a healthier, densely packed, and desired level of confluency, ideal for subsequent assays and processing. The morphology of the population includes a mix of spindle-like morphology 201 (characterized by a wider middle with two opposing ends that become less wide as the ends are approached; other words to describe this morphology include conical, fusiform, oblong, cylindroid) and fried egg morphology 202 (characterized by a flattened and relatively rounded cytoplasmic shape with a smaller and relatively round nucleus; this morphology is also described as having prominent perinuclear halo and a centrally located and round nuclei). Such morphology variations may be indicative that the macrophages are not activated and may be better suited for subsequent applications.

[0034] FIG. 3 show the results of the experiment described in example 5 that tested cell viability of macrophages that were cultured pursuant to the disclosed method and then electroporated. Post-electroporation assessments included: macrophage viability after loading with mRNA / DNA (FIG. 3A), RNPs (FIG. 3C); transfection efficiency for mRNA / DNA (FIG. 3B) and RNPs (FIG. 3D); and knock-out efficiency for macrophages transfected with RNPs (FIG. 3E). Cell viability and gene knockout efficiency were assessed using a flow cytometer 4 days post electroporation.

[0035] FIG. 4 shows a comparison of the transfection efficiency of macrophage transfection using electroporation following traditional methods of culturingmacrophages, relative to the methods described herein. FIG. 4 further shows that cells cultured using the disclosed method (“experimental group”) exhibited higher viability and improved morphology even after electroporation compared to cells cultured using traditional methods (“control group”). FIG. 4 further demonstrates improved electroporation and transfection efficiency for macrophages cultured using the disclosed method.

[0036] Embodiments of the present disclosure are also directed towards methods of detaching macrophages grown on a vessel, like a 25ml (12.5cm2) flask, comprised of polystyrene and not treated with hydrophilic agents like plasma gas by using dissociation reagents on a temperature gradient. An embodiment of the methods disclosed here include using an enzymatic dissociation reagent or enzyme cell detachment medium, such as Accutase®, and administering it to the cell culture at a temperature of 4 °C and administering a sufficient volume to cover the surface of the flask where the cells grow, like, for example, 7 mL of Accutase® on the 25ml (12.5cm2) flask. Furthermore, embodiments of the present disclosure also include incubating the 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. Furthermore, the embodiments described herein include pipetting the flask with a 10 mL serological pipette and transferring them to an appropriate vessel or vessels, like a 10 mL falcon tube, for further processing or subsequent applications, like electroporation or transfection.

[0037] It is understood that the claimed method is scalable and is not limited to any particular cell density range. It is further understood that the cells are incubated at standard incubation parameters (37 °C with 5% CO2).

[0038] In one embodiment, the temperature of the incubator is selected from, but not limited to, a temperature range of 30 °C -40 °C and a CO2 range of 1 %— 10%.

[0039] In one embodiment, the initial concentration of myeloid cells plated can be between 1 x 104to 1 x 106cell / cm2.

[0040] In one embodiment, the growth vessel used is a non-treated culture vessel or a sterile bacterial petri vessel.

[0041] In one embodiment, the growth vessel can be comprised of the material including but not limited to, plastic, polycarbonate, thermoplastic, polyolefins, polystyrene, or other similar materials.

[0042] In one embodiment, any standard cell media can be used with the claimed method such as, but not limited to, Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 Medium, Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 (DMEM F12), TexMACS™, Minimum Essential Medium (MEM), Iscove’s Modified Dulbecco’s Medium (IMDM).

[0043] In one embodiment, agitating the media to loosen a non-adherent cell population can be accomplished using standard lab techniques such as, but not limited to, pipetting, swirling, washing, and rocking.

[0044] In one embodiment, removing the media from the growth vessel can be accomplished by pipetting, vacuuming, or pouring out the media.

[0045] In one embodiment, incubating the myeloid cell population for a time sufficient to allow a population of cells to adhere to the vessel encompasses incubating the cells for up to 40 hours.

[0046] In one embodiment, differentiation media can be added to a population of adherent myeloid cells. The differentiation media can contain M-CSF. The concentration of M-CSF in the differentiation media can be between the range of 0. 1 ng / mL to 500 ng / mL. In another embodiment, no M-CSF is added to the differentiation media.

[0047] In one embodiment, cells stimulated with differentiation media are incubated for up to 10 days.

[0048] In one embodiment, the desired level of confluency of cells reached after the addition of differentiation media can be between 50%-100%.

[0049] In one embodiment, the macrophages are detached from the cell culture vessel by adding a dissociation reagent, a first incubation period of incubating the macrophages at 4 °C for 10 minutes, then a second incubation period of 20 °C for 10 minutes, then a third incubation period of 37 °C for 30 minutes.

[0050] In one embodiment, the dissociation reagent or cell detachment medium is any enzymatic dissociation reagent such as, but not limited to, Accutase®, Trypsin, and PBS / EDTA.

[0051] In one embodiment, the dissociation reagent is between 3 °C-20 °C when added to the cell culture vessel.

[0052] In one embodiment, the first incubation period is carried out at a temperature between 1 °C and 8 °C for a time period up to 25 minutes.

[0053] In one embodiment, the second incubation period is carried out at room temperature, or at a range of temperatures between 18 °C to 25 °C for up to 25 minutes.

[0054] In one embodiment, the third incubation period is carried out at a temperature between 30 °C to 40 °C for up to 60 minutes.Examples

[0055] What follows are a series of examples that illustrate the present invention in a non-limiting manner. Accordingly, the following detailed description is not limited to the disclosed embodiments and examples. Instead, the proper scope is defined by the appended claims.

[0056] Example 1: Macrophage Differentiation Protocol. 3 x 108peripheral blood mononuclear cells (3 vials at [1 x 108cells / mL]) were thawed in 37 °C water bath to form a cell suspension. The cell suspension was slowly added to 47 mL of pre-warmed XVIVO15® media dropwise. The cells were centrifuged at 150 x g for 10 mins at 22 °C with the brake off. The media was aspirated off, which was followed by gently resuspending cell pellet in 3 mL of pre-warmed TexMACS™ media. 1 mL of the cell suspension was transferred into 29 mL of pre-warmed TexMACS™ media. The foregoing was repeated with the remaining 2 mL of cell suspension. 30 mL of cell suspension was transferred into a 150 mm polystyrene petri dish not treated with hydrophilic agents and incubated overnight at 37 °C. Next, the media was gently swirled to loosen non-adherent cells. The media was then aspirated while retaining the adherent cells, which were then washed with 25 mL of room temperature phosphate-buffered saline. To remove remaining non-adherent cells, the mix was gently swirled. This process was aspirated and repeated. 30 mL of TexMACS™ media containing 50 ng / mL of M- CSF was then added. The cells were incubated at 37 °C and cultured for 5 to 7 days todifferentiate monocytes into macrophages. On day 3 or 4, half of the media (—15 mL; leave 15 mL in dish) was removed, and 15 mL of fresh, pre- warmed TexMACS™ media containing 50 ng / mL of M-CSF (account for the 15 mL of old media) was added. Additional M-CSF was not added at least 2 to 3 days prior to detaching cells to facilitate detachment.

[0057] Example 2: Macrophage Differentiation Protocol when >50% Monocyte in PBMC Population. The percentage or ratio of monocytes in a PMBC population was determined using standard flow cytometry protocols. The flow cytometry buffer was removed from PBMC population by using standard centrifugation protocols and the cells were resuspended in 1 mL of TexMACS™. An appropriate amount of the cell suspension was transferred into 29 mL of pre-warmed TexMACS™ media such that about 2 x 108cells / cm2cells were seeded into a 150 mm polystyrene petri dish not treated with hydrophilic agents. The cells were next incubated for 36 hours to 48 hours at 37 °C with 50 ng / mL of M-CSF supplemented into the media. Next, the media was gently swirled to loosen non-adherent cells, which was followed by aspirating the media. 30 mL of TexMACS™ media containing 50 ng / mL of M-CSF was then added. The cells were incubated at 37 °C and cultured for 5 to 7 days to differentiate monocytes into macrophages. On day 3 or 4, half of the media (-15 mL; leave 15 mL in dish) was removed, and 15 mL of fresh, pre-warmed TexMACS™ media containing 50 ng / mL of M-CSF (account for the 15 mL of old media) was added.

[0058] Example 3: Protocol for Detaching Cultured Macrophage and Preparation for Transfection. On the day of electroporation, a first petri dish not treated with hydrophilic agents was used, and included swirling to loosen any dead or floating cells, followed by aspirating the media. The cells were washed with 25 mL of room temperature phosphate-buffered saline (PBS). The mixture was gently swirled to remove loose cells and extracellular proteins, which was aspirated and repeated. 15 mL of ice cold Accutase® was added to the petri dish and incubated at 4 °C for 10 mins. Successive incubation steps were performed at room temperature for 10 mins and at 37 °C for 30 mins. 10 mL serological was pipetted to detach cells and the cell suspension was diluted in at least 30 mL of pre-warmed TexMACS™ media in a 50 mL Falcon tube. The tube was loosely capped and place in a 37 °C incubator until ready to wash. The process was repeated with second and third petri dishes. The process was repeated at least once for each plate if want to increase yield. The cells were centrifuged at 150 x g for 10 mins at22 °C with the brake off. The media was aspirated, and the cells were resuspended in electroporation buffer. Cells were pooled together, and electroporation buffer was added to 1 mL. 20 pL of suspension was removed to check cell count / viability using acridine orange / propidium iodide on a hemocytometer or automatic cell counter. The cells were transferred to a 15 mL falcon tube. The original 50 mL falcon tubes were rinsed with an additional 9 mL of electroporation buffer (sequential rinses) and then 9 mL of electroporation buffer was added to the 1 mL of cell suspension in the 15 mL tube. The cells were centrifuges at 150 x g for 10 mins at 22 °C with the brake off. The electroporation buffer was aspirated, and the cells were resuspended in fresh electroporation buffer to the appropriate concentration, which were in the range of, for example, 2 x 107cells / mL to 3 x 107cells / mL. Master mixes were prepared containing appropriate cargo (mRNA, DNA, siRNA, sgRNA, proteins such as Cas9 nuclease, transposons / transposases, etc.) as needed. Processing assemblies were loaded (PA) (OC- 25x3) with 25 pL of each sample. The empty wells were filled with electroporation buffer. Cells were electroplated with appropriate electroporation protocols. 20 pL of the electroporated cells were transferred to a 12-well non-tissue culture treated plate. The cells rested at 37 °C for at least 20 mins. 1 mL of pre-warmed TexMACS™ media containing 50 ng / mL of M-CSF was then added. Examples of post-electroporation seeding densities include 2 x 10' to 6 x 105cells / mL or for example 6 x 104cells / cm2to 2 x 105cells / cm2. The cells were incubated overnight or 24 hours at 37 °C.|00059| Example 4: Post Electroporation Analysis Protocol. For each sample, the media containing any dead and / or floating cells was collected into a 15 mL tube and rinsed with 1 mL of room temperature PBS. The mixture was gently swirled and pipetted to remove loose cells and combined with the corresponding media in the 15 mL tube. At least 2 mL of pre-warmed TexMACS were added to each tube to help with Accutase® dilution and washing. The tubes were lightly capped and placed into the 37 °C incubator until remaining cells were detached. 1 mL of ice cold Accutase® was added and incubated at 4 °C for 10 mins, followed by an incubation at room temperature for 10 mins and an incubation at 37 °C for 30 mins. The cells were pipetted to detach cells, which were collected into separate samples into their corresponding 15 mL tube. The cells were washed by centrifuging cells at 150 x g for 10 mins at 22 °C with the brake off. The media was then aspirated (leaving -100 pL-500 pL to ensure no cell loss). The cells were resuspended and transferred to a 1.5 mL Eppendorf tube. 1 mL of fluorescence-activatedcell sorting (FACS) staining buffer was added with the cells then centrifuged at 300 to 500 x g for 5 mins with the brake off. The supernatant was decanted, and the cells were resuspended in remaining staining buffer. Appropriate monoclonal antibodies (mAbs) were added for flow cytometry and stain for 20 mins at room temperature. 1 mL of staining buffer was added, and cells centrifuged at 300 x g to 500 x g for 5 mins with the brake off. The supernatant was gently decanted, and cells resuspended in remaining buffer. Cells were transferred to a FACS tube and flow cytometry was run to assess for cell viability (7-AAD) and transfection efficiency based on green fluorescent protein light emission.

[0060] Example 5: Cultured Macrophages Transfected with mRNA, DNA, or RNPs. Macrophages that had been cultured and detached from culture dishes as described herein in examples 1 and 3 were loaded with cargo using electroporation. For cells loaded with mRNA or DNA, prior to electroporation, cells were washed and resuspended in electroporation buffer and combined with mRNA-GFP or DNA plasmids of different sizes (pSmall-GFP, 2 kb; pMedium-GFP, 4.2 kb; and pLarge-GFP, 9.2 kb). Cargo was added at low and high concentrations (100 pg / mL and 200 pg / mL, respectively). Two electroporation protocols were used to transfect the cells, each specific to DNA or mRNA. Twenty-four hours post-electroporation, cell viability and transfection efficiency was assessed using flow cytometry as described in example 4.

[0061] For macrophages that were loaded with ribonucleoprotein complexes (RNPs) to knock out expression of a particular gene(s), macrophages were cultured and detached from culture dishes as described herein in examples 1 and 3 with a slight modification. In this example, monocytes were differentiated into macrophages for 3 days. While cells were being washed, RNPs were prepared by complexing recombinant Cas9 protein with guide RNAs (sgRNA) targeting either B2M or SIRPa. After the RNPs were prepared, cells were resuspended in electroporation buffer and combined with B2M RNPs, SIRPa RNPs, or B2M and SIRPa RNPS (multiplex) and the cells were then electroporated. Four days after electroporation, cell viability and gene knockout efficiency was assessed by flow cytometry as described in example 4.

[0062] Post-electroporation viability for macrophages loaded with mRNA DNA cargo (FIG. 3A) and RNPs (FIG. 3C) demonstrate that the culturing and detaching method described herein results in high cell viability post-electroporation (both high and lowenergy electroporation protocols, irrespective of cargo. Post-electroporation assessment of transfection efficiency for loading of mRNA / DNA (FIG. 3B) and RNPs (Fig. 3D) show that macrophages cultured using the methods described herein can be efficiently transfected with cargo. Further, FIG. 3E demonstrates that there were no significant differences in knock-out efficiency when a single gene or two genes were knocked out in macrophages cultured and detached as described herein.

[0063] Example 6: Comparing Electroporation of Macrophages Using Traditional Culturing Methods to the Disclosed Method. Cells in the control group were cultured by thawing 1 x 108PBMCs. The PBMCs were resuspended in 10 mL cell culture media (RPMI 1640, 10% FBS, 2 mM GlutaMAX™). The cells were spun down in a centrifuge for 7 minutes at 180 g. The supernatant was decanted, and the remaining cell pellet was suspended with 30 mL of culture media and seeded in a T175 cm2flask for 2 hours. The media was replaced with cell culture media supplemented with 10 ng / mL of M-CSF to remove non-adherent cells and initiate differentiation. The cells were incubated overnight and allowed to differentiate. After incubation, 20 mL of culture media supplemented with 50 ng / pL of M-CSF was added. The cells were placed back in the incubator for another 2 days. On day 5, 34 mL of media was removed and 12 mL of fresh cell culture media supplemented with 83 ng / mL of M-CSF was added and the cells were incubated for an additional 3 days. On day 7, cells were dissociated by successive PBS / EDTA washes. The harvested cells were then split into two groups. Group 1 was not electroporated, group 2 was electroporated without additional electroporated with pDNA- GFP. The macrophages in the experimental group were prepared using the protocols described in examples 1, 2, and 4, and split into the same two groups as the control. FIG. 4 demonstrates the results from the experiment showing that in both groups, the disclosed method lead to a higher population of viable macrophages. Importantly, FIG 4 shows that a higher percentage of macrophages cultured and differentiated using the disclosed methods expressed higher levels of GFP as compared to the same cells cultured and differentiated using convention methods. Furthermore, significant macrophage reattachment was observed when cells were prepared using the claimed method.

Claims

ClaimsWhat is claimed is:

1. A method of culturing myeloid cells, comprising: plating a myeloid cell population with media using a growth vessel, incubating the myeloid cell population for a time and at a temperature sufficient to allow a population of cells to adhere or attach to the vessel, agitating the media to loosen a non-adherent cell population, removing the media from the growth vessel while retaining the adherent cells, adding to the vessel differentiation media containing macrophage colonystimulating factor (M-CSF), culturing the adherent cells for a time and at a temperature sufficient to differentiate adhered cells into macrophages and increase the cell population to a desired level of confluency, removing an amount of the media with M-CSF after an incubation period, and adding media with M-CSF to the vessel.

2. The method of claim 1 , wherein the myeloid cell population post plating is at a density between 2 x 104cells / cm2to 8 x 105cells / cm2.

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

4. The method of claim 1 , wherein the growth vessel is not tissue culture treated.

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

6. The method of claim 5, wherein the media further includes serum.

7. The method of claim 1 , wherein the concentration of M-CSF in the differentiation media 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 population of cells to adhere to the vessel is about 18 to 36 hours.

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

12. The method of claim 11, wherein the desired level of confluency is between 75% and13. The method of claim 1, wherein the amount of media with M-CSF removed and added is equal to half the total volume of media with M-CSF in the growth vessel.

14. A method of detaching macrophages from a cell culture vessel comprising: adding a dissociation reagent to macrophages cultured using media and a growth vessel, incubating the macrophages at 2 °C to 6° C for up to 20 minutes, followed by incubating the macrophages at 19 °C to 23 °C for up to 20 minutes, followed by incubating the macrophages at 32 °C to 38 °C for up to 60 minutes, and transferring the detached macrophages to a vessel for subsequent processing.

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

16. The method of claim 14, wherein the growth vessel is not tissue culture treated.

17. The method of claim 14, wherein the media is any media fit for cell culture.

18. The method of claim 14, wherein the dissociation reagent is at a temperature of about4 °C when administered to the cell culture.

19. The method of claim 14, wherein the dissociation reagent is at a temperature ranging from about 4 °C to 20 °C when administered to the cell culture.

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

21. A method of culturing a population of myeloid cells wherein the population of monocytes is below 55% wherein: plating a myeloid cell population with media using a growth vessel, incubating the myeloid cell population for up to 50 hours and at a temperature sufficient to allow a population of cells to adhere or attach to the vessel with media with 50 ng / mL of M-CSF supplemented into the media, agitating the media to loosen a non-adherent cell population, removing the media from the growth vessel while retaining the population of cells that adhere or attach to the vessel, culturing the adherent cells for up to 7 days with media with 50 ng / mL of M- CSF supplemented and at a temperature sufficient to differentiate adhered cells into macrophages and increase the cell population to a desired level of confluency, removing an amount of the media with M-CSF after an incubation period, andadding media with M-CSF to the vessel.

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