Device for separating and culturing tumor tissue-derived multiple cells

Abstract

The present invention relates to a cell separation and culture device comprising a plurality of chambers and a fluid flow guide coupled to the lower portions thereof and to a method for separating and culturing cells using same. Cells in a tumor microenvironment including cancer cells can be quickly separated according to sizes from tumor tissue isolated from an individual, and the purity and yield of the separated cells are increased, thereby improving cell viability and utilization.

Description

Tumor tissue-derived multi-cell isolation and culture device

[0001] The present invention relates to a device for separating cancer tissue cells, cancer-associated fibroblasts, and immune cells according to size from tumor tissue derived from the same patient, and to a cell separation and culture method using the same.

[0002] The tumor microenvironment (TME) refers to the surrounding environment in which cancer cells grow and are transformed. This environment includes not only cancer cells but also various non-cancerous cells, such as fibroblasts, immune cells, vascular cells, and inflammatory cells, as well as various extracellular matrices, cytokines, and growth factors, among other molecules. The tumor microenvironment plays a crucial role in the development, growth, and metastasis of cancer. This environment can provide growth signals to tumor cells, protect them from immune attack, and facilitate their spread to other tissues.

[0003] However, cancer-associated fibroblasts and immune cells, which are trained by cancer cells, have different characteristics for each patient, and this does not always have a beneficial effect on the cancer. Because each patient has different characteristics, studying the tumor microenvironment, including cancer cells, is essential not only for the development of cancer treatments but also for cancer prevention and early diagnosis.

[0004]

[0005] Research into the tumor microenvironment can modulate the immune system, inhibit angiogenesis, and prevent fibroblast activation, thereby inhibiting tumor growth and slowing cancer progression. Therefore, research into the tumor microenvironment for cancer treatment continues to advance and can significantly contribute to the development of new treatment targets and methods.

[0006] Korean Patent Application No. 10-2022-0087236 discloses a method for simultaneously isolating and culturing cancer organoids from the same cancer tissue and fibroblasts and immune cells among cancer microenvironment factors. However, further research is being conducted on cell isolation and culturing techniques that can save separation and culturing time and increase cell yield and survival rate while implementing such simultaneous culturing methods.

[0007] The present inventors have studied a device and a separation and culture method that can rapidly separate various cells derived from cancer tissue with high purity according to size within a time period in which cell viability is maintained and utilize them for their intended purpose, and have completed the present invention for a multi-cell separation and culture device.

[0008] Accordingly, an object of the present invention is to provide a cell separation and culture device for isolating cells from tumor tissue isolated from an individual, comprising: an upper chamber including a membrane having through holes formed on a bottom surface; a lower chamber coupled below the upper chamber and including a porous membrane having an extracellular matrix coated on a bottom surface; and a flow-guiding section positioned below the lower chamber for controlling the flow of fluid.

[0009] Another object of the present invention is to provide a method for separating and culturing cells, comprising the steps of adding tumor tissue separated from an individual to a cell separation device and sequentially moving the separated cells to an upper chamber, a lower chamber, and a fluid flow induction unit; culturing the separated cells in the upper chamber; culturing the separated cells in the lower chamber; and separating and culturing the cells that have passed through the micropores of the lower chamber membrane.

[0010] In order to achieve the above object, the present invention provides a cell separation and culture device for isolating tumor tissue separated from an individual, comprising: an upper chamber including a membrane having through holes formed on a bottom surface; a lower chamber coupled below the upper chamber and including a porous membrane having an extracellular matrix coated on a bottom surface; and a fluid flow guide portion positioned below the lower chamber for controlling the flow of fluid.

[0011] In order to achieve another object of the present invention, the present invention provides a method for isolating and culturing cells, comprising the steps of isolating tumor tissue from an individual; adding the culture to a cell separation device and sequentially moving it to an upper chamber, a lower chamber, and a fluid flow induction unit; further culturing cells isolated from the upper chamber; culturing cells isolated from the lower chamber; and isolating and culturing cells that have passed through micropores of the lower chamber membrane.

[0012]

[0013] Hereinafter, the present invention will be described in detail.

[0014] In one aspect, the present invention provides a device for isolating and culturing cells from tumor tissue isolated from an individual, and in particular, has the characteristics of being able to rapidly isolate cells contained in the tumor tissue by size, increase cell yield, and provide high purity of the isolated cells. The tumor tissue is prepared for isolation through a preprocessing process, and may include cancer cells, tumor tissue cells or cancer organoids, fibroblasts, and immune cells, along with tumor microenvironment (TME) factors.

[0015] The cell separation and culture device of the present invention may include two or more distinct chambers. The chambers are positioned vertically. The upper chamber (110) includes an upper chamber inlet (111) for introducing a cell mixture, an upper chamber protrusion (112) formed to be positioned above the lower chamber, an upper chamber coupling portion (113) for coupling with the lower chamber, and an upper chamber bottom surface (114). The upper chamber inlet (111) and the upper chamber coupling portion (113) may form a height extending vertically from the upper chamber bottom surface (114), and the upper chamber protrusion (112) is a portion that protrudes out of the chamber to position the upper plate on top of the lower chamber and the fluid flow induction portion, and includes a protrusion longer than the diameter of the lower chamber. In addition, a through hole is formed in the upper chamber bottom surface (114), through which the cell mixture can pass and move to the lower chamber. The above-mentioned through hole is not limited in shape, and for example, the bottom surface may be a membrane in the form of a mesh. In addition, the diameter of the hole may be 5 to 50 μm, preferably 10 to 30 μm, and more preferably 15 to 20 μm, and the size may be selected depending on the size of the cell to be separated among the cells present in the cell mixture.

[0016] The lower chamber (120) of the cell separation and culture device of the present invention is coupled to the upper chamber coupling portion and includes a lower chamber coupling portion (121) through which a cell mixture is introduced from the upper chamber, a lower chamber protrusion (122) for positioning the lower chamber above the fluid flow induction portion, and a lower chamber bottom surface (123). The lower chamber bottom surface may include a porous membrane. The porous membrane may include a surface on which micropores are formed. In addition, but is not limited thereto, the diameter of the micropores may be 0.1 ㎛ to 10.0 ㎛, for example, 1 ㎛ to 10 ㎛, or 5 ㎛ to 10 ㎛, and may be variously changed depending on the purpose.

[0017] Additionally, the lower chamber may further include an extracellular matrix component or hydrogel coating on the porous membrane of the bottom surface of the lower chamber so that all or part of the cells transferred from the upper chamber can be cultured. The hydrogel is not limited to a type, but may be selected from matrigel, fibrin gel, collagen, agarose gel, alginate, HEMA gel, or a mixture thereof.

[0018] In addition, the cell separation and culture device of the present invention may include a fluid flow guide unit coupled to the lower portion of the chamber. In the present specification, the 'fluid flow guide unit' is a unit capable of controlling the flow of a fluid including a cell culture medium, and allows the cell mixture of each chamber to rapidly move through micro-holes formed on the bottom surface. In particular, the fluid flow guide unit contacts the membrane of the lower chamber and provides a pulling force when the fluid flows out through the micro-holes formed on the bottom surface of each chamber.

[0019] As an example, the fluid flow guide unit may include a hydrophilic plate coated with a hydrophilic material. The plate has a surface coated with a hydrophilic material, and the term "coating" refers to forming a new layer of a certain thickness by forming a film with a specific material on the target surface. The target surface and the modified material may be coated through various chemical bonds such as ionic bonds, covalent bonds, and hydrogen bonds, and the plate of the present invention may have the entire plate or a portion thereof, including the bottom surface that the lower chamber contacts, coated with a hydrophilic material.

[0020] In the present invention, the "hydrophilic material" provides capillary force that draws the cell mixture toward the hydrophilic plate, facilitating the flow of fluid through gravity, allowing the cell mixture transferred to the lower chamber to be quickly filtered through micropores. This enables rapid movement of the cell mixture without the use of a separate device, dramatically reducing cell separation time.

[0021] The above 'hydrophilic material' is not limited in type, and may include hydrophilic polymers such as poly-L-lysine, poly-aspartic acid, polyvinyl alcohol, polylysine, polyacrylic acid, polyacrylamide, poly(acrylonitrile-co-acrylic acid), polyethylene glycol, and polyethyleneimine, or hydrophilic proteins such as bovine serum albumin (BSA), collagen, gelatin, alginate, arginine, lysine, and histidine, and an appropriate material may be selected and used depending on the type of cell.

[0022] That is, the cell separation and culture device of the present invention can rapidly separate cells contained in tumor tissue containing tumor microenvironment factors according to their size while passing tumor tissue from a patient or individual in the order of the upper chamber to the lower chamber. For example, the largest cancer organoid or tumor tissue cells are separated in the upper chamber, and cancer-associated fibroblasts (CAFs) are separated in the lower chamber, and tumor-infiltrating lymphocytes (TILs) that have migrated through the micro-holes of the lower chamber can be obtained from a hydrophilic plate. As described above, the hydrophilic plate includes a hydrophilic coating layer, thereby further promoting the movement of the cell mixture by gravity.

[0023] As another example, the fluid flow guide unit may include a fluid guide adapter coupled to the lower chamber of the cell separation and culture device. The fluid guide adapter may include a space into which an auxiliary fluid can be injected to offset surface tension when a fluid including a cell culture solution flows out through a micro-hole of a lower chamber membrane, and may include a valve that is a passage that controls the flow of the fluid injected into the lower portion of the space and transmits it to the outside.

[0024] The above auxiliary fluid injection space is in contact with the micro-holes of the lower chamber membrane, and the valve may have a structure in which the valve gradually widens from the portion (D1) in contact with the auxiliary fluid injection space toward the discharge portion (D2). This is to fill the auxiliary fluid injection space with fluid by controlling the surface tension generated at the micro-holes of the lower chamber membrane and the upper portion of the valve.

[0025] The above fluid induction adapter eliminates the surface tension formed in the micro-holes of the lower chamber through the auxiliary fluid, allowing the cell suspension to flow out without resistance. For example, to fill the auxiliary fluid injection space of the adapter with the auxiliary fluid, the weight (F) of the fluid filled in the auxiliary fluid injection spaceH1 ) than the surface tension (F) in micropores D0 ) and surface tension (F) at the top of the valve D1 ) of the sum (F) D0 +F D1 ) should be larger, that is, the flow of fluid can be controlled as follows.

[0026] - Fluid filling: F D0 +F D1 > F H1

[0027] - Fluid leak: F D0 +F D1 < F H1

[0028] Surface tension = γ*π*D

[0029] Weight of fluid = ρ*V*g

[0030]

[0031] γ: Surface tension coefficient

[0032] ρ: density of the fluid

[0033] V: volume of fluid

[0034] g: acceleration due to gravity

[0035] D: Diameter of the upper and lower parts of the valve

[0036]

[0037]

[0038] Additionally, in order for the fluid to flow smoothly outside the adapter, the weight of the fluid filled inside the fluid induction adapter (F H2 ) is the surface tension at the bottom of the valve (F D2 ) must be greater than.

[0039] Fluid flow occurrence: ΔF D2 < ΔF H2

[0040] Fluid flow inhibition: ΔF D2 > ΔF H2

[0041]

[0042] As another aspect of the present invention, the present invention relates to a method for separating and culturing cells using the above-described cell separation and culturing device.

[0043] The above method comprises the following steps:

[0044] 1) A step of isolating tumor tissue from an object;

[0045] 2) A step of sequentially moving the tumor tissue through the upper chamber, lower chamber and fluid flow guide of the cell separation device;

[0046] 3) A step of culturing cells separated from the upper chamber;

[0047] 4) A step of culturing cells separated from the lower chamber; and

[0048] 5) A step of isolating and culturing cells that have passed through the micro-pores of the lower chamber membrane.

[0049] In one embodiment, the tumor tissue is isolated from a lesion of a cancer patient and cultured in a medium selected based on the characteristics of the tumor tissue. A medium commonly used for culturing cancer cells or cancer organoids may be selected. By directly culturing the tumor tissue isolated from the lesion of a cancer patient, factors of the tumor microenvironment (TME) can be cultured in addition to cancer cells, thereby isolating and utilizing cells suitable for cell therapy for the patient.

[0050] In the present invention, the 'tumor tissue' refers to a tissue in which a tumor has developed, and the cancer tissue may be any one selected from the group consisting of lung cancer tissue, skin cancer tissue, stomach cancer tissue, colon cancer tissue, colon cancer tissue, pancreatic cancer tissue, liver cancer tissue, thyroid cancer tissue, uterine cancer tissue, cervical cancer tissue, ovarian cancer tissue, testicular cancer tissue, prostate cancer tissue, breast cancer tissue, and oral cancer tissue, but is not limited thereto.

[0051] In the present invention, the "tumor microenvironment (TME)" may refer to surrounding tissue cells that directly or indirectly influence the formation and progression of cancer. The cancer microenvironment factors are contained in a culture of tumor tissue and may include immune cells, fibroblasts, vascular cells, or a combination thereof.

[0052] In the present invention, 'fibroblasts' may be cancer-associated fibroblasts (CAFs), which refer to fibroblasts existing in tissues surrounding cancer or malignant tumors, and are involved in tumor growth, tumor angiogenesis, tumor cell invasion, and metastasis in most cancers.

[0053] In the present invention, the 'immune cell' may be selected from the group consisting of tumor infiltrating lymphocyte (TIL), T cell, cytotoxic T lymphocyte (CTL), B cell, NK cell, mononuclear phagocyte, α / β receptor T cell and γ / δ receptor T cell or a mixture thereof.

[0054] In addition, the cell separation device of the present invention includes a plurality of chambers positioned vertically as described above, and moves the cell mixture from the upper chamber to the lower chamber through a fluid flow guide unit, i.e., in the direction of gravity, to separate cells by size from the culture through holes or micro-holes formed on the bottom surface of each chamber.

[0055] In one embodiment, the upper chamber includes a through hole with a diameter of about 15 to 20 μm in the membrane on the bottom surface, so that tissue cells, such as cancerous tissue cell clumps, larger than the diameter can be separated. The membrane on the bottom surface of the lower chamber includes micropores with a diameter of about 5 to 10 μm, which are smaller than the diameter of the upper chamber, so that fibroblasts larger than the diameter can be separated. The remaining cell mixture includes immune cells (TILs) smaller than the diameter of 5 to 10 μm, which migrate to the lower hydrophilic plate through the micropores. Through this process, cells separated at each stage can be obtained.

[0056] The method may further include a process of culturing the cells separated from the upper and lower chambers, respectively. The culturing process may be performed using a commonly known culture medium and culture method, and is not particularly limited to the method. The culturing may be performed simultaneously with the separation on an extracellular matrix or hydrogel coated on the chamber to increase the yield of cells, or may be additionally performed in a separate medium depending on the intended use.

[0057] In the present invention, the time for separation of the cell mixture while moving through the cell separation device is within 30 minutes, preferably within 20 minutes, and more preferably within 10 minutes, and as described above, the time for separation and culture of cells is saved, and cells included in the tumor microenvironment can be obtained before the viability of the cells is reduced.

[0058] The present invention relates to a cell separation and culture device, which can rapidly separate cells contained in a tumor tissue culture with high purity according to size by moving cells of a tumor microenvironment including cancer cells from an upper chamber to a lower chamber and then to a fluid flow induction section in that order from tumor tissue separated from an individual. In particular, the device of the present invention can increase the yield of each cell during the separation and culture process of cells derived together from a patient's tumor tissue, and has the effect of utilizing the cells before their viability declines.

[0059] Figure 1 is a schematic diagram showing the process of isolating cells from a patient's tissue using the cell separation and culture device of the present invention.

[0060] FIG. 2 illustrates the structure of a cell separation and culture device of the present invention. FIG. 2a illustrates a cell separation and culture device (100) including an upper chamber (110) including an upper chamber inlet (111), an upper chamber protrusion (112), an upper chamber coupling portion (113), and a membrane (114) on the bottom surface of the upper chamber; a lower chamber (120) including a lower chamber coupling portion (121), a lower chamber protrusion (122), and a membrane (123) on the bottom surface of the lower chamber; and a hydrophilic plate (130).

[0061] Fig. 2b is a detailed illustration of the structures of the upper and lower chambers in Fig. 2a, respectively, and Fig. 2c is a table showing specifications for each component of the upper and lower chambers as an example.

[0062] Figure 3 illustrates the structure of the mesh membrane constituting the bottom surface of the upper chamber and the porous membrane constituting the bottom surface of the lower chamber.

[0063] Figure 4 is a schematic diagram of the overall cell separation process using the cell separation device of the present invention.

[0064] Figure 5 is a schematic diagram showing a process for obtaining immune cells from a hydrophilic plate including a hydrophilic coating layer.

[0065] Figure 6 is a photograph showing the contact angle and the movement pattern of the cell mixture on a hydrophilic plate including a hydrophilic coating layer and a hydrophobic plate without a hydrophilic coating.

[0066] Figure 7a shows the results of comparing the migration speed and separation efficiency of a cell mixture on a hydrophilic plate including a hydrophilic coating layer and a hydrophobic plate without a hydrophilic coating.

[0067] Figure 7b shows the results of comparing the immune cell separation rates (%) of each tissue on a hydrophilic plate with a hydrophilic coating layer.

[0068] Figure 8 illustrates the process of isolating and culturing cancer-associated fibroblasts (CAFs) through collagen 1 coated on the bottom surface of the lower chamber.

[0069] Figure 9 shows the effect of shortening the adhesion time of cancer-associated fibroblasts (CAFs) by collagen 1 coating, and confirms the increased growth of cancer-associated fibroblasts compared to the uncoated condition.

[0070] Figures 10 and 11 show the results of comparing the culture results of cancer organoids separated through the cell separation device of the present invention and cancer organoids cultured using a conventional method.

[0071] Figure 12 shows the results of comparing the culture results of cancer-associated fibroblasts separated through the cell separation device of the present invention and fibroblasts cultured using a conventional method.

[0072] Figure 13 shows the results of flow cytometry analysis of tumor-infiltrating lymphocytes separated using the cell separation device of the present invention.

[0073] Fig. 14 illustrates the structure of a cell separation and culture device including a fluid induction adapter, wherein the upper chamber (110) and the lower chamber (120) have the same structure as that illustrated in Fig. 2b, and the cell separation and culture device (200) includes a fluid induction adapter (210) structure below the lower chamber. The fluid induction adapter includes an auxiliary fluid injection space (211) and a valve (212).

[0074] Figure 15 is a schematic diagram illustrating a tissue separation and culture process using a cell separation and culture device including a fluid induction adapter.

[0075] Figure 16 shows the results of confirming the optimized conditions for maintaining the flow of fluid using gravity and surface tension in a cell separation and culture device including a fluid induction adapter. Figure 16a shows the removal of surface tension formed in the micro-holes of the lower chamber membrane through the fluid, and Figure 16b shows the conditions for controlling the fluid flow by surface tension generated at the bottom of the valve.

[0076] Figure 17 shows the results of culturing tumor tissue-derived cells using a cell separation and culture device including a fluid-induced adapter. Figure 17a shows the results of culturing cancer organoids, Figure 17b shows the results of culturing fibroblasts, and Figure 17c shows the results of culturing immune cells, respectively.

[0077] Hereinafter, examples will be described in detail to specifically explain this specification. However, the embodiments according to this specification may be modified in various different forms, and the scope of this specification is not limited to the embodiments described below. The embodiments of this specification are provided to more fully explain this specification to those of average skill in the art.

[0078]

[0079] Example 1. Isolation of tumor microenvironment (TME) and cell mixture from tumor tissue

[0080] The entire process of isolating tumor tissue from a cancer patient, preprocessing, and decoupling is shown in Figure 4.

[0081] 1-1) Preprocessing process

[0082] First, tissue was excised from the patient and incubated at 37°C for 20–90 minutes with Collagenase II to degrade the extracellular matrix. The tissue was washed and centrifuged (250 rcf, 3 min, RT), and the pellet was resuspended in 5 ml of phosphate-buffered saline (PBS). The effluent containing cells was collected through a 100 μm strainer, washed, and centrifuged (250 rcf, 3 min, RT). The collected pellet was used for cell isolation.

[0083] The prepared cell pellets were separated using conventional separation methods (convectional) and decoupling culture methods, respectively.

[0084] 1-2) Cell separation and culture

[0085] Using the cell separation device of the present invention, cells were separated. First, cell clusters larger than 20 μm were separated from the upper chamber. The separated pellet was collected, washed, centrifuged (250 rcf, 3 min, RT), and resuspended in Matrigel / medium. Additional medium was added to solidify the Matrigel for 10-15 minutes at 37°C, and organoid culture was maintained for 7-14 days.

[0086] Cells larger than 8 μm were isolated from the lower chamber, and cancer-associated fibroblasts (CAFs) were cultured in a culture medium suitable for CAFs on a porous membrane coated with collagen for 7 to 14 days. CAFs were cultured in the lower chamber to reduce cell loss during transfer to another hydrophilic plate. Cells smaller than 8 μm were collected through the micropores of the lower chamber.

[0087]

[0088] Example 2. Obtaining immune cells using a hydrophilic coating plate

[0089] In Example 1, the cell suspension transferred from the upper chamber to the lower chamber contained cancer-associated fibroblasts and tumor-infiltrating lymphocytes. The surface of a hydrophilic plate positioned beneath the lower chamber was treated with 3% bovine serum albumin (BSA), a hydrophilic agent. The migration of the cell mixture loaded into the lower chamber was compared against an uncoated plate, serving as a control. (Figure 5)

[0090] As a result, as shown in Fig. 6, it was found that the cell mixture moved quickly when the plate surface-treated with 3% BSA was used. In addition, as shown in Fig. 7a, the results comparing the amount of cell mixture remaining in the actual lower chamber (transwell) over time confirmed that it decreased more quickly when the hydrophilic coating layer was included. At this time, fibroblasts, which are cells larger than 8 μm, remained in the lower chamber, and immune cells escaped and migrated to the hydrophilic plate, confirming that the separation efficiency of CAF / TIL cells was improved. (Fig. 7b)

[0091] In addition, collagen (COL-1) was coated on the bottom surface of the lower chamber above, and after moving the cell mixture as described above, the remaining fibroblasts were additionally cultured (Fig. 8). In the culture results, the attachment and growth of fibroblasts were observed over time, and as shown in Fig. 9, it was confirmed that the growth of adherent cells increased. In other words, it can be seen that the culture efficiency of adherent cells is improved when the COL-1 coating is included.

[0092]

[0093] Example 3. Confirmation of cell culture isolated from hydrophilic coated plates

[0094] 3-1) Cancer organoid culture

[0095] After pretreatment of cancer tissue isolated from the subject in the same manner as in Example 1, the pellet was resuspended in Matrigel / exclusion. The suspension was placed on a hydrophilic plate, and the Matrigel was solidified for 10-15 minutes at 37°C. The cancer organoids were maintained by culturing in a cancer organoid culture medium for 7-14 days.

[0096] As a result, as shown in Fig. 10, it was confirmed that the cancer organoids obtained through the conventional cancer organoid culture method and the cancer organoids obtained using the cell separation device of the present invention initially showed the same shape and growth rate, and it was confirmed that they did not show any difference even in long-term culture. As shown in Fig. 11, it was confirmed that they had the same shape as actual tumor tissue histologically, and it was confirmed that cancer-specific proteins were expressed identically in the cancer organoids obtained through the two methods.

[0097] 3-2) Cancer-related fibroblast culture

[0098] After preprocessing the cancer tissue isolated from the subject, a portion of the cancer tissue was placed on a hydrophilic plate coated with COL-1 (collagen-1) and cultured in a cancer-associated fibroblast (CAF) culture medium for 7 to 14 days.

[0099] As shown in Fig. 12, it was confirmed that there was no difference in the morphology of cells cultured through the conventional cancer-associated fibroblast (CAF) culture method and cells separated and cultured through the cell separation device of the present invention. In addition, in order to confirm the characteristics of cancer tissue-derived fibroblasts (CAF), the expression of markers such as αSMA, Vimentin (VIM), PRRX1, and FAP in CAF was analyzed through immunofluorescence staining. To this end, cultured CAF were fixed with 4% PFA, permeabilized with Triton X-100, and then nonspecific binding was blocked, and the primary antibody for each marker (αSMA, VIM, PRRX1, FAP) was reacted with a fluorescently labeled secondary antibody. The nucleus was stained with DAPI, and the expression and co-expression patterns of each marker were observed through a fluorescence microscope to visually evaluate the mesenchymal and activation states of CAF. As a result, it was confirmed that the protein markers indicating the characteristics of the cancer-associated fibroblasts were expressed identically in both cells obtained through the above two methods.

[0100] 3-3) Culture of tumor-infiltrating lymphocytes

[0101] Flow cytometry analysis was performed to confirm the physicochemical properties of tumor-infiltrating lymphocytes (TILs) obtained from Example 2 above.

[0102] In the results of Fig. 13, a) is the result of distinguishing lymphocytes based on the size and internal complexity of the cells using FSC-A (Forward Scatter) and SSC-A (Side Scatter), and living cells were confirmed. b) is the result of confirming that CD45 labeled with APC-Cy7 was positive, which shows that the sample is composed of lymphocytes. In addition, c) is the result of distinguishing T cells (CD3 positive) and B cells (CD19 positive), and it can be confirmed that most cells are CD3 positive, mainly composed of T cells, and B cells (CD19 positive) are relatively few. d) is the result showing the distribution of CD8 positive cells, i.e. cytotoxic T cells, and shows that CD8 positive cells (approximately 48.4%) and CD8 negative cells (approximately 51.6%) are almost equally divided among the CD3 positive cell population.

[0103] That is, the obtained sample is mostly composed of lymphocytes expressing the CD45 marker, with a high expression rate of CD3, a major T cell marker, and a low expression rate of CD19, indicating a predominance of T cells over B cells. Furthermore, the CD4 and CD8 markers are expressed at nearly equal rates, indicating that cells expressing each marker exist in roughly equal proportions.

[0104]

[0105] Example 4. Design of a cell separation device including a fluid-guided adapter.

[0106] In the cell separation and culture device described in the above Example 2 and FIG. 2, a device including a fluid induction adapter for delivering a cell culture medium to a lower chamber was designed. As confirmed in FIG. 14, the upper and lower chambers are the same, but the hydrophilic plate is replaced with a fluid induction adapter (210), and the fluid induction adapter includes an auxiliary fluid injection space (211) and a valve (212), and the valve has a structure like a passage with an upper portion narrower than the lower portion (D1 <D2)이다.

[0107] In order to form a fluid flow in the above device, the weight of the fluid contained in the loaded cell suspension and the surface tension of the micropores were adjusted to satisfy the following equation.

[0108] F D0 +F D1 > ΔF H1

[0109] (F D0 : Surface tension of micropore (D0) / F D1 : Surface tension at the top of the valve / F H1 : weight of the auxiliary)

[0110] F D2 < F H2

[0111] (F D2 : Surface tension at the bottom of the valve / ΔF H2 : Weight of cell suspension (filled throughout the adapter)

[0112] That is, in order to form a fluid flow, the weight of the fluid was adjusted to be greater than the surface tension in the hole through which it passed. To this end, when the height of the fluid injection space of the adapter was 5 to 6 mm, the diameter of the upper part of the valve was set to a range of 1 to 3 mm, and when the overall height of the adapter was 10 mm, the diameter of the lower part of the valve was set to a range of 3 to 6 mm, at which time a fluid flow could be formed.

[0113]

[0114] Example 5. Tissue separation and culture using a cell separation device including a fluid-induced adapter.

[0115] The fluid induction adapter of the above Example 4 controls the flow of fluid through surface tension. In order to isolate and culture tissue using the fluid induction adapter, the bottom surface of the lower chamber and the auxiliary fluid injection space (211) of the fluid induction adapter were positioned so as to be in contact, and PBS was injected into the fluid induction adapter through the auxiliary fluid injection space, and the injected PBS reached the bottom of the porous membrane and was maintained in complete contact with the surface. As in the above Example 1, cell suspension separated from cancer tissue was loaded into the culture chamber, and fluid flow was formed through the micro-holes (D0) formed on the bottom surface of the lower chamber according to gravity. This process is illustrated in Fig. 15.

[0116] After the fluid drained through the valve, organoids contained in the cell suspension were found in the upper chamber, cancer-associated fibroblasts in the lower chamber, and tumor-infiltrating lymphocytes in the fluid that moved through the valve, confirming that the substances contained in the cell suspension were separated according to size.

[0117] In addition, for each culture separated through a cell separation device including the fluid induction adapter, the culture was performed in the same manner as in Example 3, and the results were confirmed.

[0118] First, cancer organoids obtained from the upper chamber were confirmed as shown in Figure 17a. Tissue staining results confirmed that they were cultured in a form similar to cancer tissue.

[0119] In addition, as shown in Fig. 17b, it was confirmed that cancer-associated fibroblasts obtained from the lower chamber were successfully cultured, and the characteristics of CAFs were also confirmed in the fluorescent staining results that visually confirmed the mesenchymal and activation states of cancer-associated fibroblasts (CAFs).

[0120] Finally, tumor-infiltrating lymphocytes (TILs) were identified in cultures cultured with fluids drained through the fluid-guided adapter. Comparing FSC-A and SSC-A, viable lymphocytes were identified (a), and APC-Cy7 (CD45) was positive, indicating that the cells contained lymphocytes (b). Furthermore, comparing BV421 (CD3: T cell) and APC (CD19: B cell) revealed that most cells were CD3 positive, mainly composed of T cells, with a relatively small number of B cells (CD19 positive) (c). Finally, CD3 + CD4 among T cells + T cells and CD8 + It was confirmed that T cells were divided in almost similar proportions (d).

[0121] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than restrictive. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

[0122] [Explanation of symbols]

[0123] 100: Cell separation and culture device

[0124] 110: Upper chamber

[0125] 111: Upper chamber inlet

[0126] 112: Upper chamber protrusion

[0127] 113: Upper chamber joint

[0128] 114: Upper chamber bottom surface

[0129] 120: Lower chamber

[0130] 121: Lower chamber joint

[0131] 122: Lower chamber protrusion

[0132] 123: Lower chamber floor

[0133] 130: Hydrophilic plate

[0134] 200: Cell separation and culture device

[0135] 210: Fluid Induction Adapter

[0136] 211: Auxiliary fluid injection unit

[0137] 212: Valve

[0138] In one aspect, the present invention relates to a cell separation and culture device for separating and culturing various cell populations from tumor tissue isolated from an individual, the device comprising: an upper chamber including a membrane having through holes formed on a bottom surface; a lower chamber coupled below the upper chamber and including a porous membrane having an extracellular matrix coated on a bottom surface; and a flow-guiding section contacting below the lower chamber and controlling the flow of fluid.

[0139] As an example, the cell separation and culture device is for separating cells contained in a tumor microenvironment from tumor tissue separated from an individual according to size.

[0140] As an example, a through hole formed on the bottom surface of the upper chamber of the cell separation and culture device may have a diameter of 5 to 50 μm.

[0141] In addition, as another example, the porous membrane included in the bottom surface of the lower chamber of the cell separation and culture device has micropores formed in a size of 0.1 μm to 10.0 μm.

[0142] As an example, the bottom surface of the lower chamber may additionally include an extracellular matrix or hydrogel layer.

[0143] As an example, the hydrogel layer may be selected from matrigel, fibrin gel, collagen (collagen 1), agarose gel, alginate, HEMA gel, or a mixture thereof.

[0144] As an example, the fluid flow guide portion may be selected from a hydrophilic plate coated with a hydrophilic material or a fluid guide adapter.

[0145] Among the above fluid flow inducing parts, the hydrophilic plate may be coated with a material selected from among hydrophilic polymers including poly-L-lysine, polyaspartic acid, polyvinyl alcohol, polylysine, polyacrylic acid, polyacrylamide, poly(acrylonitrile-co-acrylic acid), polyethylene glycol, and polyethyleneimine; or hydrophilic proteins including bovine serum albumin (BSA), collagen, gelatin, alginate, arginine, lysine, and histidine.

[0146] In addition, among the fluid flow induction parts, the fluid induction adapter includes an auxiliary fluid injection space and a valve, and the valve may be positioned below the auxiliary fluid injection space and may be formed to become wider from a portion (D1) in contact with the auxiliary fluid injection space toward the bottom (D2).

[0147] As an example, a cell population applicable to the cell separation and culture device of the present invention may include tumor microenvironment factors, cancer cells, tumor tissue cells, cancer organoids, fibroblasts, or immune cells derived together from tumor tissue isolated from an individual.

[0148] As an example, the upper chamber of the cell separation and culture device of the present invention is for separating cancer cells, tumor tissue cells, or cancer organoids.

[0149] As an example, the lower chamber of the cell separation and culture device of the present invention is for separating tumor microenvironment factors or cancer-associated fibroblasts excluding immune cells.

[0150] As an example, the tumor tissue may be selected from lung cancer tissue, skin cancer tissue, stomach cancer tissue, colon cancer tissue, colon cancer tissue, pancreatic cancer tissue, liver cancer tissue, thyroid cancer tissue, uterine cancer tissue, cervical cancer tissue, ovarian cancer tissue, testicular cancer tissue, prostate cancer tissue, breast cancer tissue, and oral cancer tissue.

[0151] In another aspect, the present invention relates to a method for isolating and culturing cells from tumor tissue isolated from an individual, comprising the steps of: 1) isolating tumor tissue from an individual; 2) adding the tumor tissue to a cell separation device and sequentially moving the tumor tissue to an upper chamber, a lower chamber, and a fluid flow induction unit; 3) culturing cells isolated from the upper chamber; 4) culturing cells isolated from the lower chamber; and 5) obtaining and culturing cells that have passed through the micropores of the lower chamber membrane.

[0152] As an example, the cells separated from the upper chamber by the cell separation and culture method of the present invention are cancer cells, tumor tissue cells or cancer organoids, the cells separated from the lower chamber are tumor microenvironment factors or cancer-associated fibroblasts excluding immune cells, and the cells that passed through the micropores of the lower chamber membrane are immune cells.

[0153] As an example, the immune cell may be selected from a tumor infiltrating lymphocyte (TIL), a T cell, a cytotoxic T lymphocyte (CTL), a B cell, a NK cell, a mononuclear phagocyte, an α / β receptor T cell, and a γ / δ receptor T cell, or a mixture thereof.

[0154] As an example, the cell separation and culture method of the present invention is such that the upper chamber, lower chamber, and fluid flow guide are sequentially connected in the vertical direction to move tumor tissue in the direction of gravity.

[0155] According to the cell separation and culture method of the present invention, the migration time of the tumor tissue is within 30 minutes, and the method of the present invention has the effect of improving cell yield and cell viability.

Claims

1. A device for isolating and culturing various cell groups from tumor tissue separated from an individual, An upper chamber comprising a membrane having through holes formed on the bottom surface; A lower chamber coupled below the upper chamber and including a porous membrane coated with an extracellular matrix on the bottom surface; and A cell separation and culture device comprising a flow-guiding section that is in contact with the lower chamber and controls the flow of fluid.

2. In paragraph 1, The above cell separation and culture device is a cell separation and culture device for separating cells contained in a tumor microenvironment from tumor tissue separated from an individual according to size.

3. In paragraph 1, A cell separation and culture device, wherein a through hole formed on the bottom surface of the upper chamber has a diameter of 5 to 50 μm.

4. In paragraph 1, A cell separation and culture device, wherein a porous membrane included in the bottom surface of the lower chamber has micropores formed therein of 0.1 ㎛ to 10.0 ㎛.

5. In paragraph 1, A cell separation and culture device, wherein the bottom surface of the lower chamber additionally includes an extracellular matrix or hydrogel layer.

6. In paragraph 5, A cell separation and culture device, wherein the hydrogel layer is selected from matrigel, fibrin gel, collagen (collagen 1), agarose gel, alginate, HEMA gel, or a mixture thereof.

7. In paragraph 1, A cell separation and culture device, wherein the fluid flow induction unit is selected from among a hydrophilic plate coated with a hydrophilic material or a fluid induction adapter.

8. In paragraph 7, A cell separation and culture device, wherein the hydrophilic plate comprises a layer coated with a material selected from among hydrophilic polymers including poly-L-lysine, poly-aspartic acid, polyvinyl alcohol, polylysine, polyacrylic acid, polyacrylamide, poly(acrylonitrile-co-acrylic acid), polyethylene glycol, and polyethyleneimine; or hydrophilic proteins including bovine serum albumin (BSA), collagen, gelatin, alginate, arginine, lysine, and histidine.

9. In paragraph 7, A cell separation and culture device, wherein the fluid induction adapter includes an auxiliary fluid injection space and a valve.

10. In paragraph 9, A cell separation and culture device, characterized in that the valve is located below the auxiliary fluid injection space and widens from the portion (D1) in contact with the auxiliary fluid injection space toward the bottom (D2).

11. In paragraph 1, A cell separation and culture device, wherein the cell population includes tumor microenvironment factors, cancer cells, tumor tissue cells, cancer organoids, fibroblasts or immune cells derived together from tumor tissue isolated from an individual.

12. In paragraph 1, A cell separation device, wherein the upper chamber is for separating cancer cells, tumor tissue cells or cancer organoids.

13. In paragraph 1, A cell separation device, wherein the lower chamber is for separating tumor microenvironment factors or cancer-associated fibroblasts excluding immune cells.

14. In paragraph 1, A cell separation device, wherein the tumor tissue is any one selected from the group consisting of lung cancer tissue, skin cancer tissue, stomach cancer tissue, colon cancer tissue, colon cancer tissue, pancreatic cancer tissue, liver cancer tissue, thyroid cancer tissue, uterine cancer tissue, cervical cancer tissue, ovarian cancer tissue, testicular cancer tissue, prostate cancer tissue, breast cancer tissue, and oral cancer tissue. 15.1) Step of isolating tumor tissue from the subject; 2) A step of sequentially moving the tumor tissue to the cell separation device, the upper chamber, the lower chamber, and the fluid flow induction unit; 3) A step of culturing cells separated from the upper chamber; 4) a step of culturing cells separated from the lower chamber; and 5) A method for isolating and culturing cells cultured from tumor tissue isolated from an individual, including a step of obtaining and culturing cells that have passed through the micropores of the lower chamber membrane.

16. In paragraph 15, A method for isolating and culturing cells, wherein the cells separated from the upper chamber are cancer cells, tumor tissue cells or cancer organoids.

17. In paragraph 15, A method for isolating and culturing cells, wherein the cells separated from the lower chamber are tumor microenvironment factors or cancer-associated fibroblasts, excluding immune cells.

18. In paragraph 15, A method for separating and culturing cells, wherein the cells passing through the micropores of the lower chamber membrane are immune cells.

19. In paragraph 18, A method for isolating and culturing cells, wherein the immune cells are selected from the group consisting of tumor infiltrating lymphocytes (TILs), T cells, cytotoxic T lymphocytes (CTLs), B cells, NK cells, mononuclear phagocytes, α / β receptor T cells, and γ / δ receptor T cells, or a mixture thereof.

20. In paragraph 15, A method for separating and culturing cells, wherein the upper chamber, lower chamber, and fluid flow induction unit are sequentially connected in an up-and-down direction to move tumor tissue in the direction of gravity.

21. In paragraph 20, A method for isolating and culturing cells, wherein the migration time of the tumor tissue is within 30 minutes.

22. In paragraph 15, A method for isolating and culturing cells, wherein the method for isolating and culturing cells has the effect of improving cell yield and cell viability.

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