Method and composition for cryopreservation and culture of dendritic cells derived from lymphoid tissue

Abstract

The present invention relates to a composition for the cryopreservation of primary dendritic cells, and a method for cryopreservation and cell culture using same, and more specifically to a composition for the cryopreservation of primary dendritic cells, and a method for cryopreservation and cell culture using same, the composition comprising serum and DMSO, thereby enabling a high viability after thawing of primary dendritic cells, and being capable of inhibiting the spontaneous activation of the primary dendritic cells so as to enable same to be cultured in vitro, and being usable for immunological diagnosis and therapeutic agent development for various diseases.

Description

Method and composition for cryopreservation and culture of dendritic cells derived from lymphoid tissue

[0001] The present invention relates to a method for cryopreserving and culturing primary dendritic cells isolated from lymphoid tissue.

[0002]

[0003] Dendritic cells (DCs) are antigen-presenting cells that present antigens to T cells and are involved in the connection between the innate and adaptive immune responses. Dendritic cells are distributed in immune tissues such as the spleen and lymph nodes, as well as mucosal, intestinal, and epidermal tissues, and exist in various subtypes. Developmentally distinct from macrophages and monocytes, dendritic cell subtypes are specialized for their immunological role in antigen presentation.

[0004] In the absence of stimulation such as inflammation, dendritic cells exist in an inactive form. Inactive dendritic cells have low expression of major histocompatibility class-II (MHC-II) surface molecules, almost no expression of co-stimulatory molecules, and almost no production of inflammatory cytokines. Dendritic cells that have ingested antigens from external pathogens or damaged cells are activated. Activated dendritic cells are characterized by decreased antigen uptake capacity, increased expression of MHC-II, increased expression of co-stimulatory molecules (CD80, CD86, and CD40), secretion of pro-inflammatory cytokines, and increased migration to lymph nodes. These activated dendritic cells perform an antigen presentation function that induces T cell responses to specific antigens.

[0005] The greatest challenge in assessing the immunological function of stable dendritic cells lies in the isolation and culture of tissue-derived primary dendritic cells (DCs). Primary DCs isolated for cell culture undergo spontaneous activation, transitioning from an inactive state to an active state within 12 hours of culture. The extremely short survival time of spontaneously activated DCs precludes long-term culture. The extracellular matrix, which constitutes the spleen and lymph node tissues, regulates immune cell interactions and plays a crucial role in cell-to-cell aggregation within the tissue. Fibronectin, a major extracellular matrix protein, is expressed in lymphoid tissues. Unlike in living tissues, spontaneous activation during in vitro culture has been proposed to occur through binding or contact between dendritic cells under culture conditions. This spontaneous activation can be effectively prevented by the addition of fibronectin, a component of the extracellular matrix.

[0006]

[0007] The present invention provides a composition for cryopreservation of primary dendritic cells and tissues containing the same, and a cryopreservation method using the same.

[0008] The present invention provides a method for culturing primary dendritic cells using the above cryopreservation composition and cryopreservation method.

[0009]

[0010] 1. A method for culturing primary dendritic cells, comprising the step of cryopreserving primary dendritic cells or tissues containing the same in a composition comprising serum, DMSO and fibronectin.

[0011] 2. A method for culturing primary dendritic cells, comprising the step of cryopreserving primary dendritic cells or tissues containing the same in a composition comprising serum, DMSO and albumin.

[0012] 3. A method for culturing primary dendritic cells, further comprising the step of thawing primary dendritic cells or tissues containing them in a medium containing fibronectin, in 1 or 2 above.

[0013] 4. A method for culturing primary dendritic cells, further comprising the step of thawing primary dendritic cells or tissues containing them in a medium not containing fibronectin, in 1 or 2 above.

[0014] 5. A method for culturing primary dendritic cells, further comprising the step of thawing primary dendritic cells or tissues containing the primary dendritic cells and then co-culturing them with fibroblasts, in 1 or 2 above.

[0015] 6. A method for culturing primary dendritic cells, further comprising: a step of thawing primary dendritic cells or tissues containing the primary dendritic cells in 1 or 2 above, and then dispensing the primary dendritic cells onto a first support comprising polyvinyl alcohol (PVA) nanofibers or polycaprolactone (PCL) nanofibers; and a step of dispensing fibroblasts onto a second support comprising polycaprolactone (PCL) nanofibers to co-culture the primary dendritic cells with the fibroblasts.

[0016] 7. A method for culturing primary dendritic cells in the above 6, wherein the first support and the second support are spaced apart and stacked within an incubator.

[0017] 8. In the above 2, a method for culturing primary dendritic cells, wherein the composition further comprises fibronectin.

[0018] 9. A method for culturing primary dendritic cells in the above 1 or 2, wherein the serum is 10% to 90% FBS.

[0019] 10. A method for culturing primary dendritic cells in the above 1 or 2, wherein DMSO is 5% to 15% DMSO.

[0020] 11. A method for culturing primary dendritic cells, wherein fibronectin is included in an amount of 100 to 300 μg / mL in the above 1 or 8.

[0021] 12. A method for culturing primary dendritic cells, wherein albumin is contained in an amount of 300 to 500 mg / mL in the above 2.

[0022] 13. A composition for cryopreservation of primary dendritic cells or tissues containing the same, comprising serum, DMSO and fibronectin.

[0023] 14. A composition for cryopreservation of primary dendritic cells or tissues containing the same, comprising serum, DMSO and albumin.

[0024] 15. A composition for cryopreservation of primary dendritic cells or tissues containing them, wherein the serum comprises 10% to 90% FBS in the above 13 or 14.

[0025] 16. In the above 13 or 14, DMSO is a composition for cryopreservation of primary dendritic cells or tissues containing them, wherein DMSO is 5% to 15% DMSO.

[0026] 17. A composition for cryopreservation of primary dendritic cells or tissues containing them, wherein fibronectin is included in an amount of 100 to 300 μg / mL in the above 13.

[0027] 18. A composition for cryopreservation of primary dendritic cells or tissues containing them, wherein albumin is included in an amount of 300 to 500 mg / mL in the above 14.

[0028]

[0029] The cryopreservation composition and cryopreservation method of the present invention enable cryopreservation of primary dendritic cells isolated from the spleen, which is a living immune tissue.

[0030] The cryopreservation composition and cryopreservation method of the present invention increase the cell survival rate in a thawed state after cryopreservation of primary dendritic cells.

[0031] The cryopreservation composition and cryopreservation method of the present invention suppress self-activation of primary dendritic cells in a state in which the primary dendritic cells are thawed after cryopreservation.

[0032] The cryopreservation composition and cryopreservation method of the present invention can prevent primary dendritic cells from self-activating even in an environment (in vitro environment) without fibronectin after cryopreservation and thawing of primary dendritic cells.

[0033] The cryopreservation composition and cryopreservation method of the present invention enable in vitro culture of primary dendritic cells.

[0034] The cryopreservation composition and cryopreservation method of the present invention can implement an immunological response of primary dendritic cells in a culture state.

[0035] The cryopreservation composition and cryopreservation method of the present invention can co-culture cryopreserved primary dendritic cells with T cells so that the primary dendritic cells induce proliferation of T cells and cytokine secretion of T cells in a cultured state.

[0036] The cryopreservation composition and cryopreservation method of the present invention can contribute to the measurement of immune responses, immunological diagnosis, and development of immunological therapeutic agents for various diseases (cancer, viral infection diseases, autoimmune diseases, inflammatory diseases, organ transplantation, allergies, rheumatoid arthritis, etc.) using primary dendritic cells.

[0037]

[0038] Figure 1 shows the results of flow cytometry analysis measuring the viability of spleen dendritic cells (sDC) that were frozen for 7 days and then thawed after adding various concentrations of bovine serum (FBS) in a solution containing 10% DMSO and 10% glycerol.

[0039] Figure 2 shows the results of flow cytometry analysis of splenic dendritic cells (sDCs) after thawing, which were frozen and then thawed in a solution containing 10% DMSO and 80% FBS. CD11c was isolated from the spleen immediately after thawing. + This is the result of measuring the survival rate and the level of CD86 expression on the cell surface of freshly isolated dendritic cells (sDCs).

[0040] Figure 3 shows the results of flow cytometry analysis measuring the survival rate of splenic dendritic cells that were frozen for 7 days and then thawed in a cryopreservation composition containing bovine serum albumin (BSA) at various concentrations in a 10% DMSO and RPMI-1640 culture solution.

[0041] Figure 4 shows the results of flow cytometry analysis measuring the viability of non-cryopreserved splenic dendritic cells and the expression levels of CD11c and CD86 on the cell surface. In Example 3, splenic dendritic cells were isolated, and 200 μg / mL of fibronectin was added and cultured for 24 hours.

[0042] Figures 5 and 6 show the results of measuring the survival rate of spleen dendritic cells thawed after cryopreservation in Example 4.

[0043] Figures 7 to 10 show the results of measuring the survival rate of spleen dendritic cells thawed after cryopreservation in Example 5.

[0044] Figure 11 is a scanning electron microscope photograph of polycaprolactone (PCL) nanofibers and polyvinyl alcohol (PVA) nanofibers for co-culture of splenic dendritic cells and fibroblasts.

[0045] Figure 12 is an example of a co-culture system of splenic dendritic cells and fibroblasts.

[0046] Figure 13 shows the results of measuring the survival rate of splenic dendritic cells co-cultured with fibroblasts in Example 6.

[0047] Figure 14 shows the results of measuring the survival rate of dendritic cells isolated from the cryopreserved whole spleen cells of Example 7 after culturing them.

[0048] Figure 15 shows the results of measuring cell viability and CD86 expression after thawing dendritic cells frozen in a composition containing 10% DMSO, 80% FBS, and 200 μg / mL of fibronectin, removing dead cells, and then dispensing them onto PCL nanofiber membranes (PCL NM) and PVA nanofiber membranes (PVA NM), and culturing them with 3T3 cells for 24 hours.

[0049]

[0050] The present invention provides a composition for cryopreservation of primary dendritic cells and a cryopreservation and cell culture method using the same.

[0051] The present invention provides a composition for cryopreservation of primary dendritic cells, which comprises serum, DMSO, and fibronectin, thereby increasing the survival rate after thawing of primary dendritic cells, suppressing self-activation and enabling in vitro culture, and being usable for immunological diagnosis and development of therapeutic agents for various diseases, and a cryopreservation and cell culture method using the same.

[0052] The cryopreservation composition of the present invention comprises serum, DMSO and fibronectin.

[0053] Serum is a component of blood, the yellow liquid remaining in plasma after fibrinogen has been removed. Serum contains water, glucose, minerals, gamma globulins, and other substances. Serum contains proteins, electrolytes, antibodies, antigens, and hormones not involved in blood clotting, but excludes white blood cells, red blood cells, platelets, and clotting factors.

[0054] In one embodiment, the serum is fetal bovine serum (FBS). FBS may contain growth factors, proteins, trace elements, vitamins, hormones, and the like. It functions to preserve and maintain cells.

[0055] In one embodiment, the serum may be FCS, human AB serum, etc.

[0056] The serum may be included at a concentration of 10% to 90% (V / V). For example, the serum may be included at 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%.

[0057] The serum may be included in 100 ㎕, 150 ㎕, 200 ㎕, 250 ㎕, 300 ㎕, 350 ㎕, 400 ㎕, 450 ㎕, 500 ㎕, 550 ㎕, 600 ㎕, 650 ㎕, 700 ㎕, 750 ㎕, 800 ㎕, 850 ㎕ or 900 ㎕ per 1 mL of the cryopreservation composition.

[0058] DMSO is dimethyl sulfoxide. DMSO helps reduce cell damage caused by ice crystals during cryopreservation and storage.

[0059] DMSO may be included at a concentration of 5% to 15% (V / V). For example, DMSO may be included at a concentration of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%.

[0060] DMSO may be included in an amount of 50 ㎕, 60 ㎕, 70 ㎕, 80 ㎕, 90 ㎕, 100 ㎕, 110 ㎕, 120 ㎕, 130 ㎕, 140 ㎕ or 150 ㎕ per 1 mL of the cryopreservation composition.

[0061] Fibronectin is a glycoprotein of the extracellular matrix that inhibits the self-activation of primary dendritic cells.

[0062] Fibronectin can be included at a concentration of 100 to 300 μg / mL. Fibronectin can be included at, for example, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, or 300 μg / mL.

[0063] In one embodiment, a fibronectin stock prepared by dissolving 1-3 mg of fibronectin in 1 mL of medium may be used.

[0064] Fibronectin may be included in the cryopreservation composition at a concentration of 5% to 15% (V / V). Fibronectin may be included at 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%.

[0065] Fibronectin may be included in an amount of 50 ㎕, 60 ㎕, 70 ㎕, 80 ㎕, 90 ㎕, 100 ㎕, 110 ㎕, 120 ㎕, 130 ㎕, 140 ㎕ or 150 ㎕ per 1 mL of the cryopreservation composition.

[0066] The cryopreservation composition of the present invention may further include serum albumin.

[0067] Serum albumin may be included at 300 to 500 mg / mL. Serum albumin may be included at, for example, 300 mg / mL, 310 mg / mL, 320 mg / mL, 330 mg / mL, 340 mg / mL, 350 mg / mL, 360 mg / mL, 370 mg / mL, 380 mg / mL, 390 mg / mL, 400 mg / mL, 410 mg / mL, 420 mg / mL, 430 mg / mL, 440 mg / mL, 450 mg / mL, 460 mg / mL, 470 mg / mL, 480 mg / mL, 490 mg / mL, or 500 mg / mL.

[0068] The cryopreservation composition of the present invention may further include a medium necessary for the storage and maintenance of primary dendritic cells. The components and types of the medium are not limited to specific ones.

[0069] The cryopreservation composition of the present invention may further include a component that inhibits self-activation of primary dendritic cells.

[0070] In one embodiment, the cryopreservation composition may be comprised of 10% DMSO, 80% FBS, 10% media, and a cell pellet. 1 mL of the cryopreservation composition may comprise 100 μl of DMSO (V / V), 800 μl of FBS (V / V), and 100 μl of media and cell pellet.

[0071] In one embodiment, the cryopreservation composition may comprise 10% DMSO, 80% FBS, 200 μg / mL fibronectin, and a cell pellet. 1 mL of the cryopreservation composition may comprise 100 μl DMSO (V / V), 800 μl FBS (V / V), and 100 μl fibronectin (stock 2 mg / mL) and a cell pellet.

[0072] In one embodiment, the cryopreservation composition may be comprised of 10% DMSO, 60% FBS, 30% media, and a cell pellet. 1 mL of the cryopreservation composition may comprise 100 μl DMSO (V / V), 600 μl FBS (V / V), and 300 μl media and a cell pellet.

[0073] In one embodiment, the cryopreservation composition may be comprised of 10% DMSO, 60% FBS, 200 μg / mL fibronectin, and 20% medium and cell pellet. 1 mL of the cryopreservation composition may be comprised of 100 μl DMSO (V / V), 600 μl FBS (V / V), and 100 μl fibronectin (stock 2 mg / mL), and 200 μl medium and cell pellet.

[0074] The method for cryopreserving primary dendritic cells of the present invention comprises the steps of freezing primary dendritic cells or tissues (such as spleen) containing the cells in a state of being immersed in the cryopreservation composition, thereafter, the step of thawing the primary dendritic cells or tissues, and the step of culturing the thawed cells or cells obtained from the thawed tissues.

[0075] The present invention provides a method for in vitro culturing of primary dendritic cells, comprising the steps of freezing and preserving primary dendritic cells or tissues (such as spleen) containing the same in a cryopreservation composition, then thawing the primary dendritic cells or tissues, and culturing cells obtained from the thawed cells or tissues.

[0076] Primary dendritic cells or tissues containing them can be cultured in a medium containing fibronectin after thawing. Fibronectin can inhibit the self-activation of primary dendritic cells.

[0077] Primary dendritic cells or tissues containing them can be cultured in a medium that does not contain fibronectin after thawing. Using the cryopreservation composition of the present invention, self-activation of primary dendritic cells can be suppressed even when cultured in a medium that does not contain fibronectin.

[0078] Dendritic cells or tissues containing them can be co-cultured with fibroblasts after thawing. Co-culture with fibroblasts allows them to share beneficial substances, such as cytokines, which aids cell growth.

[0079] Primary dendritic cells or tissues containing them can be co-cultured with fibroblasts by seeding primary dendritic cells on a first support containing polyvinyl alcohol (PVA) nanofibers or polycaprolactone (PCL) nanofibers after thawing, and seeding fibroblasts on a second support containing polycaprolactone (PCL) nanofibers.

[0080] The first and second supports can be stacked and separated within the incubator, allowing the two cells to be physically separated and indirectly co-cultured.

[0081] Hereinafter, the present invention will be described in more detail with examples.

[0082]

[0083] Example

[0084] Example 1. Isolation and cryopreservation of splenic dendritic cells.

[0085] Mouse spleen dendritic cells were isolated from the spleen of C57BL / 6 mice (Orient). EDTA-BSS (Balanced Salt Solution) buffer, collagenase, and DNAse lysis solution were added. The cells were then finely chopped with surgical scissors, mixed with 6 ml of collagenase treatment solution, and incubated for 30 minutes. The separated whole spleen tissue was filtered using a 70 μM cell strainer (Falcon) and centrifuged at 1800 rpm for 5 minutes to isolate cells. Red blood cells were removed by adding red blood cell lysis buffer (Sigma Aldrich), and washed twice with RPMI-1640 medium (Wellgen) containing 10% fetal bovine serum (FBS, Sigma Aldrich). Dendritic cells were CD11c + Antibody-conjugated microbeads (Miltenyi Biotec) were reacted and isolated using CD11c Microbeads Ultrapure (MACS, Miltenyi Biotec). 1.2 × 10 8 Cells were obtained and dendritic cells were 7.5×10 6 Cells were isolated. Primary dendritic cells isolated from the spleen were 3×10 6 Depending on the number of cells, 1 mL of solution was prepared by adding different concentrations of FBS to RPMI-1640 culture medium containing 10% DMSO, and the vial was frozen and stored in a nitrogen tank for 1 week. The frozen cells were thawed in a 37°C shaking incubator before use.

[0086] In Fig. 1, the survival and apoptosis of splenic dendritic cells (sDCs) after thawing were stained with 7-aminoactinomycin-D (7-AAD) and annexin V-FITC and measured using a flow cytometer (MACSQuant VYB flow cytometer, Miltenyi Biotec.). The results measured using a flow cytometer were analyzed using FlowJo v10 (Tree Star, Ashland). When FBS was not added to the cryopreservation medium, the cell viability was less than 10%, but when the concentration of FBS was increased, the cell viability increased, and when 80% FBS was added, the viability was 62%. * indicates statistical significance (P <0.05 or less) compared to the control group (0% FBS) (n=3).

[0087]

[0088] Example 2. Measurement of dendritic cell activation using a flow cytometer

[0089] To determine whether self-activation occurred in cultured primary dendritic cells isolated from the spleen, the expression level of CD86 on the surface of dendritic cells was analyzed using flow cytometry. The level of CD86 expression was expressed as the percentage of cells stained with CD11c using a PE-conjugated CD86 antibody (CD86-PE).

[0090] In Figure 2, dendritic cells immediately after isolation from the spleen (in an unstimulated state) have very low expression of the co-stimulatory molecule CD86. In Example 1, the expression level of CD86 on the surface of dendritic cells that were frozen and then thawed in a composition containing 10% DMSO and 80% FBS was as low as 4%.

[0091] In Fig. 3, primary splenic dendritic cells (sDC) isolated from the spleen were dispensed into compositions containing 10% DMSO and various concentrations of BSA, and then frozen and preserved in a nitrogen tank for 7 days. After thawing, the cell viability was measured. When 100 mg / mL of BSA was added, the cell viability significantly increased. * indicates statistical significance (P <0.05 or less) compared to the control group (0 mg / mL BSA) (n=3).

[0092]

[0093] Example 3. Measurement of survival rate after culture of spleen dendritic cells.

[0094] CD11c isolated from spleen was added to RPMI-1640 medium containing 10% FBS at a concentration of 200 μg / mL with fibronectin (Sigma Aldrich). + 2×10 dendritic cells were cultured 5 After dispensing at / mL and culturing for 24 hours, the cell viability and CD86 expression level were measured using a flow cytometer.

[0095] As shown in Fig. 4, when 10% FBS and 200 μg / mL of fibronectin were added to RPMI-1640 medium, the cell viability was maintained at a high level of 57%, and the increase in CD86 expression was also suppressed (i.e., maintained in an inactive state). On the other hand, when 10% FBS and 200 μg / mL of fibronectin were not used, only 13% of cells survived, and cell activation was significantly increased.

[0096]

[0097] Example 4. Measurement of the viability of splenic dendritic cells thawed after cryopreservation.

[0098] Splenic dendritic cells (sDCs) were frozen for 7 days in cryopreservation compositions containing 0%, 40%, 60%, or 80% FBS, 10% DMSO, and 200 μg / mL of fibronectin. The viability of the cells obtained after thawing was measured using a flow cytometer. As a result, as shown in Fig. 5A, when 40%, 60%, or 80% FBS was included, the cell viability was maintained at high levels of 64%, 66%, and 71%, respectively.

[0099] However, when the cryopreservation composition contained FBS and fibronectin at concentrations of 0%, 40%, 60%, or 80% but did not contain 10% DMSO (i.e., contained 10% glycerol instead of 10% DMSO), the cell viability was confirmed to drop sharply to around 20%, as shown in Fig. 5A. Therefore, it was found that the cryopreservation composition must contain 10% DMSO.

[0100] In addition, when 200 ㎍ / mL of fibronectin was added to a cryopreservation composition containing 80% FBS and 10% DMSO, it was confirmed that the survival rate increased by 9% (approximately 15% based on 62%) from 62% to 71% due to the addition of fibronectin, as shown in Fig. 5B. Therefore, it was found that fibronectin had a significant effect on the cell survival rate upon thawing after cryopreservation. * indicates statistical significance as P <0.05 or less compared to the control group (-Fibronectin) (n=3).

[0101] In a cryopreservation composition comprising FBS at a concentration of 0%, 40%, 60%, or 80%, 10% DMSO, and 200 μg / mL of fibronectin, when 100 mg / mL, 200 mg / mL, or 400 mg / mL of bovine serum albumin (BSA) was added instead of FBS, the viability of primary dendritic cells thawed after cryopreservation was confirmed to be 51 to 60% (Fig. 6).

[0102]

[0103] Example 5. Measurement of the viability of dendritic cells isolated after cryopreservation and thawing of total splenocytes.

[0104] Cryopreservation compositions were prepared containing RPMI-1640 medium (RPMI-1640, 1 mM sodium pyruvate, 2% penicillin / streptomycin, 100 μM β-mercaptoethanol, 5× HEPES buffer, and 0.2% sodium bicarbonate), 10% DMSO or 10% glycerol, and 0%, 20%, 40%, 60%, or 80% FBS.

[0105] Total splenocytes isolated from each spleen were added to the above cryopreservation composition at a concentration of 3×10 7 Primary dendritic cells were obtained from the whole cells obtained by dispensing at a density of 100 μg / mL and freezing the whole spleen cells for 7 days, then thawing them using CD11c microbeads. The viability of the dendritic cells was measured by flow cytometry.

[0106] As shown in Fig. 7, when a cryopreservation composition containing 10% DMSO was used, the viability of splenic dendritic cells increased to 60% as the concentration of FBS increased. On the other hand, when a cryopreservation composition containing 10% glycerol was used, the viability of splenic dendritic cells was up to 25%.

[0107] In this example, splenic primary dendritic cells (sDC) were obtained from the whole cells isolated after freezing and storing the whole spleen cells for 7 days in a cryopreservation composition containing 200 μg / mL of fibronectin. The dendritic cells exhibited a survival rate of up to 74% (Fig. 8). The 74% survival rate represents a 14% increase compared to the case where fibronectin was not added (60% in Fig. 7). * indicates statistical significance as P <0.05 or less compared to the control group (-Fibronectin) (n=3).

[0108] For Fig. 9, cryopreservation compositions containing 10% DMSO, 20% FBS, and various concentrations of BSA in RPMI-1640 medium or DMEM medium were prepared. 1 × 10 whole spleen cells isolated from the spleen were 8 / mL in a density of 100 μg / mL, and the whole spleen cells were frozen for 7 days, then thawed to obtain primary dendritic cells. The survival rate of dendritic cells was measured using a flow cytometer. As shown in Figure 9, the cell survival rate increased similarly as the concentration of FBS increased in both types of media. For example, when 400 mg / mL of BSA was added, the survival rates of spleen cells were 65% and 67%, respectively.

[0109] When 200 μg / mL of fibronectin was added to the cryopreservation composition of Fig. 9 and the survival rate of primary dendritic cells was measured using the same method, in this case, the survival rate did not increase significantly due to the addition of fibronectin (Fig. 10).

[0110]

[0111] Example 6. Measurement of the viability of splenic dendritic cells co-cultured with fibroblasts.

[0112] (1) Manufacturing of cell support

[0113] Polycaprolactone (PCL) nanofibers and polyvinyl alcohol (PVA) nanofibers to be used as supports for co-culturing splenic dendritic cells and fibroblasts according to the present invention were produced (Fig. 11).

[0114] Polycaprolactone (PCL) nanofibers were prepared according to the method disclosed in Korean Patent No. 10-1684698. Polycaprolactone (PCL, Mw=80,000, Sigma-Aldrich) was dissolved in chloroform (Sigma-Aldrich) at a concentration of 15%. The PCL solution (5 mL) was spun at 10 kV using an electrospinning machine (NanoNC Co.) with a 25G metal syringe at a spinning distance of 20 cm and a spinning rate of 8 μl / min.

[0115] Polycaprolactone (PVA) nanofibers were also prepared according to the method of the above-mentioned Korean patent. Polyvinyl alcohol (PVA, Mw=89,000-98,000, Sigma-Aldrich) and polyacrylic acid (PAA, Mw=2000, Sigma-Aldrich) were dissolved at concentrations of 10% (w / v) and 0.2% (w / v) at 85°C for 24 h. Glutaraldehyde (GA, 2%, Sigma-Aldrich) was added to the dissolved PVA solution and mixed for 30 min to prepare an electrospinning solution. 4 mL of the electrospinning solution was electrospun at 10 kV using two 27 G metal syringes at a distance of 10 cm at a flow rate of 8 μl / min. The electrospun PVA / PAA / GA nanofibers were heat-treated at 60°C for 1 min. To crosslink the nanofibers, they were treated with hydrochloric acid vapor for 60 seconds. The PVA nanofiber membrane was immersed in a culture medium for more than 12 hours before use and sterilized by exposure to ultraviolet light for 12 hours.

[0116]

[0117] (2) Coculture of splenic dendritic cells and fibroblasts

[0118] As shown in Fig. 12, a culture system was manufactured in which PCL nanofiber membranes and PVA nanofiber membranes were attached to the upper insert of the transwell for co-culture of two cells, and PCL nanofiber membranes were attached to the base of the lower layer. NIH3T3 cells (1 × 10) which are fibroblasts were placed in the lower layer of the culture system. 6 / mL) and cultured for 3 days, then frozen and thawed dendritic cells (2 × 10 5 / mL) were dispensed and immediately assembled into two layers, followed by incubation for 24 hours. Dendritic cells were obtained after cryopreservation in a composition containing 10% DMSO, 80% FBS, and 200 μg / mL fibronectin (survival rate 60%).

[0119] And prepare a general culture plate and thaw dendritic cells (2 × 10 5 / mL) and fibroblast NIH3T3 cells (1 × 10 6 / mL) was dispensed and cultured in the same manner.

[0120] As shown in Figure 13, 2 × 10 spleen dendritic cells were cultured in a culture plate containing RPMI-1640 medium (without fibronectin). 5 When cultured for 24 hours after dispensing at a density of 100 μg / mL, the survival rate was 35% and the self-activated cells were 32%. When fibronectin was added to the culture medium at a concentration of 200 μg / mL, the survival rate increased slightly to 42%, but the self-activated cells were significantly suppressed to 1%. When PCL nanofibers and PVA nanofibers were attached to a culture plate and splenic dendritic cells were cultured, the survival rate and the degree of activation inhibition were similar to when splenic dendritic cells were cultured on a culture plate. This shows that the splenic dendritic cells according to the present invention are capable of two-dimensional culture on a plate as well as three-dimensional culture on a nanofiber support.

[0121]

[0122] Example 7. Measurement of cell viability after culturing dendritic cells isolated from cryopreserved whole spleen cells.

[0123] In Figure 14, 3×10 whole spleen cells were cultured in a composition (1 mL) containing 10% DMSO, 80% FBS, and 200 μg / mL fibronectin. 7 After freezing and preservation at / mL, thawed after 7 days and inoculated with 1×10 CD11c antibody-conjugated microbeads. 6 CD11c in dogs + Splenic dendritic cells were isolated. Dendritic cells (2 × 10) isolated from cryopreserved whole spleen cells 5 When the cells were seeded on a culture plate and cultured in RPMI-1640 medium for 24 hours, the cell viability was very low at about 22%, and 54% of the viable cells showed CD86 expression. When fibronectin was added under the culture plate culture condition, the cell viability significantly increased and self-activation was significantly inhibited.

[0124]

[0125] Example 8. Measurement of the viability of cryopreserved dendritic cells after co-culturing them with fibroblasts.

[0126] CD11c isolated from the spleen in Fig. 15 + Dendritic cells were cultured at 1×10 in a composition (1 mL) containing 10% DMSO, 80% FBS, and 200 μg / mL fibronectin. 6 After freezing and preserving at / mL, they were thawed after 7 days. The viability of thawed dendritic cells was confirmed, and dead cells were removed using Dead Cell Removal Microbeads (Miltenyi Biotec), and only surviving cells were collected at 2 × 10 5 The upper layer of PCL or PVA nanofiber membranes for co-culture was prepared by seeding the cells. NIH3T3 fibroblasts were seeded at 1 × 10 on the lower layer of PCL nanofiber membrane for co-culture. 6After seeding and culturing for 3 days, the dendritic cells were assembled with the seeded upper membrane layer and co-cultured for 24 hours. When the dendritic cells were seeded on the upper layer PCL or PVA nanofibers and co-cultured with fibroblasts on the lower layer, cell viability increased significantly and the dendritic cells were maintained in an inactive form.

[0127] When cryopreserved dendritic cells were cocultured, cell viability was not significantly increased by the addition of fibronectin. These results suggest that the survival and activity of primary dendritic cells may be stabilized by the secretion of extracellular matrix components, such as fibronectin, or other unidentified soluble factors from cocultured fibroblasts.

Claims

1. A method for culturing primary dendritic cells, comprising the step of cryopreserving primary dendritic cells or tissues containing the same in a composition comprising serum, DMSO and fibronectin.

2. A method for culturing primary dendritic cells, comprising the step of cryopreserving primary dendritic cells or tissues containing them in a composition comprising serum, DMSO and albumin.

3. A method for culturing primary dendritic cells according to claim 1 or 2, further comprising the step of culturing the primary dendritic cells or tissues containing them in a medium containing fibronectin after thawing them.

4. A method for culturing primary dendritic cells according to claim 1 or 2, further comprising the step of culturing the primary dendritic cells or tissues containing them in a medium not containing fibronectin after thawing them.

5. A method for culturing primary dendritic cells according to claim 1 or 2, further comprising the step of thawing the primary dendritic cells or a tissue containing the primary dendritic cells and then co-culturing them with fibroblasts.

6. A method for culturing primary dendritic cells, further comprising: a step of thawing the primary dendritic cells or tissues containing them according to claim 1 or 2; and a step of dispensing the primary dendritic cells onto a first support comprising polyvinyl alcohol (PVA) nanofibers or polycaprolactone (PCL) nanofibers; and a step of dispensing fibroblasts onto a second support comprising polycaprolactone (PCL) nanofibers to co-cultivate the primary dendritic cells with the fibroblasts.

7. A method for culturing primary dendritic cells according to claim 6, wherein the first support and the second support are spaced apart and stacked within an incubator.

8. A method for culturing primary dendritic cells according to claim 2, wherein the composition further comprises fibronectin.

9. A method for culturing primary dendritic cells according to claim 1 or 2, wherein the serum contains 10% to 90% FBS.

10. A method for culturing primary dendritic cells according to claim 1 or 2, wherein the DMSO is 5% to 15% DMSO.

11. A method for culturing primary dendritic cells according to claim 1 or 8, wherein the fibronectin is contained in an amount of 100 to 300 μg / mL.

12. A method for culturing primary dendritic cells according to claim 2, wherein the albumin is contained in an amount of 300 to 500 mg / mL.

13. A composition for cryopreservation of primary dendritic cells or tissues containing them, comprising serum, DMSO and fibronectin.

14. A composition for cryopreservation of primary dendritic cells or tissues containing them, comprising serum, DMSO and albumin.

15. A composition for cryopreservation of primary dendritic cells or tissues containing them, wherein the serum comprises 10% to 90% FBS according to claim 13 or 14.

16. A composition for cryopreservation of primary dendritic cells or tissues containing them, wherein the DMSO according to claim 13 or 14 is 5% to 15% DMSO.

17. A composition for cryopreservation of primary dendritic cells or tissues containing them, wherein the fibronectin according to claim 13 is contained in an amount of 100 to 300 μg / mL.

18. A composition for cryopreservation of primary dendritic cells or tissues containing them, wherein the albumin is contained in an amount of 300 to 500 mg / mL according to claim 14.

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