An electrotransfection method for DC cells

Abstract

This invention discloses a method for electroporation culture of dendritic cells (DCs), comprising the following steps: On day 0, PBMC suspension is seeded into a T175 culture flask, and a first culture medium (including basal medium, rhGM-CSF, IL-4, and VC components) is added and the flask is placed in an incubator for culture; On day 3, the first culture medium is added again and culture continues; On day 5, the culture medium in the culture flask is aspirated, and a second culture medium (including basal medium, rhGM-CSF, IL-4, IL-6, TNF-α, IL-1β, and VC) is added; On day 7, the cells are transferred to an electroporation cuvette, electroporation buffer is added, electroporation is performed, and then the second culture medium is added again and culture continues; On day 10, culture is stopped, the cells are collected, centrifuged, and the supernatant is removed to obtain DCs. Electroporation followed by culture significantly increases the viability of mature DCs, reaching approximately 90%; the mean fluorescence intensity (MFI) of mature DCs is significantly increased (up to 10⁴²¹), and the antigen expression level of DCs is good.

Description

Technical Field

[0001] This invention relates to the field of cell culture preparation technology, and in particular to a method for culturing DC cells using PBMCs via electroporation. Background Technology

[0002] Natural killer (NK) cells in the immune system attack cancer cells upon detection, simultaneously recruiting dendritic cells (DCs) to record their characteristics and relay this information to killer (T) cells in the body. Cytotoxic T cells possess a powerful ability to kill cancer cells, but they lack the ability to recognize them. Dendritic cells are the most potent professional antigen-presenting cells (APCs), often referred to as the "sentinels" of the immune system and initiators of the body's immune response. Therefore, mature dendritic cells (DCs) can "teach" T cells how to recognize cancer cells; they also promote the proliferation and differentiation of B cells, participating in humoral immune responses.

[0003] Current cell immunotherapy is developed based on the "anti-cancer" characteristics of the aforementioned types of immune cells, such as CAR-T cell immunotherapy, NK cell immunotherapy, and dendritic cell immunotherapy. Among them, dendritic cell immunotherapy is the most unique, as it acts on specific cancer cells with virtually no side effects. Furthermore, the most valuable aspect of DC vaccines lies in their ability to achieve both the elimination of cancer cells and the prevention of recurrence.

[0004] Currently, the conventional method for preparing dendritic cells (DCs) in vitro involves the directed induction, differentiation, and expansion of DC precursor cells from peripheral blood or umbilical cord blood using cytokines. Generally, isolated mononuclear cells are first added to serum-free DC culture medium, followed by the addition of appropriate amounts of cytokines such as GM-CSF and IL-4 to culture the mononuclear cells into DCs. Then, appropriate tumor antigens are added for further culture, allowing the DCs to be loaded with tumor antigens. However, this conventionally prepared DC method contains a large proportion of immature DCs, and exhibits low cell viability and antigen expression, making it unsuitable for in vitro culture and widespread clinical application. Summary of the Invention

[0005] Based on the above problems, the present invention provides a method for culturing DC cells using electroporation. This method can obtain mature DC cells with high activity and high antigen expression, and the electroporation culture efficiency is high, which has clinical application value.

[0006] The technical solution of the present invention is as follows:

[0007] A method for electroporation culture of DC cells includes the following steps:

[0008] On day 0, PBMCs were resuspended in basal medium and inoculated into T175 culture flasks, and supplemented with the first culture medium and 5% blood substitute; the T175 culture flasks were placed in an incubator at 37°C and 5% CO2 for static culture; wherein, the first culture medium includes the basal medium, and the basal medium is supplemented with rhGM-CSF at a final concentration of 500~1500 IU / mL, IL-4 at a final concentration of 50~1500 IU / mL and VC at a final concentration of 50~1500 IU / mL;

[0009] On the third day, the first culture medium and 5% blood were added to the culture flask, and the culture was continued.

[0010] On day 5, the culture waste liquid in the culture flask was aspirated, and a second culture medium was added, with the volume of the aspirated culture waste liquid to the volume of the added second culture medium in a 1:1 ratio, and the culture continued. The second culture medium included the basal culture medium, which contained rhGM-CSF at a final concentration of 500-1500 IU / mL, IL-4 at a final concentration of 50-1500 IU / mL, IL-6 at a final concentration of 50-1500 IU / mL, TNF-α at a final concentration of 10-100 IU / mL, and VC at a final concentration of 50-1500 IU / mL.

[0011] On day 7, cells were collected and transferred to an electroporation cuvette, and electroporation buffer was added before electroporation. The electroporated cell solution was then transferred to a T25 culture flask containing 4 mL of the second culture medium, and cultured in an incubator.

[0012] On day 10, cell culture was stopped, cells were collected and centrifuged, the supernatant was removed, and the cell pellet was washed repeatedly with PBS 2-3 times to obtain DC cells.

[0013] In the above electroporation culture method for DC cells, on day 7, the cell slurry is collected and further processed as follows:

[0014] First, aspirate the culture waste liquid from the culture flask, rinse the culture flask with PBS and then discard the PBS rinse waste liquid; second, add recombinant trypsin to the culture flask and place it in an incubator to digest the cells on the inner wall of the culture flask. After the cells are digested, add the second culture medium to stop the digestion, collect the digested cells and centrifuge them, remove the supernatant and collect the cell pellet.

[0015] Preferably, in the above-mentioned electroporation culture method for DC cells, the amount of recombinant trypsin added to the digested cells is 2 mL; the digested cells are then centrifuged at 400 g for 5 min.

[0016] Preferably, in the above-described electroporation culture method for DC cells, on day 7, the electroporation buffer is any one of Opti-MEM, DMEM, or EBEL.

[0017] Preferably, in the above-mentioned electroporation culture method for DC cells, on day 7, the concentration is 1.0 × 10⁻⁶. 7 Add 100 μL of the electroporation buffer to a cell concentration of 1 cell / mL.

[0018] In the above-mentioned electroporation culture method for DC cells, on day 7, the electroporation process parameters were set as follows: voltage 220V~320V, pulse time 600μs~1600μs, interval time 612ms, and electroporation times 3 times.

[0019] Preferably, in the above-mentioned electroporation culture method for DC cells, the second culture medium needs to be preheated to 37°C on day 7.

[0020] Preferably, in the above-described electroporation method for DC cells, on day 7, after transferring the electroporated cell solution to a T25 culture flask, the following treatment is also included:

[0021] The cell culture vessel was rinsed multiple times with the second culture medium, and the rinsed cell solution was transferred to a T25 culture flask.

[0022] Preferably, in the above-described electroporation culture method for DC cells, on day 10, between stopping cell culture and collecting cells, the following treatment is also included:

[0023] First, aspirate the culture waste liquid from the culture flask, rinse the culture flask with PBS, and then discard the PBS rinsing waste liquid. Second, add 2 mL of recombinant trypsin to the culture flask and place it in an incubator to digest the cells on the inner wall of the culture flask. After the cells are digested, add the second culture medium to stop the digestion.

[0024] In the above-mentioned electroporation culture method for DC cells, the basal culture medium is any one of T551H3, DMEM, or RPMI-1640.

[0025] Compared with the prior art, the electroporation method for culturing DC cells provided by the present invention has the following advantages:

[0026] 1. During DC cell culture, electroporation followed by further culture significantly increases the viability of mature DC cells, reaching approximately 90%. Simultaneously, the vitamin C component in the DC culture medium effectively protects the viability of DC cells after electroporation.

[0027] 2. During DC cell culture, electroporation followed by culturing significantly increased the mean fluorescence intensity (MFI) of mature DC cells (up to 10⁴²¹), indicating good antigen expression in DC cells.

[0028] 3. During DC cell culture, electroporation of cells before culturing can achieve an electroporation efficiency of up to 86.01%. This is beneficial for the culture of mature DC cells (with an expansion factor of more than 2.3 times) and greatly shortens the culture time of mature DC cells. Attached Figure Description

[0029] Figure 1 Bar chart showing the expansion fold of DC cells cultured on day 10 in Examples 1 to 15;

[0030] Figure 2 The bar chart shows the DC cell viability on day 10 of Examples 1 to 15. Detailed Implementation

[0031] The preferred embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0032] I. Isolation of mononuclear cells from peripheral blood

[0033] The mononuclear cells used in this invention for culturing DC cells are obtained from peripheral blood, and the specific procedure is as follows:

[0034] Collect 100ml of peripheral blood from healthy individuals meeting the standards, add anticoagulant, and centrifuge at 600g for 10min to separate plasma (after placing the plasma at 56°C for 20min, freezing at -20°C for 15min, then centrifuging at 3000g for 10min to separate protein, filter through a 0.25uM filter, and store at -20°C for later use, or briefly at 4°C for later use); add the same volume of PBS as the plasma, spread the solution on the lymphocyte separation medium, centrifuge at 600g for 30min, aspirate the white membrane, wash twice with PBS to obtain peripheral blood mononuclear cells (PBMCs) for later use.

[0035] II. Preparation of DC culture medium

[0036] The culture medium used for culturing DC cells in this invention includes a first culture medium and a second culture medium. The first and second culture media are added according to the culture time. Both culture media contain a basal culture medium, and based on this basal culture medium, cytokines and / or nutrients are added accordingly. The specific preparation is as follows:

[0037] First culture medium: Based on the basal culture medium, with added rhGM-CSF at a final concentration of 500~1500 IU / mL, IL-4 at a final concentration of 50~1500 IU / mL, and VC at a final concentration of 50~1500 IU / mL.

[0038] Second culture medium: Based on the basal culture medium, supplemented with rhGM-CSF at a final concentration of 500~1500 IU / mL, IL-4 at a final concentration of 50~1500 IU / mL, IL-6 at a final concentration of 50~1500 IU / mL, TNF-α at a final concentration of 1000~2000 IU / mL, IL-1β at a final concentration of 10~100 IU / mL, and VC at a final concentration of 50~1500 IU / mL.

[0039] In the above DC cell culture medium preparation scheme, the basal culture medium is any one of GT-T551H3, DMEM or RPMI-1640; the same basal culture medium is used for the culture of the same batch of DCs.

[0040] Preferably, the first and second culture media may also include, but are not limited to, any of the following ratios:

[0041] Group 1

[0042] First culture medium: GT-T551H3 basal culture medium with added rhGM-CSF at a final concentration of 500 IU / mL, IL-4 at a final concentration of 1500 IU / mL and VC at a final concentration of 50 IU / mL.

[0043] The second culture medium: GT-T551H3 basal medium supplemented with rhGM-CSF at a final concentration of 500 IU / mL, IL-4 at a final concentration of 1500 IU / mL, IL-6 at a final concentration of 50 IU / mL, TNF-α at a final concentration of 2000 IU / mL, IL-1β at a final concentration of 100 IU / mL, and VC at a final concentration of 50 IU / mL.

[0044] Group 2

[0045] First culture medium: DMEM basal medium supplemented with rhGM-CSF at a final concentration of 1500 IU / mL, IL-4 at a final concentration of 50 IU / mL, and VC at a final concentration of 1500 IU / mL.

[0046] The second culture medium is DMEM basal medium supplemented with rhGM-CSF at a final concentration of 1500 IU / mL, IL-4 at a final concentration of 50 IU / mL, IL-6 at a final concentration of 1500 IU / mL, TNF-α at a final concentration of 1000 IU / mL, IL-1β at a final concentration of 10 IU / mL, and VC at a final concentration of 1500 IU / mL.

[0047] Group 3

[0048] First culture medium: RPMI-1640 basal medium supplemented with rhGM-CSF at a final concentration of 800 IU / mL, IL-4 at a final concentration of 1000 IU / mL, and VC at a final concentration of 500 IU / mL.

[0049] The second culture medium consisted of RPMI-1640 basal medium supplemented with rhGM-CSF at a final concentration of 800 IU / mL, IL-4 at a final concentration of 1000 IU / mL, IL-6 at a final concentration of 1000 IU / mL, TNF-α at a final concentration of 1500 IU / mL, IL-1β at a final concentration of 60 IU / mL, and VC at a final concentration of 500 IU / mL.

[0050] Group 4

[0051] First culture medium: RPMI-1640 basal medium supplemented with rhGM-CSF at a final concentration of 1200 IU / mL, IL-4 at a final concentration of 300 IU / mL, and VC at a final concentration of 300 IU / mL.

[0052] The second culture medium was RPMI-1640 basal medium supplemented with rhGM-CSF at a final concentration of 1200 IU / mL, IL-4 at a final concentration of 300 IU / mL, IL-6 at a final concentration of 300 IU / mL, TNF-α at a final concentration of 1600 IU / mL, IL-1β at a final concentration of 30 IU / mL, and VC at a final concentration of 300 IU / mL.

[0053] III. DC Cell Culture

[0054] On day 0, firstly, PBMCs were resuspended in basal medium, and the resulting cell suspension was inoculated into T175 culture flasks and basal medium was added for 2 hours. Subsequently, the basal medium in the culture flasks was aspirated, and according to the volume of basal medium aspirated, the first culture medium prepared by any one of the above-mentioned groups and 5% blood substitute (the volume ratio of the first culture medium to the blood substitute is 9:1, and the blood substitute is commercially available) were added at a volume ratio of 2:1. Finally, the T175 culture flasks were placed in an incubator at 37°C and 5% CO2 for static culture.

[0055] On the third day, based on the volume of culture medium in the culture flask, add the first culture medium and 5% blood substitute to the culture flask at a volume ratio of 1:1 (the volume ratio of the first culture medium to the blood substitute is 9:1), and continue culturing;

[0056] On the 5th day, the culture waste liquid in the culture bottle was aspirated. According to the volume ratio of the aspirated amount to the replenished amount of 1:1, the second culture medium prepared in the second item above (which is the same group as the first culture medium) was added accordingly, and the culture was continued.

[0057] On day 7, cells were collected and transferred to electroporation cuvettes, and electroporation buffer was added. GFP-mRNA (mRNA emits green fluorescence by translating into GFP; the expression level of GFP-mRNA can be detected by flow cytometry for easy visual observation during testing; this is only for experimental purposes and has no other technical significance) was added before electroporation. The electroporated cell solution was then transferred to T25 culture flasks containing 4 mL of secondary culture medium, and cultured in an incubator.

[0058] On day 10, cell culture was stopped, cells were collected, the supernatant was removed by centrifugation, and the cell pellet was washed repeatedly with 5 mL PBS 2-3 times to obtain highly active and mature DC cells.

[0059] Preferably, in the above-mentioned electroporation culture method for DC cells, on day 7, after collecting the cell slurry, the following treatment is also required:

[0060] First, aspirate the culture waste liquid from the culture flask, rinse the culture flask with PBS, and then discard the PBS rinse waste liquid. Second, add 2 mL of recombinant trypsin to the culture flask and place it in an incubator to digest the cells on the inner wall of the culture flask. After most of the cells (about 90-98%) have been digested, add the second culture medium to stop the digestion. Centrifuge at 400g for 5 min to digest the cells, remove the supernatant, and collect the cells.

[0061] Preferably, in the above-mentioned electroporation culture method for DC cells, on day 7, the electroporation buffer is any one of Opti-MEM, DMEM or EBEL (commercially available, manufacturer: Yida Biotechnology; model: H10305).

[0062] Preferably, in the above-mentioned electroporation culture method for DC cells, on day 7, the concentration is 1.0 × 10⁻⁶. 7 Add 100 μL of electroporation buffer to a cell concentration of 1 cell / mL, and add 1~8 μg of GFP-mRNA.

[0063] Preferably, in the above-mentioned electroporation culture method for DC cells, on day 7, the electroporation process parameters are set as follows: voltage 220V~320V, pulse time 600μs~1600μs, interval time 612ms, and electroporation times 3 times.

[0064] Preferably, in the above-mentioned electroporation culture method for DC cells, on day 7, 4 ml of the second culture medium needs to be preheated to 37°C.

[0065] Preferably, in the above-described electroporation method for DC cells, on day 7, after transferring the electroporated cell solution to a T25 culture flask, the following treatment is also included:

[0066] The cell culture vessel was rinsed multiple times with the second culture medium, and the rinsed cell solution was transferred to a T25 culture flask.

[0067] Preferably, in the above-mentioned electroporation culture method for DC cells, the following treatment is also included between stopping cell culture and collecting cells on day 10:

[0068] First, aspirate the culture waste liquid from the T25 culture flask, rinse the culture flask with PBS, and then discard the PBS rinse waste liquid. Second, add 2 mL of recombinant trypsin to the culture flask and place it in a 37°C incubator to digest the cells on the inner wall of the culture flask for 3 min. After most of the cells (about 90-98%) have been digested, add the second culture medium to stop the digestion.

[0069] The technical solution of the present invention will be described in detail below through specific embodiments or comparative examples.

[0070] In the following embodiments, the PBMCs used were obtained by peripheral blood separation as described in the first item above.

[0071] (I) Culture of DC cells

[0072] Example 1 (Comparative Example 1)

[0073] This example serves as a control group, where no electroporation treatment of DC cells is required during DC cell culture.

[0074] 1.1 Preparation of DC culture medium

[0075] First culture medium: Take 100 mL of RPMI-1640 basal culture medium and add rhGM-CSF with a final concentration of 800 IU / mL, IL-4 with a final concentration of 1000 IU / mL and VC with a final concentration of 1000 IU / mL.

[0076] Second culture medium: Take 1000 mL of RPMI-1640 basal culture medium and add rhGM-CSF with a final concentration of 800 IU / mL, IL-4 with a final concentration of 1000 IU / mL, IL-6 with a final concentration of 800 IU / mL, TNF-α with a final concentration of 500 IU / mL, IL-1β with a final concentration of 60 IU / mL, and VC with a final concentration of 1000 IU / mL.

[0077] 1.2 DC cell culture

[0078] On day 0, first, take 20 mL of RPMI-1640 and resuspend 2.0 × 10⁻⁶ cells. 7 PBMC cells were seeded; then, the cell suspension was seeded into a T175 culture flask and 20 mL of RPMI-1640 was added and cultured for 2 h; subsequently, the RPMI-1640 in the culture flask was aspirated and 9 mL of the first culture medium prepared in step 1.1 of this embodiment and 1 mL of 5% blood were added; finally, the T175 culture flask was placed in an incubator at 37°C and 5% CO2 for static culture.

[0079] On the third day, add 9 mL of the first culture medium and 1 mL of 5% blood substitute to the culture flask and continue culturing;

[0080] On day 5, the culture waste liquid in the culture bottle was aspirated, and 20 mL of the second culture medium prepared in step 1.1 of this embodiment was added to continue culturing;

[0081] On day 7, first, the culture waste in the T175 culture flask was aspirated, and the culture flask was rinsed with 5 mL of PBS, after which the PBS rinse waste was discarded. 2 mL of recombinant trypsin was added to the culture flask and the flask was placed in a 37°C incubator for digestion for 3 min. After most of the cells were digested, 10 mL of the second culture medium was added to stop the digestion. The digested cells were centrifuged at 400g for 5 min, and the supernatant was removed. Subsequently, the cells were collected and transferred to a T25 culture flask, and the second culture medium preheated to 37°C was added. Finally, the T25 flask was transferred to an incubator for further culture.

[0082] On day 10, cell culture was stopped, and the culture waste liquid was poured out from the waste liquid outlet of the T25 culture flask. Then, 5 mL of PBS was added for rinsing, and the PBS rinsing solution was poured out. Then, 2 mL of recombinant trypsin was added to the culture flask, and the cells were digested in a 37 ℃ incubator for 3 min. The cells were observed under a microscope. After the cells were digested, a second culture medium was added to stop the digestion. The cells were collected, centrifuged at 400g for 5 min, the supernatant was removed, and the cell pellet was washed repeatedly with PBS 2-3 times to obtain highly active and mature DC cells.

[0083] Example 2

[0084] 2.1 Preparation of DC culture medium

[0085] First culture medium: Take 100 mL of RPMI-1640 basal culture medium and add rhGM-CSF with a final concentration of 800 IU / mL, IL-4 with a final concentration of 1000 IU / mL and VC with a final concentration of 1000 IU / mL.

[0086] Second culture medium: Take 1000 mL of RPMI-1640 basal culture medium and add rhGM-CSF with a final concentration of 800 IU / mL, IL-4 with a final concentration of 1000 IU / mL, IL-6 with a final concentration of 800 IU / mL, TNF-α with a final concentration of 500 IU / mL, IL-1β with a final concentration of 60 IU / mL, and VC with a final concentration of 1000 IU / mL.

[0087] 2.2 DC cell culture

[0088] On day 0, first, take 20 mL of RPMI-1640 and resuspend 2.0 × 10⁻⁶ cells. 7 PBMC cells were then seeded; the resuspended cell suspension was then seeded into a T175 culture flask and 20 mL of RPMI-1640 was added and cultured for 2 h; subsequently, the RPMI-1640 in the culture flask was aspirated and 9 mL of the first culture medium prepared in step 2.1 of this embodiment and 1 mL of 5% blood were added; finally, the T175 culture flask was placed in an incubator at 37°C and 5% CO2 for static culture.

[0089] On the third day, add 9 mL of the first culture medium and 1 mL of 5% blood substitute to the culture flask and continue culturing;

[0090] On day 5, the culture waste liquid in the culture bottle was aspirated, and 20 mL of the second culture medium prepared in step 2.1 of this embodiment was added to continue culturing;

[0091] On day 7, firstly, the culture waste in the T175 culture flask was aspirated, and the flask was rinsed with 5 mL of PBS, after which the PBS rinse waste was discarded. 2 mL of recombinant trypsin was added to the culture flask, and the flask was placed in a 37°C incubator for 3 minutes for digestion. After most of the cells had digested, 10 mL of the second culture medium was added to stop the digestion. The digested cells were centrifuged at 400g for 5 minutes, and the supernatant was removed. Subsequently, the cells were collected and transferred to an electroporation cuvette, and cultured at 1.0 × 10⁶ cells / mL. 7 Add 100 μL of Opti-MEM electroporation buffer and 1 μg of GFP-mRNA to a cell concentration of 100 cells / mL, maintaining a total volume of 100 μL in the electroporation cuvette. Load the cuvette into the Yida Bioelectroporator and set the electroporation parameters as follows: voltage 220V, pulse time 1200μs, interval time 612ms, and 3 electroporations. After electroporation, transfer the cell culture to a T25 flask using a pipette tip. (Pre-add 4 ml of preheated 37℃ secondary culture medium to the T25 flask and rinse the electroporation cuvette twice with the secondary culture medium. Transfer the rinsed cell culture to the T25 flask. Finally, transfer the T25 flask to an incubator for culture.)

[0092] On day 10, cell culture was stopped, and the culture waste liquid was poured out from the waste liquid outlet of the T25 culture flask. Then, 5 mL of PBS was added for rinsing, and the PBS rinsing solution was poured out. Then, 2 mL of recombinant trypsin was added to the culture flask, and the cells were digested in a 37 ℃ incubator for 3 min. The cells were observed under a microscope. After most of the cells were digested, a second culture medium was added to stop the digestion. The cells were collected, centrifuged at 400g for 5 min, the supernatant was removed, and the cell pellet was washed repeatedly with PBS 2-3 times to obtain highly active and mature DC cells.

[0093] The differences between Examples 3 to 15 and Example 2 are as follows: the ratio and concentration of the basal culture medium and the corresponding added components in the DC culture medium are different, and the electroporation process parameters are set differently on the 7th day of DC cell culture, as detailed in Tables 1 and 2.

[0094] Table 1. Composition and concentration of DC culture medium in each embodiment.

[0095]

[0096] Table 2. Electro-electric parameter setting table for each embodiment

[0097]

[0098] (II) Detection test of DC cells

[0099] Example 16

[0100] 16.1 Electroporation efficiency of DC cells and MFI detection assay

[0101] Flow cytometry analysis: Laser power was adjusted and the optical path calibrated to ensure accurate laser illumination of the detection area. Simultaneously, the photomultiplier tube voltage was set to optimize the fluorescence signal detection range, and thresholds were set to exclude interference from cell debris and other particles. A forward scattering threshold was set based on cell size, excluding signals from particles smaller than a certain size. Sample analysis: 10 μL of DC cell culture samples from day 10 of Examples 1 to 15 were placed sequentially on the flow cytometer sample rack, and the program was started for flow cytometry analysis. Cells passed through the detection zone under hydrodynamic focusing, and the resulting fluorescence and scattered light signals were detected and collected. Signal changes could be observed in real-time on the display screen during the analysis. Through sample analysis, the electroporation efficiency and MFI (mean fluorescence intensity) of cells expressing GFP (FITC fluorescence) were determined, as shown in Table 4.

[0102] Table 3 Electro-electric efficiency and MFI of each embodiment

[0103]

[0104] As shown in Table 3, when the electroporation parameters are set as follows: voltage 280V, pulse time 1200μs, and cell mRNA density 8μg, the electroporation efficiency can reach as high as 86.01%, which is beneficial to the culture of mature DC cells and greatly shortens the culture time of mature DC cells. At the same time, the average fluorescence intensity MFI is also as high as 10131, which is beneficial to the maturation of DC cells, indicating that the antigen expression level of DC cells is good.

[0105] 16.2 Cell viability assay

[0106] DC cells cultured in Examples 1 to 15 were sampled on day 0 and day 10, with 10 μL of cell suspension taken from each sample. 10 μL of trypan blue was then added for staining. The 15 DC cell samples were then stored at 18–25°C for 4 hours. The cell count was performed using flow cytometry, and the results are shown in Table 4. Figure 1 As shown; Figure 1 The data in the table is taken from Table 4.

[0107] Table 4. Number of DC cells and cell viability in each embodiment

[0108]

[0109] From Table 4 and Figure 1 It can be seen that on day 10 of DC cell culture, compared with Example 1 (Comparative Example 1), the number of DC cells expanded in Examples 2 to 15 was significantly higher (the cell expansion fold was above 2.37); at the same time, the cell viability was also significantly better, all above 90%; therefore, PBMC electroporation induction culture of DC cells resulted in higher cell expansion fold and cell viability.

[0110] It should be understood that the above description of the preferred embodiments of the present invention is quite detailed, but it should not be considered as a limitation on the scope of patent protection of the present invention. The scope of patent protection of the present invention shall be determined by the appended claims.

Claims

1. A method for electroporation culture of DC cells, characterized in that, The steps include the following: On day 0, PBMCs were resuspended in basal medium and inoculated into T175 culture flasks, and supplemented with the first culture medium and 5% blood substitute; the T175 culture flasks were placed in an incubator at 37°C and 5% CO2 for static culture; wherein, the first culture medium includes the basal medium, and the basal medium is supplemented with rhGM-CSF at a final concentration of 500~1500 IU / mL, IL-4 at a final concentration of 50~1500 IU / mL and VC at a final concentration of 50~1500 IU / mL; On the third day, the first culture medium and 5% blood were added to the culture flask, and the culture was continued. On day 5, the culture waste liquid in the culture flask was aspirated, and a second culture medium was added to continue culturing. The second culture medium included the basal culture medium, which contained rhGM-CSF at a final concentration of 500-1500 IU / mL, IL-4 at a final concentration of 50-1500 IU / mL, IL-6 at a final concentration of 50-1500 IU / mL, TNF-α at a final concentration of 10-100 IU / mL, and VC at a final concentration of 50-1500 IU / mL. On day 7, cells were collected and transferred to electroporation cuvettes, and electroporation buffer was added before electroporation. The electroporated cell solution was then transferred to T25 culture flasks containing the second culture medium, and cultured in an incubator for an extended period. On day 10, cell culture was stopped, cells were collected and centrifuged, the supernatant was removed, and the cell pellet was washed repeatedly with PBS 2-3 times to obtain DC cells.

2. The method for electroporation culture of DC cells according to claim 1, characterized in that, On day 7, collect the cell sap and proceed with the following treatment: First, aspirate the culture waste from the T175 culture flask, rinse the culture flask with PBS, and then discard the PBS rinse waste. Second, add recombinant trypsin to the culture flask and place it in an incubator to digest the cells on the inner wall of the culture flask. After the cells are digested, add the second culture medium to stop the digestion. Collect the digested cells and centrifuge them to remove the supernatant and collect the cell pellet.

3. The method for electroporation culture of DC cells according to claim 2, characterized in that, The amount of recombinant trypsin added to the digested cells was 2 mL; the digested cells were then centrifuged at 400 g for 5 min.

4. The method for electroporation culture of DC cells according to claim 1, characterized in that, On day 7, the electroporation buffer is any one of Opti-MEM, DMEM or EBEL.

5. The method for electroporation culture of DC cells according to claim 1, characterized in that, On day 7, according to 1.0 × 10 7 Add 100 μL of the electroporation buffer to a cell concentration of 1 cell / mL.

6. The method for electroporation culture of DC cells according to claim 7, characterized in that, On day 7, the electro-polarization process parameters were set as follows: voltage 220V~320V, pulse time 600μs~1600μs, interval time 612ms, and number of electro-polarization cycles 3.

7. The method for electroporation culture of DC cells according to claim 1, characterized in that, On day 7, the second culture medium needs to be preheated to 37°C.

8. The method for electroporation culture of DC cells according to claim 7, characterized in that, On day 7, after transferring the electroporated cell culture to a T25 culture flask, the following treatments were also performed: The cell culture vessel was rinsed multiple times with the second culture medium, and the rinsed cell solution was transferred to a T25 culture flask.

9. The method for electroporation culture of DC cells according to claim 1, characterized in that, On day 10, between stopping cell culture and collecting cells, the following procedures are also included: First, aspirate the culture waste liquid from the T25 culture flask, rinse the culture flask with PBS, and then discard the PBS rinse waste liquid. Second, add 2 mL of recombinant trypsin to the culture flask and place it in an incubator to digest the cells on the inner wall of the culture flask. After the cells are digested, add the second culture medium to stop the digestion.

10. The method for electroporation culture of DC cells according to any one of claims 1 to 9, characterized in that, The basal culture medium is any one of GT-T551H3, DMEM, or RPMI-1640.

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