Gel composition as well as preparation method and application thereof

Abstract

The invention discloses a gel composition and a preparation method and application thereof.The gel composition is provided with net-shaped middle through holes, and the preparation method comprises the steps that N-isopropylacrylamide and azodiisobutylimidazole phosphate are added into deionized water to be dissolved, stirred and mixed to be uniform at the normal temperature in the state that nitrogen is continuously introduced; then adding N-acryloyloxy succinimide, continuously stirring, and bubbling with nitrogen; raising the temperature to 65 DEG C, adding sodium alginate, lycium barbarum polysaccharide, fucoidin and calcium gluconate, continuously stirring and bubbling with nitrogen; finally, stirring is finished, nitrogen continues to be introduced for 30 min, then cooling is conducted in a sterile environment at the temperature of 5-20 DEG C, and the porous gel composition is prepared. Nitrogen is introduced for bubbling to obtain the porous gel composition with a middle through hole structure, and the average pore size of through holes is 40-80 microns; the porous gel composition is beneficial to wrapping and presentation of nutrients, antigens and other tiny substances and cell growth.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a gel composition for cell culture and a preparation method thereof, and application of the gel composition in dendritic cell culture. Background Art

[0002] Dendritic cells (DC) are widely distributed in the blood, liver, spleen, lymph nodes and other non-immune organs and tissues. They are the most powerful professional antigen presenting cells (APC) known to the body. They are also the only APC that can stimulate the initial T cell response and play a key role in the immune response. DC is closely related to the occurrence and development of tumors. It can efficiently absorb, process and present antigens. Immature DC has a strong migration ability. Mature DC can effectively activate the initial T cells and is at the center of initiating, regulating and maintaining the immune response. The core of an effective anti-tumor immune response is to produce a cellular immune response with CD8+-T cells as the main body. This is also the basis of DC as an immunotherapy. Therefore, the more DCs infiltrated in most solid tumors, the better the prognosis of patients.

[0003] At present, the conventional method of preparing DC is to use cytokines to induce, differentiate and expand DC precursor cells in peripheral blood and umbilical cord blood. Mature DC can effectively activate T cells and regulate the body's immune response, but mature DC has poor loading (antigen uptake and processing) ability and a short action time.

[0004] Therefore, the present invention provides a method for culturing dendritic cells in vitro, which can effectively enhance the antigen loading capacity and immune presentation capacity of mature dendritic cells. Summary of the invention

[0005] Based on the above problems, the problem to be solved by the present invention is to provide a method for preparing a gel composition for dendritic cell culture and dendritic cell culture, which can effectively enhance the antigen loading capacity and immune presentation capacity of mature dendritic cells and shorten the culture time.

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

[0007] A method for preparing a gel composition comprises the following steps:

[0008] At room temperature, add 56.6-113.2 g of N-isopropylacrylamide and 0.08-0.17 g of azobisisobutylimidazole phosphate into a flask, add 1 L of deionized water to dissolve and stir for 30-60 min, then add 0.85-1.7 g of N-acryloyloxysuccinimide, and continue stirring for 30-60 min; during the stirring process, nitrogen is continuously introduced into the flask to bubble the composition;

[0009] Heat the flask to 65°C, add 1-10g of sodium alginate, 0.5-5g of wolfberry polysaccharide, 0.05-2g of fucoidan and 0.1-2g of calcium gluconate while stirring, and continue stirring for 1-2h; during the stirring process, continuously introduce nitrogen into the flask to bubble the composition;

[0010] After the stirring was completed, nitrogen was continuously introduced for 30 minutes, and then the flask was cooled in a sterile environment at 5-20° C. for 24 hours or 48 hours to obtain a porous gel composition.

[0011] In the preparation method of the above-mentioned gel composition, N-isopropylacrylamide is 60-100g, azobisisobutylimidazole phosphate is 0.1-0.15g, N-acryloyloxysuccinimide is 1-1.5g, sodium alginate is 2-8g, wolfberry polysaccharide is 1-4g, fucoidan is 0.1-1g and calcium gluconate is 0.5-1.5g.

[0012] The prepared gel composition has a regular distribution of the pore sizes of the mesopores through the method, and the average pore size of the mesopores is 0.2-3 um.

[0013] The present invention also provides a method for culturing dendritic cells using the gel composition prepared by the above method, comprising the following steps:

[0014] Day 0, cell seeding

[0015] Take the monocytes and add them into the culture flask containing dendritic cell culture medium, and control the cell density to 1×10 6 / mL, and cultured statically at 37°C, 5% CO2 for 4 days;

[0016] Day 5, cell culture

[0017] The culture supernatant after the 4th day of culture was discarded, and the digestion solution was added. After the digestion was completed, PBS was added and mixed evenly to obtain a digestion mixture;

[0018] Centrifuging the digestion mixture, removing the supernatant, and resuspending the cell pellet obtained by centrifugation with dendritic cell induction culture medium;

[0019] The resuspended cell suspension is inoculated into another new culture bottle, and the inner bottom surface of the new culture bottle is pretreated with the gel composition prepared by the preparation method as described in claim 1, and the culture is continued at 37° C. and 5% CO2 for 24 hours;

[0020] On day 6, cells were loaded with antigens

[0021] Centrifuge the culture fluid after the 5th day of culture and leave the new culture bottle empty; collect the cell precipitate and the centrifugal supernatant after the centrifugation;

[0022] The collected cell pellets and antigen substances were electroporated;

[0023] After the electrotransfer is completed, the electrotransfer cell solution is centrifuged, the supernatant is removed, and the cell pellet after electrotransfer and centrifugation is washed once with PBS to obtain the dendritic cells loaded with the antigen substance;

[0024] After resuspending the dendritic cells loaded with antigenic substances with the centrifugal supernatant, re-inoculate them into the empty new culture bottle, and continue to culture at 37° C. and 5% CO 2 for 24 h or 48 h;

[0025] After 24 hours or 48 hours of culture, dendritic cells with the ability to present antigen substances efficiently are obtained.

[0026] In the above dendritic cell culture method, on day 0, the dendritic cell culture medium includes RPMI-1640 serum-free culture medium, which contains GM-CSF with a final concentration of 20-200 ng / ml, rhIL-4 with a final concentration of 20-100 ng / ml, and AB serum with a final concentration of 5 v / v%.

[0027] In the above dendritic cell culture method, on the 5th day, the digestion solution is 0.05% pancreatic enzyme, and the added volume of the digestion solution is 1 / 4 of the volume of the collected culture supernatant; the added volume of the PBS is 3 times the volume of the digestion solution.

[0028] In the above dendritic cell culture method, on the fifth day, the step of pretreating the new culture bottle with the gel composition is as follows:

[0029] The gel composition prepared by the preparation method of claim 1 and the dendritic cell induction culture medium are respectively taken, and mixed in a shaker at 37° C. for 10 minutes to obtain a gel solution; wherein the volume of the gel composition and the volume of the dendritic cell induction culture medium are 10-20% and 70% of the volume of the total culture medium in the culture bottle on day 0, respectively;

[0030] The gel solution was evenly spread on the bottom surface of the new culture bottle and allowed to stand for 30 minutes before use.

[0031] In the above dendritic cell culture method, on day 5, the dendritic cell induction culture medium includes RPMI-1640 basal medium, which contains TNF-α with a final concentration of 5-40 ng / ml, rhIL-6 with a final concentration of 50-200 ng / ml, prostaglandin E2 with a final concentration of 2-50 ug / ml, and AB serum with a final concentration of 1 v / v%.

[0032] In the above dendritic cell culture method, on day 6, the electroporation treatment includes the following steps:

[0033] Resuspend the cell pellet collected by centrifugation with 100uL of electrotransfer buffer to obtain a cell suspension;

[0034] Mix the cell suspension and mRNA antigen material with a size of 2500 to 3000 bp;

[0035] Under the process conditions of setting the electroporation parameters as voltage 260V, pulse time 1000us, and interval 612ms, the cells were electroporated 3 to 5 times.

[0036] In the above dendritic cell culture method, when the cell suspension and mRNA antigen substance are added to the empty new culture bottle, the cell density is controlled to be 1×10 7 / mL, the concentration of the mRNA antigen substance is controlled to be 20ug / ml:

[0037] The gel composition provided by the present invention continuously introduces nitrogen bubbling during its preparation process to form through holes in a honeycomb structure, i.e., three-dimensional holes. The average pore size of the through holes is 0.2-5 μm, which can effectively increase the specific surface area required for cell culture and is 70 times the surface area of ​​the inner wall of an ordinary culture bottle. The porous gel composition is conducive to the encapsulation and presentation of tiny substances such as nutrients and antigens. The nutrients included in the gel, such as alginate and sugars, are released to cells through the through holes. At the same time, substances secreted by the cells can also enter the gaps in the through holes, thereby effectively improving and maintaining the activity of dendritic cells, thereby improving the uptake and presentation capabilities of dendritic cells for antigens.

[0038] During the preparation of the gel composition, nitrogen bubbling is continuously introduced during the stirring process, which can also remove the oxygen encapsulated in the gel, so that the gel composition is conducive to the storage and release of cytokines and chemical drugs, thereby promoting cell growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a scanning electron microscopy image of the middle section of the gel combination in Example 1;

[0040] Figure 2This is a scanning electron microscopy image of the middle section of the gel combination in Example 2;

[0041] Figure 3 This is a scanning electron microscopy image of the middle section of the gel combination in Example 3;

[0042] Figure 4 It is a bar graph of the proportion of DC cell markers cultured in Examples 1 to 4;

[0043] Figure 5 It is a bar graph of EGFP expression rate of DC cells loaded with antigen cultured in Examples 1 to 4;

[0044] Figure 6 The figure is a bar graph showing the viability of DC cells loaded with antigens cultured in Examples 1 to 4. DETAILED DESCRIPTION

[0045] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0046] The gel composition provided by the present invention uses N-isopropylacrylamide as a porous crosslinking agent, and is stirred and dissolved in water under the action of azobisisobutylimidazole phosphate as an initiator and N-acryloyloxysuccinimide as an accelerator. During stirring, nitrogen is continuously bubbled to form a porous gel composition with a through-hole structure, and the average pore size of the through-hole is 40-80um and the porosity is 50-70%. This gel composition can effectively increase the cell culture surface area, which is 70 times the surface area of ​​the inner wall of an ordinary culture bottle; and the pore structure of the gel composition is a through honeycomb structure, that is, a three-dimensional hole, which is conducive to the packaging and presentation of micro-substances such as nutrients and antigens. The nutrients included in the gel, such as alginate, sugars, etc., will be released to cells through the through-holes, and substances secreted by cells can also enter the gaps of the through-holes; continuous nitrogen bubbling can also remove the oxygen coated in the gel, which is conducive to cell growth.

[0047] The sodium alginate in the gel can produce ionic cross-linking links with N-isopropylacrylamide, and has stronger resistance to swelling and compression; Lycium barbarum polysaccharides and fucoidan increase the cross-linking branches, enhancing the strength and stability of the gel structure; calcium gluconate can make the gel produce a more uniform and dense porous honeycomb structure, which is beneficial to cell growth, and the storage and release of cytokines and chemical drugs.

[0048] During the preparation of the gel composition, nitrogen is continuously bubbled in while stirring, which can also remove oxygen encapsulated in the gel, making the gel composition conducive to the storage and release of cytokines and chemical drugs, thereby promoting cell growth. It can also construct through holes of different pore sizes and connect blind holes into through holes.

[0049] The specific implementation scheme of the present invention is as follows.

[0050] 1. Preparation of gel composition

[0051] S11. Prepare materials

[0052] N-isopropylacrylamide: 56.6-113.2g; azobisisobutylimidazole phosphate: 0.08-0.17g; N-acryloyloxysuccinimide: 0.85-1.7g; sodium alginate: 1-10g; wolfberry polysaccharide: 0.5-5g; fucoidan: 0.05-2g; calcium gluconate: 0.1-2g;

[0053] S12, at room temperature, add N-isopropylacrylamide and azobisisobutylimidazole phosphate prepared in step S1 to a flask (such as a three-necked flask, with a stirring port, an air inlet, and an exhaust port), add 1 L of deionized water to dissolve, stir and mix for 30 to 60 minutes, and continuously introduce nitrogen bubbling; then add N-acryloyloxysuccinimide prepared in step S1, and continue stirring and reacting for 30 to 60 minutes;

[0054] S12, heat the flask to 65°C, add the sodium alginate, wolfberry polysaccharides, fucoidan, and calcium gluconate in step S1 while stirring, continue stirring and reacting for 1 to 2 hours, and continuously introduce nitrogen bubbling;

[0055] S13, after the stirring reaction is completed, nitrogen is continuously introduced for 30 minutes, and then the flask is cooled in a sterile environment at 5-20° C. for 24 hours or 48 hours to obtain a gel composition with a through-hole structure;

[0056] S14. Collect the prepared gel composition and store it at 2-8° C. for future use.

[0057] The raw materials used in the preparation of the above-mentioned gel composition preferably have the following mass: N-isopropylacrylamide: 60-100 g; azobisisobutylimidazole phosphate: 0.1-0.15 g; N-acryloyloxysuccinimide: 1-1.5 g; sodium alginate: 2-8 g; wolfberry polysaccharide: 1-4 g; fucoidan: 0.1-1 g; calcium gluconate: 0.5-1.5 g.

[0058] Preferably, in one embodiment, the raw materials used in the preparation of the above-mentioned gel composition have the following preferred masses: N-isopropylacrylamide: 70 g; azobisisobutylimidazole phosphate: 0.12 g; N-acryloyloxysuccinimide: 1.2 g; sodium alginate: 5 g; wolfberry polysaccharide: 3 g; fucoidan: 0.5 g; calcium gluconate: 1 g.

[0059] 2. Monocyte Isolation

[0060] S21. Collect 100 mL of peripheral blood from a healthy person using a sodium heparin anticoagulant blood collection tube;

[0061] S22, transfer the blood in the blood collection tube to a 100 mL centrifuge tube filled with lymph separation fluid, and centrifuge at 800 g for 20 min at room temperature;

[0062] S23. After centrifugation, the mononuclear cells in the middle buffy coat layer were aspirated and transferred to a 50 mL centrifuge tube, and CD14 positive selection magnetic beads were used to separate the mononuclear cells with CD14+≥95%;

[0063] S24. Wash the separated monocytes with DPBS solution, and centrifuge the washed monocytes at 600g for 8 minutes at room temperature; repeat the washing and centrifugation 2 to 3 times, collect the cell precipitate, and obtain the monocytes for later use.

[0064] 3. Preparation of culture medium

[0065] S31. Preparation of dendritic cell culture medium

[0066] Take RPMI-1640 basal culture medium containing GM-CSF at a final concentration of 20 to 200 ng / ml, rhIL-4 at a final concentration of 20 to 100 ng / ml and AB serum at a final concentration of 5 v / v% for later use.

[0067] S32. Preparation of dendritic cell induction culture medium

[0068] Take RPMI-1640 basal culture medium containing TNF-α with a final concentration of 5-40 ng / ml, rh IL-6 with a final concentration of 50-200 ng / ml, prostaglandin E2 with a final concentration of 2-50 ug / ml and AB serum with a final concentration of 1 v / v%, and set aside.

[0069] IV. Expansion and Culture of Dendritic Cells

[0070] S41. Day 0, cell seeding

[0071] The monocytes obtained in step S24 of the present invention are added to a TC75 culture flask (labeled 1#), wherein the 1# culture flask contains the dendritic cell culture medium prepared in step S31 of the present invention, and the cell density in the 1# culture flask is controlled to be 1×10 6 The cells were cultured at 37°C and 5% CO2 for 4 days.

[0072] S42. Day 5, cell culture

[0073] S421, culture bottle pretreatment

[0074] S4211. The gel composition obtained in step S14 of the present invention is mixed with the dendritic cell induction culture medium prepared in step S31, and then mixed in a shaker at 37° C. for 10 minutes to obtain a gel solution; wherein the volume of the gel composition and the volume of the dendritic cell induction culture medium are 10-20% and 70% of the volume of the total culture medium in the culture bottle on day 0, respectively;

[0075] S4212. Take another new TC75 culture bottle (marked 2#), use a pipette to absorb the gel solution and spread it evenly on the bottom surface of the 2# culture bottle, let it stand for 30 minutes, and a gel film will form on the bottom surface of the 2# culture bottle.

[0076] S422, dendritic cell culture

[0077] S4221. After the 4th day of culture, the culture supernatant in the 1# culture flask in step S41 is removed with a pipette, and 0.05% pancreatic enzyme digestion solution is added to the 1# culture flask to digest the cells, and the ratio of the volume of the digestion solution added to the volume of the culture supernatant is 1:4; after the digestion is terminated, PBS with 3 times the volume of pancreatic enzyme is added, and the mixture is mixed evenly to obtain a digestion mixture;

[0078] S4222, centrifuge the digested mixture at 500 g for 6 min, remove the supernatant after centrifugation, and resuspend the cell pellet obtained after centrifugation with the dendritic cell induction culture medium prepared in step S32, and control the cell density after resuspension to be 1×10 6 / mL, and obtain cell suspension;

[0079] S4223. Inoculate the resuspended cell suspension into the 2# culture flask pretreated in step S4212, and culture it at 37° C. and 5% CO 2 for 24 hours.

[0080] S43, Day 6, dendritic cells loaded with antigens

[0081] S431, after the 5th day of culture, collect the culture fluid in the 2# culture bottle in step S4223 and centrifuge at 500g for 6 minutes, and leave the 2# culture bottle empty; after the centrifugation, collect the cell pellet and the centrifugation supernatant respectively;

[0082] S432, resuspend the cell pellet with 100uL electroporation buffer to obtain a cell suspension;

[0083] S433, selecting an mRNA antigen substance with a size of 2500 to 3000 bp and containing an EGFP reporter gene; wherein, for the detection of transfection and expression of the mRNA antigen substance, the mRNA antigen substance added here contains an EGFP reporter gene;

[0084] S434: Add the cell precipitate obtained in step S431 and the mRNA antigen material selected in step S433 into the electroporation cup and mix them together. The cell density is controlled to be 1×10 7 / mL, the concentration of mRNA antigen was controlled at 20ug / ml;

[0085] S435, electroporating the cells 3 to 5 times under the process conditions of setting the electroporation parameters to voltage 260 V, pulse time 1000 us, and interval 612 ms;

[0086] S436. After the electroporation is stopped, the cell fluid obtained by electroporation is collected and centrifuged at 500 g for 6 min. After centrifugation, the supernatant is removed and the cell pellet is washed once with PBS to obtain dendritic cells loaded with antigen substances;

[0087] S437, resuspending the dendritic cells loaded with antigen obtained in step S436 with the centrifugal supernatant collected in step S431;

[0088] S438. The dendritic cells loaded with the resuspended antigen substance are inoculated again into the vacant culture flask #2 in step S431, and the culture is continued at 37° C. and 5% CO 2 for 24 h or 48 h.

[0089] S44. Collect cells

[0090] At the end of 24h or 48h culture, i.e., on the 7th or 8th day, the supernatant was removed, the cells were washed 2-3 times with PBS, and then centrifuged at 500g for 6min, the supernatant was removed, and the cell pellet was obtained, which was the dendritic cells with the ability to present antigen substances efficiently.

[0091] The technical solution of the present invention is further described in detail below through specific embodiments.

[0092] In the following examples, the monocytes used are derived from the monocytes obtained in the second step S24 of "monocyte sorting" mentioned above.

[0093] Example 1

[0094] 1.1. Preparation of 100 mL of gel composition

[0095] At room temperature, add 80g N-isopropylacrylamide and 0.1g azobisisobutylimidazole phosphate into a three-necked flask, dissolve and stir with deionized water, add 1g N-acryloyloxysuccinimide after 45 minutes, continue stirring for 45 minutes, and bubbling with nitrogen during the reaction process;

[0096] Heat the reaction system in the flask to 65°C, add 6g sodium alginate, 0.3g Lycium barbarum polysaccharide, 1g fucoidan and 1g calcium gluconate while stirring, and continue stirring for 1.5h, bubbling with nitrogen throughout the stirring process;

[0097] After the stirring was completed, nitrogen was continuously introduced for 30 min, followed by cooling in a sterile environment at 10° C. for 36 h, and the gel composition was collected and stored at 2-8° C. for later use.

[0098] 1.2. Preparation of DC culture medium

[0099] DC culture medium: Take 1L RPMI-1640, add 100ng / ml GM-CSF, 60ng / ml rh IL-4 and 5v / v% AB serum;

[0100] DC induction culture medium: Take 1L RPMI-1640, add 20ng / ml TNF-α, 100ng / ml rhIL-6, 20ug / ml prostaglandin E2 and 1v / v% AB serum.

[0101] 1.3. DC culture

[0102] Day 0: Inoculation

[0103] Take 10 mL of the DC culture medium prepared in step 1.2 of this example and add it to a TC75 culture flask (labeled 11#), then take 1×10 7 The mononuclear cells were added into the 11# culture flask and then cultured at 37°C and 5% CO2 for 4 days.

[0104] Day 5: Cell culture

[0105] 1.5 mL of the gel composition prepared in step 1.1 of this example and 7.0 mL of the DC induction cell culture medium prepared in step 1.2 of this example were taken respectively, and mixed on a shaker at 37° C. for 10 min to obtain a gel solution;

[0106] Take another new TC75 culture bottle (marked 12#), use a pipette to absorb the gel solution and spread it on the bottom surface of the 12# culture bottle, let it stand for 30 minutes, and form a gel film on the bottom surface of the 12# culture bottle for later use;

[0107] The culture supernatant in the 11# culture flask was discarded, and 0.05% trypsin was added to the 11# culture flask to digest the cells for 5 minutes, and the volume ratio of trypsin to culture supernatant was 1:4; after the cells were digested, 3 times the volume of trypsin was added to PBS and shaken to obtain a digestion mixture;

[0108] The digestion mixture in the 11# culture flask was collected and centrifuged at 500 g for 6 min. After the centrifugation, the supernatant was removed and the cell pellet obtained after centrifugation was resuspended with the DC induction culture medium prepared in step 1.2 of this example. The cell density after resuspension was controlled to be 1×10 6 / mL, and obtain cell suspension;

[0109] The resuspended cell suspension was inoculated into the pretreated 12# culture flask, and the culture was continued at 37°C and 5% CO2 for 24 hours.

[0110] 1.4 Antigen-loaded DC

[0111] At the end of 24 h of culture (i.e., day 6), the culture fluid after the end of the 5th day of culture was collected and centrifuged at 500 g for 6 min, and the 12# culture bottle was left empty;

[0112] After centrifugation, collect the cell pellet and supernatant separately; resuspend the cell pellet with 100uL electrotransfer buffer; use the collected supernatant separately;

[0113] Select mRNA antigen containing EGFP reporter gene, and the size of mRNA antigen is 2500-3000 bp;

[0114] The cell pellet resuspended in electroporation buffer and the selected mRNA antigen were added to the electroporation cup, and the cell density was controlled at 1×10 7 / mL, the concentration of mRNA antigen was controlled at 20ug / ml; under the process conditions of setting the electrotransfer parameters as voltage 260V, pulse time 1000us, and interval 612ms, the cells were electrotransferred 3 to 5 times;

[0115] After the electroporation was stopped, the cell fluid obtained by electroporation was collected and centrifuged at 500 g for 6 min. After centrifugation, the supernatant was removed and the cell pellet was washed once with PBS to obtain dendritic cells loaded with antigen substances.

[0116] Resuspend the dendritic cells loaded with antigen substances using the centrifugal supernatant collected above;

[0117] The dendritic cells loaded with antigens were resuspended and inoculated into an empty 12# culture flask again, and the culture was continued at 37°C and 5% CO2 for 48 hours.

[0118] 1.5. Collect cells

[0119] At the end of 48h culture (i.e., day 8), the culture supernatant was removed, and the cells in the 12# culture flask were washed 2 to 3 times with PBS, followed by centrifugation at 500g for 6min, and the supernatant was removed to obtain the cell pellet, which is the dendritic cells with the ability to present antigen substances efficiently.

[0120] Example 2

[0121] 2.1. Preparation of 100 mL of gel composition

[0122] At room temperature, add 56.67g N-isopropylacrylamide and 0.17g azobisisobutylimidazole phosphate into the flask, dissolve with deionized water and stir to mix, add 0.85g N-acryloyloxysuccinimide after 30 minutes and continue stirring for 30 minutes, and use nitrogen bubbling during the reaction process;

[0123] Heat the reaction system in the flask to 65°C, add 1g sodium alginate, 0.5g Lycium barbarum polysaccharide, 2g fucoidan and 2g calcium gluconate while stirring, continue stirring for 1h, and keep bubbling with nitrogen during the stirring process;

[0124] After the stirring was completed, nitrogen was continuously introduced for 30 min, followed by cooling in a sterile environment at 5° C. for 48 h, and the gel composition was collected and stored at 2-8° C. for later use.

[0125] 2.2 Preparation of DC culture medium

[0126] DC culture medium: Take 1L RPMI-1640, add 20ng / ml GM-CSF, 100ng / ml rh IL-4 and 5v / v% AB serum;

[0127] DC induction culture medium: Take 1L RPMI-1640, add 40ng / ml TNF-α, 50ng / ml rhIL-6, 2ug / ml prostaglandin E2 and 1v / v% AB serum.

[0128] 2.3 DC culture

[0129] Day 0: Inoculation

[0130] Take 10 mL of the DC culture medium prepared in step 2.2 of this example and add it to a TC75 culture flask (labeled 21#), then take 1×10 7 The mononuclear cells were added into the 21# culture flask and then cultured at 37°C and 5% CO2 for 4 days.

[0131] Day 5: Cell culture

[0132] 10 mL of the gel composition prepared in step 2.1 of this example and 70 mL of the DC induction cell culture medium prepared in step 2.2 of this example were taken respectively, and mixed on a shaker at 37° C. for 10 min to obtain a gel solution;

[0133] Take another new TC75 culture bottle (marked 22#), use a pipette to absorb the gel solution and spread it on the bottom surface of the 22# culture bottle, let it stand for 30 minutes, and form a gel film on the bottom surface of the 22# culture bottle for later use;

[0134] The culture supernatant in the 21# culture flask was discarded, and 0.05% trypsin was added to the 21# culture flask to digest the cells for 5 minutes, and the volume ratio of trypsin to culture supernatant was 1:4; after the cells were digested, 3 times the volume of trypsin was added to PBS and shaken to obtain a digestion mixture;

[0135] The digestion mixture in the 21# culture flask was collected and centrifuged at 500 g for 6 min. After the centrifugation, the supernatant was removed and the cell pellet obtained after centrifugation was resuspended with the DC induction culture medium prepared in step 2.2 of this example. The cell density after resuspension was controlled to be 1×10 6 / mL, and obtain cell suspension;

[0136] The resuspended cell suspension was inoculated into the pretreated 22# culture flask, and the culture was continued at 37°C and 5% CO2 for 24 hours.

[0137] 2.4 Antigen-loaded DC

[0138] At the end of 24 h of culture (i.e., day 6), the culture fluid after the end of the culture on day 5 was collected and centrifuged at 500 g for 6 min, and the 23# culture bottle was left empty;

[0139] After centrifugation, collect the cell pellet and supernatant separately; resuspend the cell pellet with 100uL electrotransfer buffer; use the collected supernatant separately;

[0140] Select mRNA antigen containing EGFP reporter gene, and the size of mRNA antigen is 2500-3000 bp;

[0141] The cell pellet resuspended in electroporation buffer and the selected mRNA antigen were added to the electroporation cup, and the cell density was controlled at 1×10 7 / mL, the concentration of mRNA antigen was controlled at 20ug / ml; under the process conditions of setting the electrotransfer parameters as voltage 260V, pulse time 1000us, and interval 612ms, the cells were electrotransferred 3 to 5 times;

[0142] After the electroporation was stopped, the cell fluid obtained by electroporation was collected and centrifuged at 500 g for 6 min. After centrifugation, the supernatant was removed and the cell pellet was washed once with PBS to obtain dendritic cells loaded with antigen substances.

[0143] Resuspend the dendritic cells loaded with antigen substances using the centrifugal supernatant collected above;

[0144] The dendritic cells loaded with antigens were resuspended and inoculated into an empty 22# culture flask again, and the culture was continued at 37°C and 5% CO2 for 24 hours.

[0145] 2.5. Collect cells

[0146] At the end of 24h culture (i.e., day 7), the culture supernatant was removed, and the cells in the 12# culture flask were washed 2-3 times with PBS, followed by centrifugation at 500g for 6min, and the supernatant was removed to obtain the cell pellet, which is the dendritic cells with the ability to present antigen substances efficiently.

[0147] Example 3

[0148] 3.1. Preparation of 100 mL of gel composition

[0149] At room temperature, add 113.2g N-isopropylacrylamide and 0.08g azobisisobutylimidazole phosphate into a flask, dissolve and stir with deionized water, add 0.17g N-acryloyloxysuccinimide after 60min, and continue stirring for 60min. During the reaction, stir and bubble with nitrogen.

[0150] Heat the reaction system in the flask to 65°C, add 10g sodium alginate, 5g Lycium barbarum polysaccharide, 0.05g fucoidan and 0.1g calcium gluconate while stirring, continue stirring for 2h, and keep bubbling with nitrogen during the stirring process;

[0151] After the stirring was completed, nitrogen was continuously introduced for 30 min, followed by cooling in a sterile environment at 20° C. for 24 h, and the gel composition was collected and stored at 2-8° C. for later use.

[0152] 2.2 Preparation of DC culture medium

[0153] DC culture medium: Take 1L RPM I-1640, add 200ng / ml GM-CSF, 20ng / ml rh I L-4 and 5v / v% AB serum;

[0154] DC induction culture medium: Take 1L RPMI-1640, add 5ng / ml TNF-α, 200ng / ml rhIL-6, 50ug / ml prostaglandin E2 and 1v / v% AB serum.

[0155] 3.3 DC culture

[0156] Day 0: Inoculation

[0157] Take 10 mL of the DC culture medium prepared in step 3.2 of this example and add it to a TC75 culture flask (labeled 31#), then take 1×10 7 The mononuclear cells were added into the 31# culture flask and then cultured at 37°C and 5% CO2 for 4 days.

[0158] Day 5: Cell culture

[0159] 20 mL of the gel composition prepared in step 3.1 of this example and 70 mL of the DC induction cell culture medium prepared in step 3.2 of this example were taken respectively, and mixed on a shaker at 37° C. for 10 min to obtain a gel solution;

[0160] Take another new TC75 culture bottle (labeled 32#), use a pipette to absorb the gel solution and spread it on the bottom surface of the 32# culture bottle, let it stand for 30 minutes, and form a gel film on the bottom surface of the 32# culture bottle for later use;

[0161] The culture supernatant in the 31# culture flask was discarded, and 0.05% trypsin was added to the 31# culture flask to digest the cells for 5 minutes, and the volume ratio of trypsin to culture supernatant was 1:4; after the cells were digested, 3 times the volume of trypsin was added to PBS and shaken to obtain a digestion mixture;

[0162] The digestion mixture in the 31# culture flask was collected and centrifuged at 500 g for 6 min. After the centrifugation, the supernatant was removed and the cell pellet obtained after centrifugation was resuspended with the DC induction culture medium prepared in step 3.2 of this example. The cell density after resuspension was controlled to be 1×10 6 / mL, and obtain cell suspension;

[0163] The resuspended cell suspension was inoculated into the pretreated 32# culture flask and continued to be statically cultured at 37°C and 5% CO2 for 24 hours.

[0164] 3.4 Antigen-loaded DCs

[0165] At the end of 24 h of culture (i.e., day 6), the culture fluid after the end of the 5th day of culture was collected and centrifuged at 500 g for 6 min, and the 33# culture bottle was left empty;

[0166] After centrifugation, collect the cell pellet and supernatant separately; resuspend the cell pellet with 100uL electrotransfer buffer; use the collected supernatant separately;

[0167] Select mRNA antigen containing EGFP reporter gene, and the size of mRNA antigen is 2500-3000 bp;

[0168] The cell pellet resuspended in electroporation buffer and the selected mRNA antigen were added to the electroporation cup, and the cell density was controlled at 1×10 7 / mL, the concentration of mRNA antigen was controlled at 20ug / ml; under the process conditions of setting the electrotransfer parameters as voltage 260V, pulse time 1000us, and interval 612ms, the cells were electrotransferred 3 to 5 times;

[0169] After the electroporation was stopped, the cell fluid obtained by electroporation was collected and centrifuged at 500 g for 6 min. After centrifugation, the supernatant was removed and the cell pellet was washed once with PBS to obtain dendritic cells loaded with antigen substances.

[0170] Resuspend the dendritic cells loaded with antigen substances using the centrifugal supernatant collected above;

[0171] The dendritic cells loaded with antigens were resuspended and inoculated into an empty 32# culture flask again, and the culture was continued at 37°C and 5% CO2 for 24 hours.

[0172] 3.5. Collect cells

[0173] At the end of 24h culture (i.e., day 7), the culture supernatant was removed, and the cells in the 32# culture flask were washed 2 to 3 times with PBS, followed by centrifugation at 500g for 6min, and the supernatant was removed to obtain the cell pellet, which is the dendritic cells with the ability to present antigen substances efficiently.

[0174] Example 4 (Comparative Example 1)

[0175] The difference between this embodiment and embodiments 1 to 3 is that no gel combination is prepared and used during the DC culture process.

[0176] 4.1. Preparation of DC culture medium

[0177] DC culture medium: Take 1L RPMI-1640, add 100ng / ml GM-CSF, 60ng / ml rh IL-4 and 5v / v% AB serum;

[0178] DC induction culture medium: Take 1L RPMI-1640, add 20ng / ml TNF-α, 100ng / ml rhIL-6, 20ug / ml prostaglandin E2 and 1v / v% AB serum.

[0179] 4.2 DC culture

[0180] Day 0: Inoculation

[0181] Take 10 mL of the DC culture medium prepared in step 4.2 of this example and add it to a TC75 culture flask (labeled 41#), then take 1×10 7 The mononuclear cells were added into the 41# culture flask and then cultured at 37°C and 5% CO2 for 4 days.

[0182] Day 5: Cell culture

[0183] The culture supernatant in the 41# culture flask was discarded, and 0.05% trypsin was added to the 41# culture flask to digest the cells for 5 minutes, and the volume ratio of trypsin to culture supernatant was 1:4; after the cells were digested, PBS with a volume of 3 times that of trypsin was added and shaken to obtain a digestion mixture;

[0184] The digestion mixture in the 41# culture flask was collected and centrifuged at 500 g for 6 min. After the centrifugation, the supernatant was removed and the cell pellet obtained after centrifugation was resuspended with the DC induction culture medium prepared in step 4.1 of this example, and the cell density was controlled to be 1×10 6 / mL, and obtain cell suspension;

[0185] The resuspended cell suspension was inoculated into another new culture bottle (labeled #42), and the culture was continued at 37°C and 5% CO2 for 48 hours.

[0186] 4.3 Antigen-loaded DCs

[0187] At the end of 24 h of culture (i.e., day 6), the culture fluid after the end of day 5 culture was collected and centrifuged at 500 g for 6 min, and the 42# culture bottle was left empty;

[0188] After centrifugation, collect the cell pellet and supernatant separately; resuspend the cell pellet with 100uL electrotransfer buffer; use the collected supernatant separately;

[0189] Select mRNA antigen containing EGFP reporter gene, and the size of mRNA antigen is 2500-3000 bp;

[0190] The cell pellet resuspended in electroporation buffer and the selected mRNA antigen were added to the electroporation cup, and the cell density was controlled at 1×10 7 / mL, the concentration of mRNA antigen was controlled at 20ug / ml, and the electroporation parameters were set as voltage 260V, pulse time 1000us, and interval 612ms, and the cells were electroporated 3 to 5 times;

[0191] After the electroporation was stopped, the cell fluid obtained by electroporation was collected and centrifuged at 500 g for 6 min. After centrifugation, the supernatant was removed and the cell pellet was washed once with PBS to obtain dendritic cells loaded with antigen substances.

[0192] Resuspend the dendritic cells loaded with antigen substances using the centrifugal supernatant collected above;

[0193] The dendritic cells loaded with antigens were resuspended and inoculated into an empty 42# culture flask again, and the culture was continued at 37°C and 5% CO2 for 48 hours.

[0194] 4.4. Collecting cells

[0195] At the end of 48h culture (i.e., day 8), the culture supernatant was removed, and the cells in the 42# culture flask were washed 2 to 3 times with PBS, followed by centrifugation at 500g for 6min, and the supernatant was removed to obtain the cell pellet, which is the dendritic cells with the ability to present antigen substances efficiently.

[0196] 2. Gel combination and dendritic cell test

[0197] 1. Scanning electron microscope imaging detection of gel composition

[0198] The gel composition samples in Examples 1 to 3 were dried and solidified naturally. The solidified bodies were then placed in liquid nitrogen for 5 minutes, taken out and naturally fractured, and observed and photographed using a scanning electron microscope (model: three-dimensional imaging field emission scanning electron microscope (VolumeScope 2SEM, manufacturer: Thermo Scientific Fisher) (magnification 6500 times). Figures 1 to 3 As shown. Figures 1 to 3 It can be seen that the gel composition fault surface (and inside the gel composition) is distributed with honeycomb-like through holes that are connected to each other, and the average pore size is 0.2 to 3 μm.

[0199] 2. Gel composition porosity test

[0200] The gel compositions prepared in Examples 1 to 3 were weighed in a water-containing state (wet weight G1), and weighed after being naturally dried at room temperature to remove moisture and solidify (dry weight G0), and the results were recorded as shown in Table 1. The porosity (p) was calculated according to the weight measurement method. The porosity calculation formula is: p = {(G2-G1) / G1} × 100%, and the calculated porosity results are shown in Table 1.

[0201] Table 1 Porosity vehicle data record table

[0202]

[0203] As shown in Table 1, the porosity of the gel compositions obtained in Examples 1 to 3 is between 58% and 63%. Therefore, during the stirring reaction of N-isopropylacrylamide, azobisisobutylimidazole phosphate and N-acryloyloxysuccinimide, nitrogen is continuously introduced for bubbling, so that the oxygen encapsulated in the gel can be removed and a porous scaffold structure can be constructed. The porous gel composition is conducive to the storage and release of cytokines and chemical drugs, thereby promoting cell growth.

[0204] 3. Flow cytometry phenotyping of dendritic cells

[0205] Take the DC cells in Examples 1 to 4, sample them on the 8th day of culture, add PBS solution, and adjust the cell concentration to 1×10 6 / mL. Using flow cytometry, the immature DC cell (DC precursor cell) marker CD80, mature DC cell marker CD11 c and CD86 were detected by phenotypic flow cytometry, and the HLA-DR concentration reflecting DC maturity was also detected, as shown in Table 2 and Figure 4 As shown; Figure 4 The figures are drawn based on the data in Table 2.

[0206] Table 2 Statistical table of DC cell marker ratios

[0207]

[0208] From Table 1 and Figure 4 It can be seen that the ratio of markers CD11c and CD86, and the ratio of HLA-DR, etc. respectively represent the expression levels of mature DC cells. In Examples 1 to 4, the ratio of DC precursor cell marker CD80 in Example 4 is lower than the double positive ratio of mature DC cell markers CD11c and CD86, especially the ratio of HLA-DR in Example 4 is much lower than the ratio of HLA-DR in Examples 1 to 3.

[0209] This is because: in Examples 1 to 3 of the present invention, the inner bottom wall of the culture bottle is pre-treated with a gel combination to form a layer of gel film, and the gel film has honeycomb-shaped through holes of 0.2 to 5 μm inside, which can effectively increase the specific surface area required for cell culture. This porous gel composition is conducive to the packaging and presentation of tiny substances such as nutrients and antigens. For example, alginate, sugars, etc. are released to cells through the through holes. At the same time, substances secreted by cells can also enter the gaps in the through holes, effectively promoting the storage and release of cytokines and chemical drugs, etc., so as to effectively improve and maintain the activity of dendritic cells, thereby promoting the rapid proliferation and growth of cells.

[0210] 4. Detection of EGFP expression rate of antigen-loaded dendritic cells

[0211] The antigen-loaded dendritic cells cultured in Examples 1 to 4 were sampled 24 h and 48 h after electroporation, washed once with PBS, centrifuged at 400 g for 4 min, and directly loaded for flow cytometry. The fluorescence channel corresponding to EGFP was selected, and the DCs without antigen loading were used as controls to draw gates to detect the EGFP expression rate. The detection results are shown in Tables 3 and Figure 5 As shown, Figure 5 So Figure 3 Draw based on data.

[0212] Table 3 EGFP expression rate of antigen-loaded dendritic cells

[0213]

[0214] From Table 3 and Figure 5It can be seen that the EGFP expression rate of DC cells cultured in Examples 1 to 3 is much higher than the EGFP expression rate of DC cells cultured in Example 4. This shows that the mRNA antigen containing the EGFP reporter gene transferred into DC cells will express the EGFP protein, and the positive test means that the mRNA transfer rate is higher. From the results, the EGFP expression rate of Examples 1 to 3 is higher, indicating that more DC cells are transferred with a large amount of antigen mRNA, which further illustrates that the DC culture method of preparing the porous gel composition by passing nitrogen and pretreating the culture bottle with the composition can promote the rapid proliferation and growth of DC cells and greatly improve the viability of DC cells.

[0215] 5. Antigen-loaded dendritic cell viability detection

[0216] The antigen-loaded dendritic cells cultured in Examples 1 to 4 were sampled 24 h and 48 h after electroporation, washed once with PBS, centrifuged at 400 g for 4 min, incubated with 7-AAD antibody for 5 min, and tested by flow cytometry. DCs without 7-AAD antibody were gated as controls, and cell counts were performed to calculate cell viability as shown in Tables 4 and Figure 6 As shown; Figure 6 The data are drawn based on Table 4.

[0217] Table 4 Cell viability of anti-DC cells electroporated for 24h and 48h

[0218]

[0219] From Table 4 and Figure 6 It can be seen that the viability of DC cells cultured in Examples 1 to 3 (above 92%) is much higher than that of DC cells cultured in Example 4 (78.43%, 75.65%), which means that a layer of gel film is formed at the bottom of the culture bottle after the pretreatment of the gel combination, and there are honeycomb through holes of 0.2 to 5 μm inside the gel film. This porous gel composition is conducive to the storage and release of cytokines and chemical drugs, and further promotes rapid cell proliferation, resulting in a higher cell viability, and the cell viability also exceeds the cell viability specified in the clinical application standard (≥90%).

[0220] It should be understood that the above description of the preferred embodiments of the present invention is relatively detailed and cannot be regarded as limiting the scope of patent protection of the present invention. The scope of patent protection of the present invention shall be based on the attached claims.

Claims

1. A method for preparing a gel composition, characterized in that: The steps include: At room temperature, add 56.6-113.2 g of N-isopropylacrylamide and 0.08-0.17 g of azobisisobutylimidazole phosphate into a flask, add 1 L of deionized water to dissolve and stir for 30-60 min, then add 0.85-1.7 g of N-acryloyloxysuccinimide and continue stirring for 30-60 min; during the stirring process, continuously bubble nitrogen into the flask; Heat the flask to 65°C, add 1-10g of sodium alginate, 0.5-5g of wolfberry polysaccharide, 0.05-2g of fucoidan and 0.1-2g of calcium gluconate while stirring, and continue stirring for 1-2h; during the stirring process, continuously bubble nitrogen into the flask; After the stirring was completed, nitrogen was continuously introduced for 30 minutes, and then the flask was cooled in a sterile environment at 5-20° C. for 24 hours or 48 hours to obtain a porous gel composition.

2. The method for preparing the gel composition according to claim 1, characterized in that: N-isopropylacrylamide is 60-100g, azobisisobutylimidazole phosphate is 0.1-0.15g, N-acryloyloxysuccinimide is 1-1.5g, sodium alginate is 2-8g, wolfberry polysaccharide is 1-4g, fucoidan is 0.1-1g and calcium gluconate is 0.5-1.5g.

3. The gel composition obtained by the preparation method according to claim 1 or 2, characterized in that: The gel composition has regular distribution of mesopores, and the average pore diameter of the mesopores is 0.2-3 μm.

4. A method for culturing dendritic cells, characterized in that: The method comprises the following steps: Day 0, cell seeding Take the monocytes and add them into the culture flask containing dendritic cell culture medium, and control the cell density to 1×10 6 / mL, and cultured statically at 37°C, 5% CO2 for 4 days; Day 5, cell culture The culture supernatant after the 4th day of culture was discarded, and the digestion solution was added. After the digestion was completed, PBS was added and mixed evenly to obtain a digestion mixture; Centrifuging the digestion mixture, removing the supernatant, and resuspending the cell pellet obtained by centrifugation with dendritic cell induction culture medium; The resuspended cell suspension is inoculated into another new culture bottle, and the inner bottom surface of the new culture bottle is pretreated with the gel composition prepared by the preparation method as described in claim 1, and the culture is continued at 37° C. and 5% CO2 for 24 hours; On day 6, cells were loaded with antigens Centrifuge the culture fluid after the 5th day of culture and leave the new culture bottle empty; collect the cell precipitate and the centrifugal supernatant after the centrifugation; The collected cell pellets and antigen substances were electroporated; After the electrotransfer is completed, the electrotransfer cell solution is centrifuged, the supernatant is removed, and the cell pellet after electrotransfer and centrifugation is washed once with PBS to obtain the dendritic cells loaded with the antigen substance; After resuspending the dendritic cells loaded with antigenic substances with the centrifugal supernatant, re-inoculate them into the empty new culture bottle, and continue to culture at 37° C. and 5% CO 2 for 24 h or 48 h; After 24 hours or 48 hours of culture, dendritic cells with the ability to present antigen substances efficiently are obtained.

5. The method for culturing dendritic cells according to claim 4, characterized in that: On day 0, the dendritic cell culture medium includes RPMI-1640 serum-free culture medium, which contains GM-CSF with a final concentration of 20-200 ng / ml, rhIL-4 with a final concentration of 20-100 ng / ml, and AB serum with a final concentration of 5 v / v%.

6. The method for culturing dendritic cells according to claim 4, characterized in that: On the fifth day, the digestion solution was pancreatic enzyme with a concentration of 0.05%, and the added volume of the digestion solution was 1 / 4 of the volume of the collected culture supernatant; the added volume of the PBS was 3 times the volume of the digestion solution.

7. The method for culturing dendritic cells according to claim 4, characterized in that: On day 5, the steps of pretreating the new culture flask with the gel composition are as follows: The gel composition prepared by the preparation method of claim 1 and the dendritic cell induction culture medium are respectively taken, and mixed in a shaker at 37° C. for 10 minutes to obtain a gel solution; wherein the volume of the gel composition and the volume of the dendritic cell induction culture medium are 10-20% and 70% of the volume of the total culture medium in the culture bottle on day 0, respectively; The gel solution was evenly spread on the bottom surface of the new culture bottle and allowed to stand for 30 minutes before use.

8. The method for culturing dendritic cells according to claim 4 or 7, characterized in that: On day 5, the dendritic cell induction culture medium includes RPMI-1640 basic culture medium, which contains TNF-α with a final concentration of 5-40 ng / ml, rhIL-6 with a final concentration of 50-200 ng / ml, prostaglandin E2 with a final concentration of 2-50 ug / ml, and AB serum with a final concentration of 1 v / v%.

9. The method for culturing dendritic cells according to claim 4, characterized in that: On day 6, the electroporation process included the following steps: Resuspend the cell pellet collected by centrifugation with 100uL of electrotransfer buffer to obtain a cell suspension; Mix the cell suspension and mRNA antigen material with a size of 2500 to 3000 bp; Under the process conditions of setting the electroporation parameters as voltage 260V, pulse time 1000us, and interval 612ms, the cells were electroporated 3 to 5 times.

10. The method for culturing dendritic cells according to claim 9, characterized in that: When the cell suspension and mRNA antigen material are added to the empty new culture bottle, the cell density is controlled to be 1×10 7 / mL, and the concentration of the mRNA antigen substance was controlled to be 20ug / ml.

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