A kind of cell cross-linking agent material for three-dimensional hepatocyte spheroid culture
A technology of cross-linking agent and hepatocytes, applied in animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problem of weak binding ability of cross-linking agent to cells, low bridging rate of cross-linking agent, and insufficient cell binding force. and other problems, to achieve the effect of retaining liver targeting ability, improving hydrophilicity, and promoting cell aggregation.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0027] Embodiment 1, liver cell cross-linking agent ALG-GA-N (CH 3 ) 2 preparation of
[0028] Step 1, GA hydrophilic modification:
[0029] GA-N(CH 3 ) 2 Synthesis
[0030] Dissolve 5.3g DCC and 3.4g HOBt in dichloromethane and stir at room temperature for 30min. Add 10gGA (Compound 1) to the solution and stir for 20min. After that, 2.3mL N,N-dimethylethylenediamine was added, and stirred at room temperature for 12h. The reaction solution uses a chromatographic column after utilizing a rotary evaporator to remove the solvent (volume ratio: CH 2 Cl2:MeOH:NH 3 h 2 O=15:1:1) for purification, then dissolve the product in ethyl acetate, let it stand at 0°C for 2h, filter, evaporate the filtrate to dryness, and obtain a white powder product, namely compound 2 (10.6g, 92% ).
[0031] Step 2, hepatocyte cross-linking agent ALG-GA-N (CH 3 ) 2 (DS=12%) Preparation:
[0032] 1st, scu-GA-N (CH 3 ) 2 Synthesis
[0033] 3.2g compound GA-N (CH 3 ) 2 (Compound 2) was diss...
Embodiment 2
[0044] Embodiment 2, preparation of hepatocyte cross-linking agent SCTS-GA-N(CH3)2:
[0045] 1. Synthesis of SCTS
[0046] A schematic diagram of the synthesis of chitosan sulfate is attached image 3 As shown, add 45mL of HSO to a 500mL three-neck flask 3 Cl, under ice-bath conditions, slowly drop 90 mL of 98% concentrated sulfuric acid into the three-neck flask, and stir for 15 min. Increase the stirring speed, and slowly add 3g of chitosan solids into the reaction system. After about 10 minutes, when the system is stable, remove the ice-water bath; after 2 hours of reaction at room temperature, stop the reaction; Precipitate in ether (order storage at -26°C for 3h). Then, the precipitate was suction-filtered under reduced pressure, washed once with diethyl ether, and then washed several times with acetone, the solid was collected and dissolved with an appropriate amount of distilled water, and the pH was adjusted to neutral with 1M NaOH; then the sample solution was tran...
Embodiment 3
[0049] Embodiment 3, cell uptake experiment:
[0050] (1) Take the HepG2 cells in the logarithmic growth phase, digest with trypsin for 1 min, add 1 mL of DMEM cell culture medium to neutralize the trypsin, blow the cells to peel them off from the culture dish, transfer them to a centrifuge tube, and centrifuge Discard the medium, pipette 1mL of new DMEM medium again, pipette to disperse the cells evenly, count, and count 2×10 cells per well. 5 The density of cells was seeded into a 12-well cell culture dish, and the cells were pipetted evenly with a 1mL pipette gun, at 37°C, 5% CO 2 Cultivate in the incubator for 24h.
[0051] (2) Remove the medium with a pipette gun and wash once with PBS buffer solution. Then, 1 mL of the nanoparticle solution mixed in a volume ratio of 1:1 was added. Place it in the cell culture phase at 37°C for 4h.
[0052] (3) Remove the culture medium with a pipette gun, wash with PBS buffer solution three times, 5min each time, 1mL / plate, and fina...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More