Cell cross-linking agent material for three-dimensional hepatocyte sphere 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.
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Embodiment 1
[0027] Example 1. ALG-GA-N (CH 3 ) 2 Preparation
[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 for 30min at room temperature. Add 10 g of GA (Compound 1) to the solution and stir for 20 min. Then, 2.3 mL of N,N-dimethylethylenediamine was added, and the mixture was stirred at room temperature for 12 hours. After the reaction solution is used to remove the solvent by the rotary evaporator, use the chromatographic column (volume ratio: CH 2 Cl2:MeOH:NH 3 H 2 O=15:1:1). After purification, the product was dissolved in ethyl acetate, allowed to stand at 0°C for 2h, filtered, and the filtrate was evaporated to dryness and vacuum dried to obtain a white powdery product, compound 2 (10.6g, 92%) ).
[0031] Step 2. ALG-GA-N (CH 3 ) 2 (DS=12%) Preparation:
[0032] No. 1, scu-GA-N(CH 3 ) 2 Synthesis
[0033] 3.2g compound GA-N(CH 3 ) 2 (Compound 2) was dissolved in 30mL pyridine, 4.7g succini...
Embodiment 2
[0044] Example 2. Preparation of hepatocyte cross-linking agent SCTS-GA-N(CH3)2:
[0045] 1. Synthesis of SCTS
[0046] The synthesis diagram of chitosan sulfate is attached image 3 As shown, add 45mL of HSO to a 500mL three-necked bottle 3 Cl, under ice bath conditions, slowly add 90 mL of 98% concentrated sulfuric acid dropwise to the three-neck flask, and stir for 15 min. Increase the stirring speed, slowly add 3g of chitosan solid to the reaction system, about 10 minutes later, when the system is stable, remove the ice-water bath; after reacting at room temperature for 2h, stop the reaction; slowly add the reaction solution dropwise to 1000mL of ice Precipitate in ether (-26°C and store for 3h). Then, the precipitate was filtered under reduced pressure, washed with ether once, and then washed with acetone several times. The solids were collected and dissolved with an appropriate amount of distilled water, and the pH was adjusted to neutral with 1M NaOH; then the sample soluti...
Embodiment 3
[0049] Example 3. Cell uptake experiment:
[0050] (1) Take the HepG2 cells in the logarithmic growth phase, digest them with trypsin for 1 min, add 1 mL of DMEM cell culture medium to neutralize the trypsin, pipette the cells to separate them from the culture dish, transfer them to a centrifuge tube, and centrifuge Discard the medium, re-pipette 1mL of new DMEM medium, pipette to disperse the cells evenly, count, and press 2×10 per well 5 The density of each cell is inoculated into a 12-well cell culture dish, and the cells are evenly pipetted with a 1mL pipette. At 37℃, 5% CO 2 Cultivate for 24h in an incubator.
[0051] (2) Remove the medium with a pipette, 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 4 hours.
[0052] (3) Remove the culture medium with a pipette, wash three times with PBS buffer solution, 5 min each time, 1 mL / plate, and finally remove all...
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