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Preparation method and application method of double network hydrogel for three-dimensional cell culture

A three-dimensional culture and hydrogel technology, which is applied in general culture methods, cell culture supports/coatings, biochemical equipment and methods, etc., can solve the problem of balancing cell compatibility, mechanical strength and long-term stable culture , can not promote cell spreading, poor cell adhesion and other problems, to achieve the effect of promoting cell adhesion and proliferation, improving degradation rate and high mechanical strength

Active Publication Date: 2022-03-29
SOUTHEAST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] It is worth noting that a single HA hydrogel makes the cell shape always spherical in 3D culture due to poor cell adhesion, which cannot promote cell spreading.
[0005] GelMA is beneficial to cell growth and proliferation due to its excellent cell adhesion and cytocompatibility similar to gelatin, but its biggest disadvantage is its fast degradation rate.
Therefore, it is difficult for single-network hydrogels to simultaneously meet the requirements of cell compatibility, mechanical strength and long-term stable culture.

Method used

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  • Preparation method and application method of double network hydrogel for three-dimensional cell culture
  • Preparation method and application method of double network hydrogel for three-dimensional cell culture
  • Preparation method and application method of double network hydrogel for three-dimensional cell culture

Examples

Experimental program
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Effect test

Embodiment 1

[0032] (1) Preparation of Sortase A Enzymatic Crosslinking Substrate Methacrylated Hyaluronic Acid (HAMA-P)

[0033] Weigh 23.55 mg of HAMA (n(-C=C-)=50 μmol, 1 equiv) and completely dissolve it in 8 mL of ultrapure water, add 16 mL of 300 mM triethanolamine (TEOA) buffer solution (pH 8.0) and mix well, pass through N 2 15min to remove the oxygen in the solution. Subsequently, weigh 65mg P 1 (75μmol, 1.5equiv) or 76mg P 2 (75μmol, 1.5equiv) was added to the above mixture, at room temperature, N 2 The reaction was continuously stirred in the atmosphere for 24h. After the reaction was completed, a large amount of absolute ethanol was added to precipitate the reaction product, centrifuged at 5000 rpm / min for 15 min, and the obtained precipitate was dissolved in ultrapure water again. The above purification steps were repeated twice. Finally, the product was dialyzed against ultrapure water (Mw 3500 Da) for 4 days at room temperature, freeze-dried for 3 days, and stored at -...

Embodiment 2

[0042] The difference between (1) in this embodiment and embodiment 1 is: take 32.5mg P 1 (37.5μmol, 0.75equiv) or 38mg P 2 (3.75μmol, 0.75equiv) was added to the above-mentioned TEOA mixed solution containing HAMA, at room temperature N 2 Reaction in the atmosphere for 24h. HAMA-P 1 / P 2 The double bonds are not completely replaced, and can be further cross-linked by ultraviolet light to form a single-component hyaluronic acid double network hydrogel, which will not be described in this invention. Others are the same as in Example 1.

Embodiment 3

[0044] The difference between (2) in this embodiment and Example 1 is: take 1% (w / v) of HAMA-P and 5% (w / v) of GelMA and dissolve them completely in the DPBS buffer containing 0.5% LAP , adding 80 μM Sortase A enzyme, forming a gel for about 1 min, and then cross-linking with ultraviolet light for 10 s to prepare a double network hydrogel.

[0045] Others are the same as in Example 1.

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Abstract

The invention discloses a preparation method and an application method of a double-network hydrogel for three-dimensional cell culture, which belongs to the field of biomedical materials. Firstly, a methacrylated hyaluronic acid grafted with a Sortase A enzyme-specific substrate short peptide is synthesized. (HAMA) conjugate (HAMA‑P), the substrate is enzymatically cross-linked with a certain concentration of Sortase A to obtain an injectable hyaluronic acid single network hydrogel. The method has the advantages of readily available raw materials, mild reaction conditions, short reaction time, and the like. Then, enzymatically and optically double-crosslinked hyaluronic acid-gelatin double network hydrogels were prepared. (GelMA) hydrogel as the second reinforcement network. The double-network hydrogel prepared by the invention provides a suitable scaffold and three-dimensional microenvironment for cell adhesion and growth, and has good application prospects in injectable tissue engineering, 3D printing, and three-dimensional cell culture.

Description

technical field [0001] The invention belongs to the field of biomedical materials, and in particular relates to a preparation method and an application method of a double-network hydrogel for three-dimensional cell culture. Background technique [0002] Hydrogels are widely used as matrix materials to support tissue regeneration, as delivery vehicles for cells or drugs, or for three-dimensional cell culture applications. Hyaluronic acid hydrogel (HA) has the characteristics of good biocompatibility, not easy to degrade, high swelling ratio and structure highly similar to extracellular matrix, etc. It is a promising three-dimensional cell culture scaffold material. [0003] At present, HA hydrogels are mainly modified by chemical functional groups and cross-linked by adding cross-linking agents to form hydrogels with specific shape, mechanical hardness and biological activity. For example, -SH was covalently cross-linked to the HA backbone by reaction with dihydrazine sulfat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08J3/075C08J3/24C08J3/28C08L89/00C08L5/08C12N5/00
CPCC08J3/075C08J3/24C08J3/28C12N5/0068C12N5/0062C08J2389/00C08J2405/08C08J2305/08C12N2513/00C12N2533/80C12N2533/54
Inventor 黄宁平李小佩
Owner SOUTHEAST UNIV
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