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3D printing bio-ink as well as preparation and application thereof

A bio-ink and 3D printing technology, applied in the field of 3D bio-printing, can solve the problems of long-term existence of bio-ink, poor stability of cytokines, unsatisfactory 3D cell model culture in vitro, etc., to achieve strong toughness, maintain integrity, The effect of improving the utilization rate

Active Publication Date: 2022-04-12
SHANGHAI CHEERMORE BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, most of the bio-inks have good mechanical properties and biocompatibility, but in the face of in vitro 3D cell model culture, how to construct complex extracellular matrix has become one of the urgent problems to be solved. At present, many scholars will grow some cells Factors are added to bioink, but the stability of cytokines is poor and cannot exist in bioink for a long time. Therefore, the cultivation of many in vitro 3D cell models is not satisfactory

Method used

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  • 3D printing bio-ink as well as preparation and application thereof
  • 3D printing bio-ink as well as preparation and application thereof
  • 3D printing bio-ink as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 A kind of 3D printing bio-ink and preparation method thereof

[0044] The preparation process is as figure 1 shown.

[0045] ①Dissolve 0.85g of sodium alginate powder in 5ml of deionized water, sterilize by high-pressure steam after dissolution, and store at 4°C.

[0046] ② Prepare 5% (m / v) hyaluronic acid aqueous solution and 3% (m / v) chitosan aqueous solution at 4°C, take 2ml of each, mix according to the volume ratio of 1:1, add 500μL of 50mg / ml collagen I and 500 μL 50mg / ml fibronectin, stored at 4°C.

[0047] ③Extract the cell membranes of HSF and HaCaT cells using the membrane protein cell fraction extraction kit (Shanghai Beyontine Biotechnology Co., Ltd. P0033), adjust the number of cells to obtain a cell membrane of 50 mg / ml, and add 500 μL of 10 mg / ml laminin , 500 μL of 10 mg / ml elastin and 500 μL of 10 mg / ml sphingosine phosphate were mixed with 500 μL of HSF cell membrane and 500 μL of HaCaT cell membrane, and left at 4°C for 15 minutes to ...

Embodiment 2

[0055] Example 2 The finished product of 3D cell printing

[0056] 1. Select human skin fibroblasts (HSF) and human immortalized keratinocytes (HaCaT) that have been subcultured for 24-48 hours, digest them with Trypsin-EDTA and centrifuge for 5 minutes, and resuspend HaCaT with the upper cell bioink prepared in Example 1. cells so that the concentration of HaCaT cells in the bioink was 1 x 10 6 cells / ml, resuspend HSF cells with the lower cell bioink prepared in Example 1, so that the concentration of HSF cells in the bioink is 5.5×10 7 cells / ml (be careful to blow gently to avoid air bubbles, which will affect printing), and put them into low-temperature barrels to prepare for printing. At the same time, other bioinks were used to prepare the control group, as follows:

[0057] Experimental group: the bioink prepared in Example 1 was used.

[0058] Control group 1: using the cell membrane encapsulation system removed from step ③ in Example 1, and the remaining bioinks pre...

Embodiment 3

[0066] Example 3 Finished HE staining of 3D cell printing

[0067] 1. Take the printed product of step 2 in Example 2 (the lower layer is a printed tissue composed of human skin fibroblasts (HSF) and the upper layer is composed of human immortalized keratinocytes (HaCaT), soak and culture in DMEM medium for 7 days, and replace it with Epi -life medium for gas-liquid critical culture, HaCaT exposed culture, HSF submerged culture, in order to promote the formation of cuticle. The printed tissues at the 12th week after printing (that is, 11 weeks of air-liquid critical culture) were embedded in O.C.T.Compound, fixed at -80°C for 24 hours, and taken out to prepare slices.

[0068] 2. Cool down the freezer slicer (Leica CM1950) and blades in advance, wait until the temperature of the box is -18°C, and the temperature of the quick-freezing table is -25°C to start slicing, and cut slices with a thickness of 8 μm.

[0069] 3. Immediately fix the slices, and fix with AAF mixed fixativ...

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Abstract

The invention belongs to the technical field of 3D biological printing, and particularly relates to 3D printing biological ink as well as preparation and application thereof. The biological ink comprises a sodium alginate solution, a hyaluronic acid-protein-chitosan system and a cell membrane wrapping system. The toughness of the bio-ink is improved, a hyaluronic acid-protein-chitosan matrix is constructed, meanwhile, fibronectin, laminin and elastin are added, and the proteins not only can stably exist in the bio-ink, but also can promote the growth of in-vitro cells. By applying the cell membrane wrapping technology, the utilization rate of the protein is greatly improved. According to the invention, laminin, elastin and sphingosine phosphate wrapped by the cell membrane are added into the bio-ink, so that the acting protein and the printing cell are specifically combined better, the degradation rate of the protein is reduced, long-term protein nutrition support is provided for in-vitro culture of a printing structure, and the integrity of a printing result can be maintained for a long time.

Description

Technical field: [0001] The invention belongs to the technical field of 3D bioprinting, and in particular relates to a 3D printing bioink and its preparation and application. Background technique: [0002] 3D printing (3DP), a type of rapid prototyping technology, also known as additive manufacturing, is a method based on digital model files, using bondable materials such as powdered metal or plastic, and constructing them by layer-by-layer printing. object technology. The technology has applications in jewelry, footwear, industrial design, architecture, engineering and construction (AEC), automotive, aerospace, dental and medical industries, education, geographic information systems, civil engineering, firearms, and others. Today, 3D printing is one of the representative cutting-edge technologies, and its application value has been recognized by many people in the industry. In the medical field, 3D printing has gradually penetrated into many subdivided application scenari...

Claims

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Application Information

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IPC IPC(8): A61L27/20A61L27/22A61L27/38A61L27/56A61L27/60C12N5/00C12N5/071
Inventor 朱才彬
Owner SHANGHAI CHEERMORE BIOLOGICAL TECH CO LTD
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