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Silkworm cocoon imitating support and preparation method and application thereof

A silkworm cocoon and solution technology, applied in the field of silkworm cocoon scaffolds, can solve the problems of inability to combine cell preservation with cell therapy, restrict the direct application of resuscitated cells, reduce the effect of cell cryopreservation, etc., achieve high safety and effectiveness, and promote adhesion. and growth, high cell viability

Pending Publication Date: 2022-05-13
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, 3D scaffolds composed of natural or synthetic polymers suffer from poor thermal conductivity and poor mechanical properties, which reduce the cryopreservation effect of cells.
In addition, traditional 3D scaffolds are poorly resistant to ice crystals and suffer from mechanical or structural damage after cryopreservation, leading to poor cell survival
Most importantly, most scaffold materials cannot combine cell preservation well with cell therapy due to their poor biocompatibility, which greatly limits the direct clinical application of resuscitated cells.

Method used

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  • Silkworm cocoon imitating support and preparation method and application thereof
  • Silkworm cocoon imitating support and preparation method and application thereof
  • Silkworm cocoon imitating support and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] (1) Cut the silkworm cocoons into small pieces, and boil them with a 0.02M sodium carbonate solution prepared with deionized water. Boil for 1 hour, separate the supernatant and undissolved matter, wash the undissolved matter with water, 3 times, 20 minutes each time. After drying, put it into lithium bromide solution (9.3M) at a ratio of 1:50 (g / mL) and place it at 60°C for 4 hours. After dissolution, the solution turned clear amber. Dialysis (M w 12000) for 2 days, 9000rpm, centrifuge for 20 minutes, discard the precipitate, and freeze-dry for 3 days to obtain silk fibroin.

[0032] (2) supernatant in step (1) is dialyzed in water (M w 12000) for 2 days, remove the sodium carbonate solution, freeze-dry and collect to obtain sericin.

[0033] (3) The silk fibroin prepared in step (1) is dissolved in hexafluoroisopropanol to make a solution of 7.5% w / v

[0034] (4) Add 5% w / w polyalanine to the solution prepared in step (3) and stir for 4 hours.

[0035] (5) Add...

Embodiment 2

[0040] The neutrophils were co-cultured with the imitation silkworm cocoon scaffold prepared in Example 1 for 30 minutes, and 1 mL of cell freezing solution was used to replace the medium and transferred to a cryopreservation tube. Then, the cryovials were placed in a freezer box (Nalgene, at a freezing rate of 1°C / min in a -80°C freezer) overnight, and then stored in liquid nitrogen for 2 days. After resuscitating in a water bath at 37°C, the cells were digested and collected, and the cells were stained with a live dead cell staining kit. The experimental results showed that the proportion of living cells was 69% ( image 3 ).

Embodiment 3

[0042] Human umbilical cord blood mesenchymal stem cells were co-cultured with the imitation silkworm cocoon scaffold prepared in Example 2 for 30 minutes, and 1 mL of cell freezing solution was used to replace the culture medium and transferred to a cryopreservation tube. Then, the cryovials were placed in a freezer box (Nalgene, at a freezing rate of 1°C / min in a -80°C freezer) overnight, and then stored in liquid nitrogen for 2 days. After resuscitating in a water bath at 37°C, the cells were digested and collected, and the cells were stained with a live dead cell staining kit. The experimental results showed that the proportion of living cells was 90.9%.

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Abstract

The invention discloses a silkworm cocoon-imitating bracket as well as a preparation method and application thereof. The silkworm cocoon-imitating bracket disclosed by the invention has a unique core-shell structure, can be optimized according to chemical and physical characteristics, and can be used for carrying out low-temperature preservation on biological samples with minimum low-temperature damage. According to the invention, adhesion and growth of cells can be promoted, rapid and uniform rewarming of cryopreserved cells can be realized, and activity and proliferation of the cells can be maintained to the greatest extent after freezing and thawing, so that immune cells and stem cells can be effectively cryopreserved. The tissue engineering scaffold constructed by the stem cell-silkworm cocoon-imitating scaffold retains a high cell survival rate after cryopreservation, and can be directly implanted into a damaged part to promote tissue regeneration and repair. Therefore, the silkworm cocoon imitating scaffold has high safety and effectiveness in the aspect of long-term preservation of cell and tissue engineering scaffolds, and has wide application prospects in the fields of basic research, cell therapy, tissue engineering and the like.

Description

technical field [0001] The present invention relates to material preparation and biomedical fields such as surface modification, cell cryopreservation, tissue repair and disease treatment, and especially relates to a new bottom-up preparation technology integrating electrospinning, in-situ surface functionalization and cryoforming. Obtain a cocoon-like holder. Background technique [0002] Cell therapy, which involves introducing healthy cells into a patient's body to relieve troublesome physical conditions, has gradually become an effective alternative to traditional treatments, providing new treatment options for humans. However, there are still some obstacles hindering the widespread clinical application of cell therapy, such as ethical controversies related to embryonic stem cells, cases of treatment fraud, and cell biosafety for patients. In particular, cells are living biological species, and it is difficult to obtain enough high-quality cells for patient transplantat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): D01F8/02D01F8/12D01F8/16D06M15/15A61L27/38A01N1/02D06M101/10D06M101/34D06M101/30
CPCD01F8/02D01F8/12D01F8/16D06M15/15A61L27/3834A01N1/0263D06M2101/10D06M2101/34D06M2101/30
Inventor 陈建美宁兴海
Owner NANJING UNIV
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