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Double-layer gel system carrying mesenchymal stem cell spheres as well as preparation method and application of double-layer gel system

A double-layer gel and mesenchymal stem cell technology, applied in the field of double-layer gel system carrying mesenchymal stem cell spheres and its preparation, can solve the problems of aggravated injury, cell apoptosis, difficulty in generating neuron regeneration, and axon extension and functional recovery, etc., to achieve the effect of enhancing curative effect, promoting paracrine function, and good synergistic therapeutic effect

Pending Publication Date: 2022-12-02
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These reactive oxygen species will extensively undergo peroxidation reactions with proteins, lipids, etc., eventually leading to cell apoptosis and further aggravating damage.
At the same time, the limited regenerative capacity of the CNS makes it difficult to produce effective neuronal regeneration, axon extension, and functional recovery in the chronic phase of spinal cord injury.
At present, there are no reports on the joint construction of active oxygen scavenging gel, GelMA gel and three-dimensional MSC balls for the repair of spinal cord injuries

Method used

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  • Double-layer gel system carrying mesenchymal stem cell spheres as well as preparation method and application of double-layer gel system
  • Double-layer gel system carrying mesenchymal stem cell spheres as well as preparation method and application of double-layer gel system
  • Double-layer gel system carrying mesenchymal stem cell spheres as well as preparation method and application of double-layer gel system

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The preparation of embodiment 1 MSC ball

[0042] Dissolve low-melting point agarose in PBS to obtain an agarose solution with a mass concentration of 2%, and put it into a high-pressure steam sterilizer for sterilization. Add the agarose solution dropwise to a round-bottomed 96-well plate while hot, 50 μL per well, wait for cooling and solidification, and store at 4°C for later use.

[0043] After digesting and counting MSCs, they were planted in a 96-well plate pre-coated with agarose at a density of 15,000 per well, 150 μL per well. The 96-well plate was placed in a centrifuge and centrifuged at 1000rpm for 8min to aggregate the MSCs.

[0044] The MSCs were cultured in a cell incubator for 24 hours to obtain MSC spheres.

[0045] like figure 1 As shown, the prepared MSC spheres showed a good spherical shape, and the dense connection between MSCs could be seen on the surface.

[0046] like figure 2 It was shown that compared with the same amount of MSCs, the pre...

Embodiment 2

[0047] The construction of embodiment 2 double-layer gel system

[0048]Dissolve 0.5g of 4-(bromomethyl)phenylboronic acid and 0.1g of N,N,N',N'-tetramethyl-1,3-propanediamine in 10mL of dimethylformamide, under oil bath conditions The reaction was stirred at constant temperature (60° C.) for 24 h. After the reaction, the reactant was poured into 1000 mL pre-cooled tetrahydrofuran to obtain a white flocculent precipitate, which was collected by centrifugation (2000 rpm, 3 min). The product was washed three times with 20 mL of tetrahydrofuran, and dried in a vacuum oven for 48 hours to obtain the active oxygen responsive cross-linking agent TPA. image 3 It is the chemical structure of TPA and its TPA NMR spectrum. The characteristic peaks in the hydrogen spectrum conform to the structure of TPA, indicating the successful synthesis of TPA. Figure 4 It shows that the solid gel can be formed quickly after mixing equal volumes of TPA and polyvinyl alcohol. According to the re...

Embodiment 3

[0053] Example 3 Double-layer gel system carrying MSC spheres

[0054] Collect 12 MSC spheres (containing 15,000 cells) in an EP tube, remove the supernatant, add 20-30 μL of GelMA solution, mix well, perform UV cross-linking for 10-15 seconds, and then wrap the outer layer of active oxygen scavenging gel with A double-layer gel system carrying MSC spheres can be obtained by wrapping on the inner layer gel in the same way.

[0055] like Figure 8 As shown, the bilayer gel system can effectively carry MSC spheres.

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Abstract

The invention discloses a preparation method of a double-layer gel system carrying mesenchymal stem cell spheres, which comprises the following steps: carrying out oil bath stirring reaction on 4-(bromomethyl) phenylboronic acid and N, N, N ', N'-tetramethyl-1, 3-propane diamine in dimethylformamide; pouring the reactant into tetrahydrofuran to obtain a white precipitate which is a cross-linking agent TPA with active oxygen responsiveness; dropwise adding polyvinyl alcohol into a long strip shape, and dropwise adding TPA to obtain active oxygen scavenging gel on the outer layer; adding methylacryloylated gelatin GelMA into water to obtain a GelMA solution, then adding the GelMA solution into the MSC spheres, mixing, and carrying out ultraviolet crosslinking to obtain inner-layer GelMA gel carrying the MSC spheres; and wrapping the outer-layer active oxygen scavenging gel on the inner-layer GelMA gel carrying the MSC balls in a wrapping manner to obtain the double-layer gel system. The invention also discloses the double-layer gel system obtained by the method and application of the double-layer gel system in preparation of products for treating spinal cord injury. The double-layer gel system is good in biocompatibility, excellent in mechanical property and good in repeatability, and has a better synergistic treatment effect.

Description

technical field [0001] The invention belongs to the fields of preparation material preparation and regenerative medicine, and in particular relates to a double-layer gel system carrying mesenchymal stem cell spheres, a preparation method and application thereof. Background technique [0002] Central nervous system (CNS) injury diseases, such as traumatic brain injury and spinal cord injury, will lead to the death of a large number of nerve cells, microglia, and astrocytes in the damaged area, resulting in temporary or permanent neurological dysfunction in patients. Cause organ dysfunction, paralysis and even death. In the acute stage of spinal cord injury, CNS ischemia, massive cell death, inflammatory cell infiltration, and a large amount of reactive oxygen species are produced during these processes. These reactive oxygen species will extensively undergo peroxidation reactions with proteins, lipids, etc., eventually leading to cell apoptosis and further aggravating damage...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/06A61K35/28A61K31/14A61K47/42A61K47/32A61P25/00
CPCA61K9/06A61K35/28A61K31/14A61K47/42A61K47/32A61P25/00A61K2300/00
Inventor 高建青曹坚周彦军
Owner ZHEJIANG UNIV
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