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Suspension cultivation method of human cranial mesenchymal stem cell-derived neuron-like cells

A technology of neuron-like cells and mesenchymal stem cells, applied in the field of suspension culture of neuron-like cells

Active Publication Date: 2019-01-04
ZHEJIANG PROVINCIAL PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cell transplantation therapy provides a new direction for the treatment of the above diseases, but because the nerve cells used for cell transplantation therapy cannot be obtained and exogenous nerve cells are difficult to culture and expand, therefore, it is necessary to find a method that can construct neural stem cells under in vitro conditions. The perfect three-dimensional tissue carrier to maintain the differentiation and proliferation of cells, and at the same time, the culture method that can promote tissue regeneration and function in the transplanted defect tissue has become the key to treatment

Method used

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  • Suspension cultivation method of human cranial mesenchymal stem cell-derived neuron-like cells
  • Suspension cultivation method of human cranial mesenchymal stem cell-derived neuron-like cells
  • Suspension cultivation method of human cranial mesenchymal stem cell-derived neuron-like cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Isolation of Human Calvaria Mesenchymal Stem Cells

[0021] will be 10cm 3 Skull bone slices were minced with a medical electric drill saw, transferred to a 50mL centrifuge tube, added 25mL of normal saline, mixed well, placed in a low-temperature shaker, at 8°C, with a speed of 100-200 rpm, Shake for 15 minutes, filter with a 200-mesh sieve, centrifuge the filtrate at 1000 rpm for 6 minutes, remove the supernatant, suspend the precipitate with cell culture medium, take a small amount of suspension for counting by an automatic hematology analyzer, and measure 2×10 5 / cm 2 Inoculation density to 25cm 2 Place in a plastic cell culture bottle at 37°C in an incubator containing 5% CO2. After 48 hours, pour it out to remove unattached cells, replace with fresh culture medium, and then change the medium every 2 days. When the cells grow to 90% confluent, Digested and passaged to P3 passage, obtained fibrous human skull mesenchymal stem cells with a single shape....

Embodiment 2

[0022] Example 2: Human calvarial mesenchymal stem cells differentiated into neuron-like cells in vitro

[0023] The P3 generation human calvarial mesenchymal stem cells obtained by the method in Example 1 were inoculated into the induction medium, 37°C, 5% CO 2 Induced culture in the incubator for 21 days, wherein the medium was changed every 2 days, the induced cells were collected for identification, and mature neuron-like cells were obtained.

[0024] The composition of the final concentration of the induction solution: 1×N2, 1×B27, 50 ng / ml b-FGF, and the solvent is DMEM / F12 basal culture medium (purchased from Corning Company). N2 and B27 were purchased from Gibco, and b-FGF was purchased from Peprotech.

Embodiment 3

[0025] Example 3: Culture and proliferation of neuron-like cells derived from human skull mesenchymal stem cells

[0026] The neuron-like cells obtained by the method in Example 1 were inoculated into suspension gel-DMEM medium prepared with different concentrations of gellan gum, and placed at 37°C / 5%CO 2 Routinely culture and proliferate in an incubator, and collect cells for observation and detection.

[0027] Preparation of suspension gel: finished gellan gum (Gelrite @ ) after heating to prepare a solution, add calcium chloride solution to cool, mix with DMEM / F12 basic culture medium and place in a culture container. Finished gellan gum (Gelrite @ ) are commercially available.

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Abstract

The invention relates to a suspension cultivation method of human cranial mesenchymal stem cell-derived neuron-like cells. The suspension cultivation method comprises the following steps: extracting mesenchymal stem cells by taking human skull fragments or scraps as tissue sources, performing cultivation and incubation, and inoculating to induced liquid by the density of 10<4> to 10<6> pieces / cm<2>; heating a finished product gellan gum to more than 90 DEG C and preparing a solution with the mass percentage ratio of 1 to 3 percent for later use; observing the density and the pore diameter of anetwork structure in suspending gel prepared from a gellan gum solution with different concentration by an electron microscope or a laser scanning confocal microscope; and transferring the selected neural stem cells into a suspending gel-DMEM culture medium to complete cultivation and proliferation. The suspension cultivation method has the following beneficial effects: by the method, the technical defects of the existing laboratory condition and the restriction of objective factors such as gravity are remedied; the biological behavior and the functional morphological characteristic of the neural stem cells are combined; and the suspending gel-DMEM culture medium which is prepared from the gellan gum subjected to modification treatment can effectively induce to differentiate human cranialmesenchymal stem cells.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a suspension culture method of neuron-like cells derived from human skull mesenchymal stem cells. Background technique [0002] Traumatic brain injury, cerebrovascular accident and spinal cord injury are common and frequently-occurring diseases worldwide, and they are also the primary factors leading to post-injury disability. Although great progress has been made in the first aid and early rehabilitation of traumatic brain injury and cerebrovascular accident, there is still a lack of effective treatment methods for neurological deficits in the later stages of traumatic brain injury and cerebrovascular accident. Cell transplantation therapy provides a new direction for the treatment of the above diseases, but because the nerve cells used for cell transplantation therapy cannot be obtained and exogenous nerve cells are difficult to culture and expand, therefore, it is necessa...

Claims

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Application Information

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IPC IPC(8): C12N5/0793
CPCC12N5/0619C12N2506/1353C12N2533/70
Inventor 卢刚麻育源
Owner ZHEJIANG PROVINCIAL PEOPLES HOSPITAL
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