Method for freezing preservation nervous stem cells

A neural stem cell and cryopreservation technology, applied in the field of biotechnology and tissue engineering, can solve the problems of time and space matching, source and quantity can not meet the demand, etc., achieve good integrity, reduce osmotic shock, and reduce damage

Inactive Publication Date: 2007-11-14
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, it has been tried to transplant neural stem cells into striatum and other cranial neuropathy areas and injured spinal cord in various animal models and human body, and achieved certain curative effect, but it should be widely used in the field of clinical treatment. The quantity is still far from meeting the demand
Moreover, there is also the problem of matching the transplanted cells from the patient to be transplanted in time and space

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Fixation of neural stem cells in collagen

[0022] Type I rat tail collagen was purchased from Upstate Company, and the collagen was dissolved in 0.2% (v:v) sterile acetic acid solution with a concentration of 100 mg / 28.1 ml and a pH of 3-4. First, adjust the pH of the collagen to 7.4 with 0.5 N NaOH; then, centrifuge the collected neurospheres (the diameter of "neurospheres" for cryopreservation should not exceed 100 μm), count the number of neurospheres under a microscope, and use 2X cell culture medium ( DMEM:F12:1640 / 1:1:1; N2; bFGF; EGF) were resuspended. Take the neurosphere suspension and mix equal volumes of collagen, so that the density of the collagen-neurosphere mixture is 1×10 4 pieces / ml. All solutions containing collagen were placed in an ice bath to prevent solidification of the collagen. The collagen-neurosphere mixture was transferred to 2ml cryopreservation tubes, 250 μl per tube (the thickness of the gel was about 3 mm), and the collagen...

Embodiment 2

[0023] Embodiment 2: drawing the standard curve of the concentration change in the import process of cryoprotectant DMSO

[0024] The concentration of DMSO can be obtained by measuring its osmotic pressure value. First, prepare cryoprotectant solution: cryoprotectant is DMSO, solvent is neural stem cell culture fluid (DMEM:F12:1640 / 1:1:1; N2; bFGF; EGF), DMSO concentration is from 0% to 20% (v / v), the interval is 2.5% (v / v), the osmotic pressure of all cryoprotectant solutions prepared is measured with an osmometer, each data point is measured repeatedly 3 times, the average value and variance are calculated, and the regression equation is obtained , to draw a standard curve.

Embodiment 3

[0025] Embodiment 3: The osmotic equilibrium time determination of cryoprotectant DMSO in neurosphere-collagen gel

[0026] Add 250 μl of neurosphere-collagen mixture (gel thickness is about 3 mm) to the cryovial, and the density of neurospheres is 1×10 4 pcs / ml; after the collagen solidified into a gel at 37°C for 2 hours, 250 μl of 15% DMSO solution was quickly added to it at one time, and the timer was started at the same time. 10 μl samples were taken at 0 min, 3 min, 5 min, 10 min, 15 min, 20 min, 40 min, and 60 min, and the osmotic pressure was measured with an osmometer at room temperature (15°C). Then draw the change curve of osmotic pressure according to the measurement results. When the osmotic pressure changes very little and almost no longer changes, it is regarded as osmotic equilibrium, and the time point when the osmotic equilibrium starts is the osmotic equilibrium time. A control group was set up in the experiment, and the density of neurospheres in the cont...

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Abstract

The invention provides a neural stem cell cryopreservation method, which belongs to cell biotechnology field. Its characteristic is fixes directly the cell embedding in certain density collagen culture medium, saves in the tube to become the rubber frozen; Through the experiment determined that freezing protecting agent DMSO inducts the time best in the collagen - cell compound;15%2XDMSO will induct in the collagen - cell compound, the room temperature is balanced 15 minutes ; frozen pipe will be diverted to control speed cooling frozen boxes, boxes and frozen at -85 degree C refrigerator placed to ensure that the rate of cooling -1 degree C / min, four hours after the turn -196 degree C liquid nitrogen preservation. The effect of this invention and the benefits of this method is to reduce the infiltration process of DMSO on both sides of the membrane pressure differential caused injury. After the recovery, the cell - collagen compound maintains the good integrity, the cellular form and activeness is good, may use in vivo transplant directly.

Description

technical field [0001] The invention belongs to the fields of biotechnology and tissue engineering, and relates to a method for cryopreserving cells. Background technique [0002] Tissue engineering technology is to inoculate normal tissue cells cultured and expanded in vitro into a biomaterial that has excellent cytocompatibility and can be degraded and absorbed by the body to form a complex, and then implant the cell-biomaterial complex into human tissue The damaged parts of organs, while being gradually degraded and absorbed by the body, the cells continue to proliferate and differentiate to form new tissues with the same shape and function as the corresponding tissues and organs, so as to achieve the purpose of repairing trauma and reconstructing functions. [0003] Collagen, because of its non-antigenicity and good biocompatibility, can not only promote cell growth and metabolism, participate in tissue healing, induce tissue regeneration, but also, after implanted in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06A01N1/02
Inventor 葛丹马学虎刘天庆崔占峰高志新李香琴
Owner DALIAN UNIV OF TECH
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