Construction method of human nerve stem cell bank

Abstract

The invention discloses a construction method of a human nerve stem cell bank. The method comprises the following steps: carrying out human urine separation to obtain human kidney epithelial cells, subculturing the human kidney epithelial cells, partially cryopreserving the human kidney epithelial cells, inducing the subcultured human kidney epithelial cells to form nerve stem cells, separating the nerve stem cells obtained after induction, carrying out amplification culturing, identifying the nerve stem cells, cryopreserving the confirmed nerve stem cells, coding and warehousing. The method avoids the use of fetal bovine serum in the whole process, eliminates the introduction of foreign proteins, and reduces hidden troubles; the method allows the successfully induced stem cells with activity to be preserved in order to form the bank, and the stem cells can be provided for clinic use within 3d; and a cryopreservation mode in the method is characterized in that nerve bulbs with a certain size are cryopreserved, a protection agent is introduced step by step in the cryopreservation process, and the cooling rate is set in grading, so recovered nerve stem cells still have high cell viability and activity, and have significantly higher than cell viability and activity than the recovered nerve stem cells preserved through current routine slow low-temperature cryopreservation methods.

Description

technical field

[0001] The invention relates to a method for constructing a cell bank, in particular to a method for constructing a human neural stem cell bank. Background technique

[0002] Neural Stem Cell (NSC for short) is a kind of stem cell existing in embryonic or adult brain, spinal cord and other tissues. It is a kind of mother cell with division potential and self-renewal ability, which can be differentiated into Various types of cells in nervous tissue such as neurons, astrocytes, and oligodendrocytes.

[0003] The regenerative repair properties of neurons have always been an insurmountable obstacle in medical science. The structure of the central nervous system is fragile and vulnerable, and it determines intelligent activities, so central nervous system diseases and their sequelae (including Parkinson's disease, Alzheimer's disease, stroke, malignant glioma, and central and peripheral nerve damage, etc.) ) has always been a persistent disease affecting human h...

Examples

Embodiment 1

[0033] Example 1 Construction method of human peripheral blood immune cell bank

[0034] (1) Isolation of human kidney epithelial cells: Take 150-200ml of mid-section urine into a sterile container, and add an appropriate amount of double antibody in advance to the sterile container; transfer the above urine into a 50ml centrifuge tube, and centrifuge at 400g for 10min; after centrifugation, discard For the supernatant, resuspend the cells with PBS containing double antibodies or normal saline, and then centrifuge at 400g for 10min; 0.1% gelatin pre-coated T25 culture flask; place the culture flask inoculated with cells at 37°C, 5% CO 2 and the oxygen concentration of the incubator is always controlled to be 5.0%; wherein: the formula of the kidney epithelial cell culture medium is that every 100ml culture medium comprises: 5ml Ultroser G (Pall Company, product number 15950-017), 0.5ml Non-essential amino acids (Life technologies company, product number 11140050), 0.5ml Gluta...

Embodiment 2

[0044] Example 2 Cryopreservation of human induced neural stem cells

[0045] (1) Take the induced P4 neural stem cells that can be passaged, transfer them to a 50ml centrifuge tube, centrifuge them (50g, 3-5min), remove the supernatant, and add 1ml of Accutase cells to the centrifuge tube The digestion solution was digested at 37°C for 5 minutes.

[0046] (2) After terminating the digestion with DMEM / F12 medium, centrifuge at 50g for 3-5min; discard the supernatant, collect the cells, and base the cell concentration on cryopreservation at 5×10 6 cells / ml cryopreservation solution to calculate the volume of cryopreservation solution to be used; suspend the cells with the above-mentioned volume of cryopreservation solution, mix well, and distribute into cryopreservation tubes; the composition of the cryopreservation solution is 10% DMSO + 90% FBS.

[0047] (3) Transfer the cryopreservation tube to the programmed cooling device, the cooling rate of the programmed cooling devic...

technical field

[0001] The invention relates to a method for constructing a cell bank, in particular to a method for constructing a human neural stem cell bank. Background technique

[0002] Neural Stem Cell (NSC for short) is a kind of stem cell existing in embryonic or adult brain, spinal cord and other tissues. It is a kind of mother cell with division potential and self-renewal ability, which can be differentiated into Various types of cells in nervous tissue such as neurons, astrocytes, and oligodendrocytes.

[0003] The regenerative repair properties of neurons have always been an insurmountable obstacle in medical science. The structure of the central nervous system is fragile and vulnerable, and it determines intelligent activities, so central nervous system diseases and their sequelae (including Parkinson's disease, Alzheimer's disease, stroke, malignant glioma, and central and peripheral nerve damage, etc.) ) has always been a persistent disease affecting human h...

Examples

Embodiment 1

[0033] Example 1 Construction method of human peripheral blood immune cell bank

[0034] (1) Isolation of human kidney epithelial cells: Take 150-200ml of mid-section urine into a sterile container, and add an appropriate amount of double antibody in advance to the sterile container; transfer the above urine into a 50ml centrifuge tube, and centrifuge at 400g for 10min; after centrifugation, discard For the supernatant, resuspend the cells with PBS containing double antibodies or normal saline, and then centrifuge at 400g for 10min; 0.1% gelatin pre-coated T25 culture flask; place the culture flask inoculated with cells at 37°C, 5% CO 2 and the oxygen concentration of the incubator is always controlled to be 5.0%; wherein: the formula of the kidney epithelial cell culture medium is that every 100ml culture medium comprises: 5ml Ultroser G (Pall Company, product number 15950-017), 0.5ml Non-essential amino acids (Life technologies company, product number 11140050), 0.5ml Gluta...

Embodiment 2

[0044] Example 2 Cryopreservation of human induced neural stem cells

[0045] (1) Take the induced P4 neural stem cells that can be passaged, transfer them to a 50ml centrifuge tube, centrifuge them (50g, 3-5min), remove the supernatant, and add 1ml of Accutase cells to the centrifuge tube The digestion solution was digested at 37°C for 5 minutes.

[0046] (2) After terminating the digestion with DMEM / F12 medium, centrifuge at 50g for 3-5min; discard the supernatant, collect the cells, and base the cell concentration on cryopreservation at 5×10 6 cells / ml cryopreservation solution to calculate the volume of cryopreservation solution to be used; suspend the cells with the above-mentioned volume of cryopreservation solution, mix well, and distribute into cryopreservation tubes; the composition of the cryopreservation solution is 10% DMSO + 90% FBS.

[0047] (3) Transfer the cryopreservation tube to the programmed cooling device, the cooling rate of the programmed cooling devic...

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