Passage method and application of human induced pluripotent stem cells

A pluripotent stem cell and cell technology, applied in the field of single cell passage and culture of human induced pluripotent stem cells, can solve the problems of difficulty in large-scale expansion, easy differentiation of hiPSCs, low efficiency of cryopreservation and recovery, and achieve the effect of improving efficiency.

Active Publication Date: 2014-12-03
SHENZHEN SANQI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the above-mentioned problems in the prior art, the present invention provides a single-cell subculture and culture method of human induced pluripotent stem cells (hiPSCs), which can rapidly expand hiPSCs, keep the cells in a good growth state, and overcome the HiPSCs are easy to differentiate, difficult to expand on a large scale, low in cryopreservation and recovery efficiency, easy to carry out nucleofection and single cell cloning, and bring great convenience to operations such as gene modification

Method used

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  • Passage method and application of human induced pluripotent stem cells
  • Passage method and application of human induced pluripotent stem cells
  • Passage method and application of human induced pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 hiPSCs single cell passage

[0041] 1) Spread ECM on a 6-well plate 1-2 hours before cell subculture.

[0042] 2) Take out the cells with a confluence rate of about 75%-85% from the incubator, wash once with DMEM / F12 medium, add Accutase digestive enzyme and place in 37°C with 12% volume fraction of CO 2 Digest in the incubator, when the cells are basically digested, add mTeSR1 to stop the digestion.

[0043] 3) Centrifuge at 200g for 3 minutes, discard the supernatant, resuspend the cells in mTeSR1 containing Y27632, and gently pipette evenly.

[0044] 4) Count the cells with a hemocytometer, inoculate them on a 6-well plate pre-coated with ECM at a density of 20,000-30,000 / cm2, and place them in CO at 37°C and a volume fraction of 12%. 2 Incubator cultivation.

[0045] 5) The next day, observe the cell density and adhesion under the microscope ( Figure 1A ). If there is contact between the cells and the cell density is appropriate, remove Y27632 and repla...

Embodiment 2

[0048] Example 2 hiPSCs single cell clone

[0049] 1) Take out cells with a confluence rate of about 75%-85% from the incubator, add Accutase digestive enzyme and place in 37°C, 8-15% volume fraction of CO 2 Digest in the incubator, when the cells are basically digested, add mTeSR1 to stop the digestion.

[0050] 2) Trypan blue staining, use a hemocytometer to count the number of viable cells, use mTeSR1+Y27632 inhibitor to dilute the cell suspension to 5-15 cells / ml, add 100ul of the cell suspension to the ECM-coated 96-well plate / Well, put in 8-15% CO 2 cultured in an incubator.

[0051] 3) After 10 days of microscopic examination, the cloned hiPSCs were passaged and expanded to obtain single-cell-derived hiPSCs.

Embodiment 3-h

[0052] Example 3- Identification of pluripotency after single-cell passage of hiPSCs

[0053] After hiPSCs were single-cell passaged for 20 passages using the method in Example 1, some cells were taken for EB formation experiments and Teratomas formation experiments.

[0054] 1) Embryooid body formation

[0055] Resuscitate hiPSCs on the ECM, and transfer the cells to the feeder for culture when the cells grow to 75%. After the clonal group grows on the feeder for 5-6 days, transfer the clonal group to suspension culture ( Figure 2A ). After 8 days of suspension culture, EB balls were transferred to gelatin-coated 6-well plates for adherent growth. After 8 days of EB ball adherent growth, the RNA of adherent cells was collected, and the expression of three germ layer markers was detected by qPCR ( Figure 2B ). The results showed that compared with hiPSCs, the cells differentiated by EBs increased in the three germ layer cell markers, and compared with hiPSCs, there was ...

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Abstract

The invention relates to a passage method and an application of human induced pluripotent stem cells. The method solves the problems of easy cell differentiation, low transfection efficiency and difficult gene modification of traditional clone block passage methods. The method is a single cell passage and culture method, and comprises the following steps: fully digesting pluripotent stem cell clones to singe cells by using digestive enzyme added with a ROCK inhibitor; counting by using a blood counting chamber or a cell counter; spreading cells on an ECM coated culture plate according to a density of 20000-30000 cells / cm<2>; culturing the induced pluripotent stem cells by using a medium containing the ROCK inhibitor culture conditions comprising a temperature of 37DEG C and containing 8-15% of CO2; and passing 20 generations to obtain induced pluripotent stem cells with unchanged pluripotency including the expression of pluripotency genes, in vivo and in vitro differentiation potential and the like.

Description

technical field [0001] The invention relates to a subculture method of human induced pluripotent stem cells, in particular to a single cell subculture and culture technology of human induced pluripotent stem cells. Background technique [0002] In 2006, Shinya Yamanaka of Kyoto University in Japan first reported the research on induced pluripotent stem cells in the world-renowned academic journal Cell. They cloned the genes of four transcription factors, Oct3 / 4, Sox2, c-Myc and Klf4, into viral vectors, and then introduced them into mouse fibroblasts, and found that they could be induced to transform, and successfully obtained embryonic stem cells (embryonic stem cells, ES cells) are called induced pluripotent stem cells (iPSCs, induced pluripotent stem cells). The resulting iPSCs were similar to embryonic stem cells in terms of morphology, gene and protein expression, epigenetic modification status, cell doubling ability, embryoid body and teratoma generation ability, and ...

Claims

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Application Information

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IPC IPC(8): C12N5/10
Inventor 杨佳银刘雨晴
Owner SHENZHEN SANQI BIOTECH
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