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Preparation method of low-immunogenicity iPSC cell, low-immunogenicity iPSC cell and composition

A technology of immunogenicity and composition, applied in the field of cells, can solve the problems of difficult standardization, high-throughput production, cumbersome steps, etc., and achieve the effects of excellent differentiation effect, high positive expression rate, and time reduction.

Active Publication Date: 2022-07-01
HELP STEM CELL INNOVATIONS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing technology such as [3] discloses the method of obtaining such cells, but the steps are cumbersome, and the introduction of three kinds of editing requires at least two steps of transfection and two clone selection and identification, and it is difficult to achieve standardization and high-throughput production

Method used

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  • Preparation method of low-immunogenicity iPSC cell, low-immunogenicity iPSC cell and composition
  • Preparation method of low-immunogenicity iPSC cell, low-immunogenicity iPSC cell and composition
  • Preparation method of low-immunogenicity iPSC cell, low-immunogenicity iPSC cell and composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Construction of the vector

[0069] 1) Selection of CRISPR gene editing vector: The plasmid / vector that expresses Cas9 and sgRNA at the same time is selected from PX458 obtained from Addgene;

[0070] 2) Insertion of the target sgRNA into PX458: The sequence between the two BbsI sites (245 and 267) was replaced by the corresponding sgRNA sequence by conventional molecular cloning methods, that is, the corresponding target sgRNA vector was obtained.

[0071] A total of 4 sgRNA plasmids were constructed in the experiment, and the tables corresponding to their numbers and sgRNA sequences are shown in Table 1:

[0072] Table 1 4 sgRNAs

[0073] name / number use for site sequence B2M-sgRNA CRISPR vector B2M SEQ ID NO: 2 CIITA-sgRNA CRISPR vector CIITA SEQ ID NO: 8 AAVS1-sgRNA CRISPR vector AAVS1 SEQ ID NO: 21 AAVS1-CD47 sequence Homologous recombination template vector AAVS1 (insert CD47) SEQ ID NO: 20

[0074]...

Embodiment 2

[0076] Example 2 Electrotransfection

[0077] 2.1 iPSCs were cultured to 90% confluency using standard methods, iPSCs were cultured to 90% confluence using standard methods, cells were digested and counted.

[0078] 2.2 After counting the cells, remove 1×10 6 -1×10 7 cells (in this example, 1 × 10 6 ), centrifuge at low speed, take out the centrifuge tube to the biological safety cabinet, unscrew the cap of the tube, and discard the supernatant. By definition, low-speed centrifugation is a centrifugation with a rotational speed of 8,000 r / min or less and a relative centrifugal force of 10,000 × g or less.

[0079] 2.3 Absorb 0.5ug-5ug (preferably 0.5ug-2ug, in this example, 1ug) of the above four plasmids respectively, and add them into the electroporation buffer. The four plasmids absorb 0.5ug-5ug respectively, and can be designed as 0.5ug, 0.6ug, 0.7ug, 0.8ug, 0.9ug, 1ug, 1.1ug, 1.2ug, 1.3ug, 1.4ug, 1.5ug, 1.6ug, 1.7 ug, 1.8ug, 1.9ug, 2ug, 2.1ug, 2.2ug, 2.3ug, 2.4ug, 2....

Embodiment 3

[0081] Example 3 Electric transfer to pick clones

[0082] 3.1 Change 2ml Stemflex medium every day, after two days of culture, digest, count, and inoculate 5000 cells in a 10cm dish (Matrigel was pre-coated, and 10ml Stemflex+Rocki medium was added), Rocki in Stemflex+Rocki medium The concentration is 10 μM.

[0083] 3.2 After about 7 days of culture, single clones were picked in the laminar flow workbench. A total of 48 clones were picked and inoculated in two 24-well plates (Matrigel was pre-laid, and 500ul Stemflex+Rocki medium was added to each well). Continue to culture for about 7 days to verify positive clones.

[0084] 3.3 Identification of triple edited clones.

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Abstract

The invention belongs to the technical field of cells, and relates to a low-immunogenicity iPSC cell preparation method, a low-immunogenicity iPSC cell and a composition, the preparation method comprises the following steps: using a CRISPR system: introducing plasmid containing Cas9 protein and sgRNA targeting B2M site into the cell to knock out B2M protein; the method comprises the following steps: introducing plasmids of sgRNA containing a Cas9 protein and a targeted CIITA site into a cell so as to knock out the CIITA protein; introducing a plasmid which contains Cas9 protein and a targeted AAVS1 site and is inserted into a multi-nucleic acid sequence sgRNA for coding an extracellular fragment of CD47 protein into the cell; editing of the three genes is achieved only through one-step electrotransfection operation. The construction of the low-immunogenicity iPSC cell line is realized by adopting a rapid iPSC cell editing mode.

Description

technical field [0001] The invention belongs to the field of cell technology, and relates to a preparation method of low immunogenic iPSC cells, low immunogenic iPSC cells and compositions. Background technique [0002] Induced pluripotent stem cells (iPSCs) were originally developed by Japanese scientist Shinya Yamanaka in 2006 by using a viral vector to transfer a combination of four transcription factors (Oct4, Sox2, Klf4 and c-Myc) into adult somatic cells. A pluripotent stem cell similar to embryonic stem cells obtained by its reprogramming. [0003] Human induced pluripotent stem cells have the following characteristics: no ethical issues; easy source of cells, available from adult cells (such as skin, blood); strong differentiation ability, able to differentiate into different functional cells; infinite expansion, which can reduce costs and high cell consistency. [0004] Human induced pluripotent stem cell-derived or derived cell products hold promise for clinical ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/113C12N15/55C12N15/12C12N15/90
CPCC12N5/0696C12N15/113C12N9/22C12N15/907C07K14/70503C12N2310/20C12N2510/00C12N15/1138C07K14/70539C07K14/47C07K14/005C12N2506/45Y02A50/30
Inventor 葛文雪陈涛涛王嘉显
Owner HELP STEM CELL INNOVATIONS CO LTD
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