Method for establishing iPS
An erythroid culture and nucleated cell technology, applied in the field of establishing iPS cells, can solve the problems of increasing the use cost, being unsuitable for extensive and large-scale clinical application, increasing the risk of disease treatment, and achieving the effect of simplifying the culture system
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0040] Example 1: A method for establishing iPS using peripheral blood cells without gene insertion, without FBS, and without Feeder:
[0041] 1. Collect peripheral blood from healthy volunteers, separate mononuclear cells and myeloid CD33 + Cell or Lymphoid CD3 + CD19 + cells and pre-stimulated culture
[0042] Collect 10ml of venous blood from healthy volunteers, dilute it with PBS-EDTA 1:1, slowly add it to 10ml Ficoll, centrifuge at 400g for 30 minutes, and carefully collect the mononuclear cell layer (buffy coat) after discarding the supernatant.
[0043] Tianjin Blood Center purchased fresh white blood cells, and collected mononuclear cells by Ficoll method as above.
[0044] After the collected mononuclear cells were counted, they were cultured with erythroid induction medium for 8 days.
[0045] Erythroid induction media include:
[0046] Serum free medium (SFM) 100ml
[0047]
[0048] Note: After SFM configuration, filter and sterilize with 0.2μm filter, stor...
Embodiment 2
[0063] Embodiment 2, analysis of teratomas in NOD / SCID mice
[0064] Select hiPS cells whose karyotype is identified as normal chromosome, count, 5x10 6 Add 100 μl of Matrigel solution to the cells and mix well. Planted subcutaneously in the groin of NOD / SCID mice with a 1ml syringe. Subcutaneous tumors can be seen in mice at about 1 month old. The excised tumor tissue was fixed in 10% formaldehyde solution for 24 hours, then embedded in paraffin, sectioned and stained with H.E. to determine the morphology of the tumor. Such as Figure 4 As shown, iPS cells can be injected into the subcutaneous inguinal of NOD / SCID mice to generate teratomas, and H&E staining of tissue sections identified three germ layer histological forms, indicating that iPS has the pluripotency to differentiate into three germ layer cells.
Embodiment 3
[0065] Example 3, analysis of iPSCs genome non-integration
[0066] After 1, 10 and 15 passages of iPS clones, the cells were collected and the total DNA was extracted using Qiagen DNeasy kit. The DNA of the cells before electroporation was used as a negative control, and one copy of pCEP-OS mixed in the genome was used as a positive control. The SYBR dye method was used to detect the residual amount of Episomal plasmid in iPS cells after multiple passages by real-time PCR. The primers of CEP plasmid DNA can detect both free and integrated forms at the same time. The primer sequences are as follows:
[0067] use
[0068] Primers were set for the specific sequence of the episomal plasmid, and PCR was used to identify whether there was an insertion sequence in the 10-generation iPS cell line, and no transgene was found in 6 different cell lines, such as Figure 6 shown. Demonstrate that iPS is an insertion-free iPS cell line.
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More