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In vitro chondrocyte telomere extension and proliferation culture medium, kit and application

A telomere lengthening and chondrocyte technology, applied in the field of cell biology, can solve problems such as affecting the quality of life, inability to walk, stiffness, etc., and achieve the effects of increasing telomerase activity, prolonging telomere length, and increasing proliferation rate

Active Publication Date: 2020-04-21
THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Patients often feel pain and stiffness, and eventually develop into severe arthritis, or even be unable to walk, which seriously affects the quality of life
[0007] Since mature cartilage tissue is a tissue without nerves, blood vessels, and lymphatic vessels, and chondrocytes are sparsely distributed in cartilage tissue, the self-renewal speed of cartilage tissue is slow and the self-repair ability is limited
In particular, the proliferation rate and self-repair ability of chondrocytes in elderly patients are very low, which greatly limits the application of senile cartilage injury treatment

Method used

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  • In vitro chondrocyte telomere extension and proliferation culture medium, kit and application
  • In vitro chondrocyte telomere extension and proliferation culture medium, kit and application
  • In vitro chondrocyte telomere extension and proliferation culture medium, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1 Telomerase mRNA and complete culture medium process chondrocytes of elderly patients

[0065] 1. Telomerase mRNA must first be synthesized in vitro. Plasmid pBABE-neo-hTERT (plasmid 1774, Addgene, Cambridge, MA, USA) was amplified by PCR (primers were SEQ ID No.2 and SEQ ID No.3), and the TERT CDS region containing telomerase was amplified DNA (SEQ ID No.4). Then the 3'UTR (SEQ ID No.5) and 5'UTR (SEQ ID No.6) were amplified from human genomic DNA, and the polyA-containing sequence (SEQ ID No.7) was synthesized from the whole sequence, using overlapping PCR Methods All of them were connected to the DNA of the TERT CDS region to form a fragment: 5'UTR-TERT CDS-3'UTR-sequence containing polyA. Then, the fragment was double-digested with restriction endonucleases EcoR1 and SalI and ligated to the same digested plasmid Pcmv6-XL4. The telomerase plasmids with the correct sequence verified by sequencing, the target gene fragments in these plasmids are lineariz...

Embodiment 2

[0069] Example 2 Detection of chondrocyte proliferation, telomerase activity, telomere length and cell metabolism after telomerase mRNA and complete medium treatment

[0070]In this example, 4 groups of experiments are set up, namely control, hTERT, Compound and hTERT+compound, wherein control represents cells without any treatment, using basal medium (DMEM / F12 (Hyclone) + 5% human AB serum (Innovative)) Culture; hTERT represents cells transfected with telomerase mRNA, cultured in basal medium; Compound represents cells not transfected with telomerase mRNA, cultured in complete medium supplemented with various small molecule compounds; hTERT+compound represents cells Transfect telomerase mRNA and culture in complete medium. The primary chondrocytes were the zeroth generation, and at the third generation, the cells were planted in the wells of a 6-well plate, with 20,000 P3 generation chondrocytes per well.

[0071] When the cell density reaches nearly 100%, digest the cells w...

Embodiment 3

[0078] Example 3 Assembly of Human-derived Tissue Engineered Regenerated Cartilage

[0079] After the chondrocytes of elderly patients treated with telomerase mRNA and cultured in the complete medium containing various small molecular compounds are expanded to a sufficient number and in good growth state, it is necessary to prepare cell suspensions on collagen membrane scaffolds. After assembly and compounding, when the chondrocytes are completely attached to the collagen membrane, the finished human tissue engineered regenerated cartilage is made and transported to the hospital for treatment.

[0080] Firstly, AM medium (DMEM / F12 (Hyclone)+10% patient's autologous serum) was prepared. The cultured chondrocytes were taken for cell digestion, digested with 0.05% trypsin at 37°C for 5 min, and added complete medium to terminate the digestion. Centrifuge at 300g for 5 minutes at room temperature, resuspend the cells in AM medium, and count the cells. Centrifuge at 1500rpm for 5...

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Abstract

The invention discloses a culture medium, a kit and application for in-vitro chondrocyte telomere multiplication culture. The culture medium is prepared by adding serum, vitamin C having a final concentration of 5-50mu g / ml, B18R or p65i having a final concentration of 0.01-2mu g / ml, rapamycin having a final concentration of 0.1-50ng / ml and resveratrol having a final concentration of 0.1-50mu M into a DMEM / F12 or RPMI1640 basic culture medium, wherein the serum is human AB serum having a volume final concentration of 1-20 percent or fetal calf serum having a volume final concentration of 1-20 percent. By using the culture medium or kit for in-vitro chondrocyte telomere multiplication culture, chondrocyte can be subjected to telomerase mRNA transfection in vitro, so that telomere of chondrocyte is lengthened in subsequent culturing, and multiplication and vitality of chondrocyte, especially senile patients, can be remarkably improved. The culture medium or kit can be used for improving or treating cell function decline caused by senescence, and can be practically applied to regenerative medicine treatment.

Description

technical field [0001] The invention relates to the field of cell biology, in particular to a culture medium, kit and application for chondrocyte telomere extension and proliferation in vitro. Background technique [0002] Telomeres are protective caps at the ends of chromosomes, which are special structures composed of short, repeated non-transcribed sequences (TTAGGG) and some binding proteins, which can protect the stability of the genome and prevent the loss of genetic material at the ends of chromosomes. Cell expansion progressively affects telomere length. When the telomere is shortened to a certain extent, its protection will disappear, which will cause cell aging and apoptosis. [0003] Telomerase is the enzyme that synthesizes telomeres and maintains their length. Telomerase consists of two parts, an RNA template subunit, the telomerase mRNA component, and a protein catalytic unit, the telomerase reverse transcriptase. Not all body cells have telomerase. Embryon...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10
CPCC12N5/0655C12N2500/38C12N2501/727C12N2501/999C12N2510/00
Inventor 吴海涛
Owner THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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