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Cryopreservation solution for tendon stem cells from achilles tendon of human and preparation method of cryopreservation solution

A tendon stem cell and cryopreservation technology, applied in the field of stem cells, can solve problems such as being unsuitable for clinical application, affecting treatment results, contaminating allergens, etc., to avoid the introduction of heterologous substances, high clinical safety, and cell damage. small effect

Active Publication Date: 2016-01-06
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Currently commonly used cell cryopreservation solutions or commercially available cell cryopreservation solutions are usually composed of fetal bovine serum and dimethyl sulfoxide, but fetal bovine serum is a heterogeneous substance with complex components and introduces contamination and allergen risk, not suitable for clinical application
Especially in cell therapy, the presence of heterologous proteins may cause unknown adverse reactions and seriously affect the treatment results

Method used

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  • Cryopreservation solution for tendon stem cells from achilles tendon of human and preparation method of cryopreservation solution
  • Cryopreservation solution for tendon stem cells from achilles tendon of human and preparation method of cryopreservation solution
  • Cryopreservation solution for tendon stem cells from achilles tendon of human and preparation method of cryopreservation solution

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1, human Achilles tendon-derived tendon stem cell cryopreservation solution

[0024] Tendon stem cells were cultured in L-DMEM containing 10% FBS at 37°C, 5% CO 2 Cultivate under the condition of 2-3 days, subculture at 1:5; take the tendon stem cells in the logarithmic growth phase, make cell suspension with cell culture medium, adjust the cell concentration to 1×10 5 cell / mL, take 15mL of cell suspension and inoculate it on a 10cm-diameter petri dish, at 37°C, 5% CO 2 The cell culture medium is L-DMEM containing 1% FBS.

[0025] When culturing to the 24th hour, collect the supernatant for later use, and replace the new cell culture medium to continue culturing; when cultivating to the 48th hour, collect the supernatant again, and centrifuge the supernatant collected twice at 2000RPM for 5min, Remove cell debris and store at a constant temperature of 4°C for later use. Prepare cryopreservation solution for cryopreserved cells and conventional cell cryopre...

Embodiment 2

[0029] Example 2. Cryopreservation, resuscitation and viability of tendon stem cells derived from human Achilles tendon

[0030] The final density of cryopreserved tendon stem cells derived from human Achilles tendon is 1-5×10 6 cell / mL, cryopreservation volume is 1.5mL / tube, cryopreservation according to the following procedures: first place at 4°C for 2h, then place in liquid nitrogen port for 30min, and finally put into liquid nitrogen (-196°C).

[0031] After 2 weeks of cryopreservation, the cells were resuscitated. Each group resuscitated 3 tubes. The cryopreservation tubes were taken out of the liquid nitrogen and placed directly in warm water at 37°C, and shaken from time to time to melt as soon as possible in a short time. Centrifuge in a centrifuge for 5 minutes, discard the supernatant, add 1mL cell culture medium to each tube, count and calculate the average value. Specifically, the assay methods for cell count and cell viability are as follows: 1. trypsinize the a...

Embodiment 3

[0035] Example 3, Proliferation Ability of Cells After Recovery

[0036] Take a 12-well culture plate, inoculate each well with the recovered cells at a density of 10,000 cells / well, add 1 mL of L-DMEM solution containing 10% FBS to each well, and place at 37°C, 5% CO 2Cultured in an incubator, the medium was changed every three days. From the 3rd day (the newly recovered cells need about 24 hours to adapt to the environment), take out the plate every 24 hours, randomly select 3 wells, aspirate the old culture medium and wash it with PBS, add trypsin to digest the cells, stop the digestion, and prepare single cells Suspension, blow evenly, take 10 μL of cell suspension and 10 μL of 0.4% trypan blue, mix well, add the sample to the blood cell counting plate, count the number of cells in the large squares at the four corners of the counting plate under a 10-fold microscope, and count the number of cells As shown in Table 3, the cell growth curve is drawn with time as the horizo...

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Abstract

The invention relates to the technical field of stem cells, in particular relates to a cryopreservation solution for tendon stem cells from an achilles tendon of a human and a preparation method of the cryopreservation solution. The cryopreservation solution for the tendon stem cells from the achilles tendon of the human consists of a conditional culture solution and dimethyl sulfoxide. The preparation method of the cryopreservation solution for the tendon stem cells from the achilles tendon of the human comprises the following steps: (1) cell culture; (2) preparation of the conditional culture solution; and (3) mixing of the conditional culture solution and dimethyl sulfoxide at a volume ratio of 9 to 1. According to the cryopreservation solution, the cost of cell cryopreservation can be reduced to a certain extent, and introduction of heterologous substances can be avoided; compared with the conventional cell cryopreservation solution, the cryopreservation solution disclosed by the invention is higher in clinical safety. The cryopreservation solution for the tendon stem cells from the achilles tendon of the human is lower in cell damage effect in a cell cryopreservation process; meanwhile, the cell survival rate is higher; the cryopreservation effect is obviously higher than that of the conventional cell cryopreservation solution; the cryopreservation solution can be used for long-time storage and application of the tendon stem cells from the achilles tendon of the human.

Description

technical field [0001] The invention relates to the technical field of stem cells, in particular to a cryopreservation solution of tendon stem cells derived from human Achilles tendon and a preparation method thereof. Background technique [0002] Stem cells are a type of pluripotent cells with self-renewing ability. Under certain conditions, it can differentiate into a variety of functional cells, which are called "universal cells" in the medical field. Stem cells are divided into embryonic stem cells (embryonic stem cells, ES cells) and adult stem cells (somatic stem cells) according to their developmental stages. Stem cells are divided into three categories according to their developmental potential: totipotent stem cells (TSC), pluripotent stem cells (pluripotent stem cells) and unipotent stem cells (unipotent stem cells) (multipotent stem cells). [0003] Necrosis of the femoral head is a common and frequently-occurring disease in orthopedics, with a long course of di...

Claims

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Application Information

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IPC IPC(8): A01N1/02
Inventor 陈海佳王一飞葛啸虎黄幸李平
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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